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LIST OF ABSTRACTS
SCIENTIFIC SESSION: TISSUE ENGINEERING
TE.1. Emerging stem cell based technologies in drug discovery and therapy of neurological disorders .......................9
TE.2. Primordial germ cells and avian oviduct cells as a tool for in vivo production of human
biopharmaceuticals ...........................................................................................................................................10
TE.3. Strategies for optimizing cell and tissue culture: best practices, recommendations and threats .........................11
TE.4. Hepatogenic differentiation ability of human adipose tissue- derived mesenchymal stem cells
and their potential for liver disease treatment....................................................................................................12
TE.5. The bad seeds: Neural stem cells and the origin of malignant glial tumors .......................................................13
TE.6. hPSC & AdMSC cultivation in Eppendorf Stirred-Tank Bioreactors ................................................................14
SCIENTIFIC SESSION: BIOMATERIALS
B.1. Resorbable micro- and nanoparticles as a platform for targeted therapies in skeletal system ..............................16
B.2. Collagen meniscus implants ................................................................................................................................18
B.3. Cell-biomaterial interaction as a player in tissue engineering ..............................................................................19
B.4. Polymers from natural sources for biomedical applications ................................................................................20
B.5. Advance your genomic research ..........................................................................................................................21
SCIENTIFIC SESSION: REGENERATIVE MEDICINE
RM.1. Cell therapy for cardiovascular diseases - ready for clinical application ..........................................................22
RM.2. Modelling cardiovascular disease: a new approach with pluripotent stem cells ...............................................23
RM.3. Management of corneal limbal stem cell deficiency with cultivated epithelial cells ........................................24
RM.4. Medical applications of epidermal progenitor cells ..........................................................................................25
RM.5. Regenerative medicine in urogynecology: are we ready? ................................................................................26
RM.6. Treatment of knee osteoarthritis by microfragmented adipose tissue ...............................................................27
RM.7. Cell technologies for treatment of the consequences of civil and combat bone lesions ....................................28
ORIGINAL PAPER SESSION
O.1. Application of 3D polyethersulfone membranes for repair of articular cartilage defects.....................................30
O.2. New approach in evaluation of transplanted cells fate after transurethral injection in large animal model..........31
O.3. Differentiation potential of Pax7-/- pluripotent stem cells in regenerating skeletal muscle .................................32
O.4. Microparticles with vancomycin immobilized on porous scaffolds for prevention of implant-related
infections in bone tissue ......................................................................................................................................33
O.5. Keratin associeted proteins as a new type of biodressing supported wound healing ...........................................35
O.6. Autotransplantation of the adipose tissue derived stem cells for diabetic foot ulcers healing ..............................37
O.7. Under the surface: from hair to digit regeneration unveiling regenerative potential of distinct skin stem
cells ....................................................................................................................................................................38
YOUNG SCIENTIST’S SESSION
Y.1.
Y.2.
Y.3.
Y.4.
In vitro model for probiotics selection based on immune-related gene expression in chickens ..........................40
DT40 cell line as a tool for functional analyses of chicken immune response ....................................................41
Influence of cross-linking on the amniotic membrane ........................................................................................43
Collagen and elastin hydrolysates hydrogels cross-linked by dialdehyde starch and pectin ...............................44
Central European Conference on Regenerative Medicine 2015
Y.5. Interactions of modified with ascorbic acid poly(ester urethane)s with phosphate buffered saline (PBS)
and their hemocompatibility...............................................................................................................................45
Y.6. Scanning Electron Microscopy of microporous poly(ester urethane)s modified with and ascorbic acid
for tissue scaffolds .............................................................................................................................................46
Y.7. Ti/TiO2 nanotube platform for the electrochemical detection of tumor necrosis factor (TNFα) .........................47
Y.8. Factors affecting status of isolated human hepatocytes ......................................................................................48
Y.9. Growth properties and pluripotency marker expression of spontaneously formed three-dimensional
aggregates of human adipose-derived stem cells ................................................................................................49
Y.10. Wound repair with cryopreserved cord blood serum treatment .........................................................................50
Y.11. Poly(ester urethane)s modified with ascorbic acid as useful biomaterials in electrospinning process ...............51
Y.12. Microporous hydroxyapatite material for tissues regeneration in periodontitis in dogs - a clinical study..........52
Y.13. Composites of chitosan/collagen/hyaluronic acid with addition of nanohydroxyapatite ...................................53
POSTER SESSION
P.1. Relationship between neural differentiation, hypoxia and epigenetic regulation in HUCB-NSC at low
and ambient oxygen concentration ......................................................................................................................55
P.2. Cell-penetration in model microorganisms by selected phthalocyanine photosensitizers ....................................56
P.3. Goat animal model - an attractive alternative for testing the effects of cell transplantation into the urethra ........57
P.4. Surface properties of thin films based on collagen from scales of northern pike (Esox lucius) ............................59
P.5. Polyurethanes as biomaterials for cardiovascular system regeneration and restoration - recent trends
and future directions ............................................................................................................................................60
P.6. Polysaccharide-protein hydrogels as scaffolds for corneal epithelial culture .......................................................61
P.7. Lysophosphatidylcholine modulates activity of pancreatic cells ..........................................................................62
P.8. Mechanical properties of collagen/silk fibroin composites ..................................................................................63
P.9. Fabrication techniques of polyurethanes designed for tissue engineering – recent developments and future
prospective ...........................................................................................................................................................64
P.10. Calcite scaffolds for tissue engineering..............................................................................................................65
P.11. Alumina-polyurethane composite for medical application .................................................................................66
P.12. Nucleoside 5’-O-thiotriphosphates as promising agents in wound healing ........................................................68
P.13. Biological activity of selected phthalocyanines .................................................................................................69
P.14. Synthesis and photodegradation of bis[4-(dimethylamino)-phenyl]squaraine in organic solvents .....................70
P.15. Modern 3D bioprinting capabilities as the future of regenerative medicine? .....................................................71
P.16. Effect of C60 fullerene aqueous colloid solution on biochemical parameters of liver and visco-elastic
membrane properties of rats’ erythrocytes ........................................................................................................72
P.17. Enzymatically induced mineralization of gellan gum for bone tissue engineering applications: impact
of manufacturing conditions on mineralization effectiveness.............................................................................74
P.18. Characterization of thin chitosan/hyaluronic acid blend films ...........................................................................76
P.19. Synthesis and properties of magnetic nanoparticles with polymer coating for bioligands immobilization.........77
P.20. Expansion and directed differentiation of human endometrial stromal cells under conventional
and low-oxygen cell culture conditions .............................................................................................................78
P.21. Neural crest-derived stem cells from bulge region of whisker follicle: potential for multilineage
differentiation and regeneration of injured peripheral nerve .............................................................................80
P.22. Biomedical (nano)engineering – nanofibers used in regenerative medicine.......................................................81
P.23. What are the potential prospective applications of stem cells in clinical dermatology and plastic surgery?
The scientific report from in vivo studies. ..........................................................................................................83
P.24. Is amniotic membrane a promising material for regeneration of the urinary tract? ............................................84
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P.25. Influence of surface heterogeneities on collagen adsorption and osteoblast-like cells adhesion
and proliferation ................................................................................................................................................85
P.26. Autotransplantation of the adipose tissue derived stem cells for venous stasis ulcers healing ...........................87
P.27. Review of 3D printing applications in medicine ................................................................................................88
P.28. Chemical synthesis and analysis of peptides modified on their C-terminus with 4-amidinobenzylamine
(Amba) ...............................................................................................................................................................89
P.29. Biocompatible poly-L-lactic acid/hydroxyapatite nanofibrous composite for bone tissue regeneration .............90
P.30. Implantation of xenopericardium and aortic valve leaflets .................................................................................92
TRICOMED LECTURER
T.L. New generation mesh implants used in urogynaecology .....................................................................................94
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SCIENTIFIC SESSION: TISSUE ENGINEERING
Saturday, 14 March 2015
Chair: Prof. Samy L. Habib MS, PhD; Prof. Piotr Trzonkowski MD, PhD;
Krzysztof Kobielak MD, PhD
TE.1. Emerging stem cell based technologies in drug discovery and therapy
of neurological disorders
Prof. Leonora Bużańska PhD, DSc
Stem Cell Bioengineering Laboratory, Mossakowski Medical Research Centre,
Polish Academy of Sciences, Warsaw, Poland
Developing reliable human-based in vitro systems to study disease models as well
as drug toxicity and their mode of action is a major challenge for establishing new
and safe therapies. The emerging technologies involved in the creation of such
systems combine advancement in stem cell research with bioengineering
of the stem cell niche. This allows creating “biomometic” paradigm mimicking
in vivo-like environments. The strategy to create such advanced research systems
isbidirectional. The first approach is to establish “microscale” microenvironments
allowing to test cellular cultures even at the single cell resolution, on the high
content/throughput platforms. Such platforms are already implemented for drug
and toxicity screening, but also for basic research to test response of cells
representing relevant human disease models with specific genetic background.
The second approach is to obtain in “macroscale” in vitro biomimetic systems
that allow to create and grow complicated human tissues, organoids and even
whole organs. Such systems should provide a structural and logistic 3D template
for cell differentiation and functional assembly as well as environmental control
by molecular and physical signaling. In the latter approach stem cells
are cultivated on the scaffolds within bioreactors that provide in vivo-like
environments, producing differentiated, mature tissues implemented in tissue
engineering and new treatment modalities for regenerative medicine.
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Both “micro” and “macro” research systems are endorsed by personalized
medicine strategy, where stem cells for testing and the treatment are obtained
from the patient. In that respect the induced Pluripotenet Stem Cell (iPSC)
and direct lineage conversion technologies provide a giant advancement step
for pharmacology and regenerative medicine applications, enabling personalized
testing, diagnosis and treatment.
Acknowledgements. Sponsored by statutory funds 20/2015 to MMRC.
TE.2. Primordial germ cells and avian oviduct cells as a tool for in vivo
production of human biopharmaceuticals
Prof. Marek Bednarczyk PhD, DSc
Department of Animal Biochemistry and Biotechnology, UTP University
of Science and Technology, Bydgoszcz, Poland
Transgenic avian bioreactors have been proposed as a powerful way
of addressing the growing need for recombinant biopharmaceutical production
(Kawabe and Kamihira, 2011). However, despite many advantages, production
of transgenic avian species has been delayed against mammalian models mainly
due to physiological and developmental differences between these groups.
This suggests that there is a strong need for more intensive basic research
on the development of technology platform based on the methods of genetic
engineering, molecular biology, cell manipulation and embryology, which will
become a tool for the expression of recombinant therapeutic proteins
in the oviduct of hen - bioreactor. Generation of transgenic chickens through
chimeric intermediates, produced by the transfer of primordial germ cells (PGCs)
has been attempted in our laboratory. The main elements of this strategy involve:
plasmid construction, isolation of donor embryonic cells, their transfection
in vitro, injection into the circulatory system of recipient embryos, raising
chimeras and identification of germline chimeras /transgene carriers/. These
chimeric chickens are then tested for germline transmission and used to create
fully transgenic animals through breeding strategies. To improve production
of recombinant proteins in eggs, we have undertaken studies on the efficiency
of various elements belonging to the above technology. The basis of new
approach to chicken transgenesis, elaborated in our laboratory are:
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 the methods of isolation, purification and transfection of chicken PGCs
(Chojnacka-Puchta et al., in press),
 the method of chicken oviduct epithelial cells (COEC) in vitro culture –
in vitro assay for testing tissue-specific vectors for expression of recombinant
proteins in oviduct of transgenic hens, (Kasperczyk et al., 2012; Stadnicka
et al., 2014),
 the constructs with the optimal combination of promoter/enhancer/regulatory
units (Chojnacka-Puchta, 2012).
Generation of transgenic birds by non-viral, cell-based method
and the effectiveness of the main steps of this strategy will be presented
and discussed in detail.
Acknowledgements. Research partially funded by the National Centre
for Research and Development, grant No. PBS3/A8/30/2015, and by National
Science Center grant No. NR 2011/03/N/NZ9/03814.
TE.3. Strategies for optimizing cell and tissue culture: best practices,
recommendations and threats
Dominika Biskup MSc & Jakub Bidas MSc,
SANLAB J. Kaczorek, M. Bińczak sp.j.
Incubators have long been classified as commodity equipment within
a laboratory. By supporting both clinical and non-clinical applications from
research to development, manufacturing and quality control, they play a vital role
throughout many testing processes. The use of cell culture incubators
for cultivation of cells and tissues for human use in transfusion, regenerative
medicine and cell therapy is set to expand.
Although, occasionally taken for granted, tightly controlled, reliable incubation
system can be one of the most valuable pieces of equipment in a laboratory.
The ability to culture a healthy cell line can make the difference between
a successful and failed investigation, and influence the results of research
projects.
Incubators continue to evolve from basic laboratory equipment to sophisticated
and precise environment for cell culturing. Panasonic continues to be
on the forefront of such advancements with multiple innovations that were first-in
class within this field.
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As the sole, authorized distributor of the Panasonic Japanese biomedical
equipment in Poland, SANLAB will introduce the latest innovations in the field
of work optimization presenting the most advanced model of CO2 incubator
MCO-170AIC. The following issues will be considered:
Consistent Environment
- stable CO2, humidity, and temperature levels
- ideal simulated in vivo conditions within the chamber
- recovery of the interior conditions after each door opening
- patented DHA Direct Heat and Air Jacket heating system
- unique Dual Detector IR Sensor
Contamination Control
- Panasonic's exclusive InCu saFe® copper-enriched stainless steel alloy
- patented, ozone-free, programmable SafeCell® UV lamp
- H2O2 decontamination system
- integrated shelf supports
- condensation management system
Increasing Work Efficiency
- data logging features / compliance to GLP and SOP
During conference, the actual unit of MCO-170AIC incubator will be presented
at the SANLAB's booth so you could easily familiarize with all the features
described. You are invited!
TE.4. Hepatogenic differentiation ability of human adipose tissue- derived
mesenchymal stem cells and their potential for liver disease treatment
Agnieszka Banaś PhD1, Takumi Teratani2, Takahiro Ochiya2
1
Center for Medical and Natural Sciences Research and Innovation, Faculty
of Medicine, University of Rzeszów, Rzeszów, Poland
2
Division of Molecular and Cellular Medicine, National Cancer Center Research
Institute, Tokyo, Japan
Regenerative medicine holds promise for the recovery of damaged organs
and tissues by the development of stem cell- based therapy. For example, even
though an injured liver is highly regenerative, many debilitating diseases lead to
hepatocyte dysfunction and organ failure. For this reason, liver is one target
for which the development of stem cell-based therapy is of great significance.
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Liver transplantation is the only effective treatment for severe liver injuries.
However, because of organ rejection and the limited number of donors,
alternative therapeutic approaches are needed. Transplantation of hepatocytes
or liver devices generated in vitro from stem cells could offer a potentially
unlimited and minimally invasive source of cells for hepatocyte replacement
and regeneration, therefore, might be superior to whole organ transplantation.
Mesenchymal stem cells (MSCs), largely present in the adult human body,
represent an attractive tool for the establishment of a stem cell-based therapy
for liver diseases. Recently, the therapeutic potential and immunomodulatory
activity of MSCs have been revealed. MSCs are adherent, fibroblast – like cells
positive for unspecific markers: CD29, CD44, CD73, CD90, CD105 and negative
for: CD11a, CD11b, CD14, CD19, CD34, CD45, CD79a and HLA-DR.
Adipose tissue-derived mesenchymal stem cells (AT-MSCs), so-called adiposederived stem cells or adipose stromal cells, are especially attractive in the context
of future clinical applications, because of their high accessibility with minimal
invasiveness and possible autologous transplantation.
The goal of this study is to present: in vitro hepatic differentiation of human
AT-MSCs; characterization of AT-MSc- derived hepatic-like cells with respect
to their hepatic markers and functions; and the therapeutic potential of AT-MSCs/
AT-MSCs – derived hepatic-like cells in vivo by their transplantation into nude
mice with CCl4-caused liver injury. Our results suggest that AT-MSCs represent
an attractive tool for studies on stem cell therapy for liver disease.
TE.5. The bad seeds: Neural stem cells and the origin of malignant glial
tumors
Jarosław Maciaczyk MD, PhD
University Medical Center Düsseldorf, Germany
Despite modern multimodal treatment including surgery, radiation and alkylating
agent-based chemotherapy, glioblastoma multiforme (GBM) has still a dismal
clinical prognosis with a median survival of 14-16 months after initial diagnosis.
Even though the cells of origin and sequence of molecular events leading
to the tumor development remain yet undiscovered, increasing experimental data
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and clinical observations suggest a pivotal role of neural stem cells (NSC)
and/or glial restricted progenitors (GRP) in this process. For that reason, precise
decipherment of the cytoarchitecture, local environment and region-specific
regulation of the neurogenic niches in adult central nervous system provides
valuable insights in brain tumor carcinogenesis. Moreover, recent discovery
of brain tumor stem cells (BTSC), responsible for initiation of malignant gliomas,
their propagation and resistance towards standard therapy, has a significant
impact on the current view on brain tumor pathophysiology. BTSC with apparent
similarity to neural stem cells show often an aberrant activation of stem cell
specific signaling pathways, such as Notch, WNT/β catenin or SHH, leading
to continuous proliferation and initiation of altered differentiation programs.
Experimental inhibition of abnormally activated stem-cell signaling pathways
in brain tumor models results in a specific depletion of otherwise therapy resistant
stem cell compartment. Therefore, thorough understanding of these molecular
regulatory mechanisms in context of BTSC biology seems to be compulsory
for the development of novel, cancer stem cell-targeted and patient-tailored
therapeutic approaches. The relationship between NSC and brain tumors appears
to be far more complex. Owing the natural tropism of NSC towards intracranial
pathologies, including brain tumors, the application of neural stem cells following
their ex vivo engineering might open a new avenue for cell-based therapy
in the treatment of patients suffering from malignant gliomas.
TE.6. hPSC & AdMSC cultivation in Eppendorf Stirred-Tank Bioreactors
Andreas Tack1, Ma Sha2, Robert Zweigerdt3
1
Eppendorf AG Bioprocess Center, Juelich, Germany
Eppendorf Inc., Enfield (CT), United States of America
3
Hannover Medical School (MHH), Hannover, Germany
2
Scientific progresses in the field of stem cell research and development during
the last decade created a growing demand for high cell numbers for various
applications in drug discovery and cell therapy. However commercialization
of these applications needs to overcome given limitations of established
cultivation methods. T-Flasks and other cultivation systems are widely used
in stem cell research, but are limited in terms of control and scalability. Stirredtank bioreactors, well-established in traditional cell culture, have the potential
to streamline stem cell development through precise control, automation,
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and comprehensive documentation. Single-use technologies reduce labor time
and simplify validation.
Induced pluripotent stem cells – Parallel cultivation in mini scale
Researchers at Hannover Medical School (Germany) cultivated human induced
pluripotent stem cells (hiPSC) in suspension using stirred-tank, single-use mini
bioreactors. While small working volumes save valuable resources, these
bioreactors offer industry standard online monitoring and control of key process
parameters such as pH, dissolved oxygen, and biomass formation. Their
use resulted in massively increased cell counts while maintaining pluripotency
levels needed for further investigations.
Mesenchymal stem cells – Clinical-scale production using microcarriers
Differentiation capabilities of mesenchymal stem cells from different sources
are limited but their availability makes them perfect candidates for clinical
applications in regenerative medicine.
Additional to regulatory restrictions the high amounts of cells needed for a single
patient still is one of the limiting factors to make next generation personalized
medicine feasible. Researchers at the Eppendorf Application Lab (Enfield, CT,
USA) were able to produce up to 1 billion adipose-derived mesenchymal stem
cells (AdMSCs) in a single run in a lab scale, microcarrier based single-use
bioreactor process. With this approach cell material for clinical applications
can be produced within short time and patient-specific without the need for laborintensive individual cultivation steps.
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SCIENTIFIC SESSION: BIOMATERIALS
Chair: Prof. Krzysztof Pielichowski PhD, DSc; Prof. Artur P. Terzyk PhD, DSc;
Prof. Andrzej Chwojnowski PhD, DSc
B.1. Resorbable micro- and nanoparticles as a platform for targeted
therapies in skeletal system
Prof. Elżbieta Pamuła PhD, DSc
Department of Biomaterials, Faculty of Materials Science and Ceramics,
AGH University of Science and Technology, Kraków, Poland
Corresponding author: [email protected]
Introduction. Skeletal system diseases such as osteoporosis and osteomielitis
have become socioeconomic challenge of our aging society affecting millions
of people worldwide [1,2]. Bone infections are treated with antibiotics
(gentamicin, vancomicin) usually via parenteral route [3], while antiosteoporosis
medicaments, i.e. bisphosphonates and calcitonin are administered orally
and intranasally [4,5]. Unfortunately all these routes are characterized
by insufficient biodistribution and systemic toxicity.
Aim of the study. Our approach is to design local and long-lasting delivery
systems of medicaments used in the treatment of bone tissue diseases. To assure
sustained drug release we entrapped drug molecules into long biodegrading
poly(lactide-co-glycolide) (PLGA) spherical micro- and nanocarriers.
Such loaded capsules were then diversely processed in a way to obtain different
forms of biomaterials (injectable or implantable) that meet requirements
of complex therapies.
Materials and methods. PLGA micro- and nanoparticels were produced
by double emulsification. Their morphology (AFM, optical microscopy),
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physicochemical properties (size, ζ potential, solubilization) and drug release
kinetics were assessed. Then the carriers were suspended in hydrogels of protein
and polysaccharide origin intended to be administered by injection directly
to affected area in bone tissue. The second approach was to immobilize
the carriers within highly porous PLGA scaffolds, while the third was based
on freeze-drying of spherical carriers/hydrogel composites to produce implantable
forms of biomaterials. The systems were studied with respect to surgical
handiness, mechanical properties (compression tests, rheology) and drug release
kinetics. The systems delivering antibiotics were tested with Staphylococus
spp.via Kirby-Bauer method, while those loaded with antiosteoporosis drugs
with cells responsible for bone remodeling (osteoblasts and osteoclasts).
Results and conclusions. The results showed that by changing shear rate
in the second step of emulsification it was possible to obtain drug-loaded PLGA
particles of defined size (micro- or nanometric). The carriers released the drugs
in a sustainable manner, which was even prolonged when the carriers were
suspended in hydrogels or processed into implantable composites. The systems
containing antibiotics showed antimicrobial activity against classical strains
of S. aureus and S. epidermidis and their clinical isolates form infected joins.
The systems containing bisphosphonates and calcitonin downregulated
osteoclasts and simultaneously did not affect osteoblasts functions. Proposed
processing methods preserved biological activity of encapsulated drugs and thus
the systems may constitute a promising solution in site-specific therapies of bone
tissues pathologies.
Acknowledgements. This study was supported from National Science Centre,
Poland (2012/05/B/ST8/00129).
References
1. Acros D. et al., Acta Biomater 10(2014)1793-1805.
2. Campoccia D. et al., Biomaterials 34(2013) 8533-8554.
3. Movrakanasa T.A. et al., Can J Diabetes 39(2015)18-20.
4. Bae J., Park J.W., Int J Pharm 480(2015)37-47.
5. Amaro M.I. et al., Int J Pharm 483(2015)6-18.
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B.2. Collagen meniscus implants
Łukasz Paczesny MD, PhD
Orvit Clinic Toruń, Poland
Meniscus function is crucial factor for knee biomechanics. The menisci play
an important role in shock absorption, synovial fluid distribution, proprioception
and joint stabilization. Meniscus tear is one of the most frequent knee trauma.
Destruction of the meniscus tissue causes disturbance of load distribution
on the cartilage surfaces and decreases knee stability. This leads to degenerative
changes of the knee joint and promotes osteoarthritis. In the recent 30 years many
meniscus saving surgical techniques were introduced. Nowadays meniscus tears
should be sutured arthroscopically, however there are many meniscus tears that
cannot be treated in this way because of tear localization or massive meniscal
tissue damage. In the mid 80-ies Stone, Steadman et al. developed a Collagen
Meniscus Implant. After 6 years of in vitro studies, they introduced it in clinical
practice, with first paper published in 1993. The structure of the CMI collagen
implant, surgical technique and tissue ingrowth phases are discussed on the basis
of recently published long –term studies and author’s own experience.
The advantages of the collagen implant include: good clinical outcome, slow
but confirmed meniscus-like tissue formation on the scaffold and long term
observations availability. The weaknesses of this treatment option include: slow
meniscus-like tissue formation with persisting abnormal MRI signal, the need
to remove most of the meniscus in the implantation site, even undamaged
meniscus tissue, very delicate implant structure resulting in very long rehab
protocol. The author’s own “surgeon’s dream” regarding future of meniscus
regenerative medicine would be a glue-like scaffold which would allow
aggressive rehabilitation, with no need to remove the healthy part of the meniscus
during the procedure and which would provide fast tissue ingrowth confirmed
by normal meniscus signal on the MRI scans.
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B.3. Cell-biomaterial interaction as a player in tissue engineering
Prof. Małgorzata Lewandowska- Szumieł PhD, DSc, FBSE
Tissue Engineering Laboratory, Department of Histology and Embryology,
Centre for Preclinical Research and Technology, Medical University of Warsaw,
Warsaw, Poland
By definition, tissue engineering combines the principles of engineering
and the life sciences toward tissue regeneration. The most classic product
of tissue engineering (TE) consists of cells and a biomaterial scaffold. Therefore,
at the very beginning of the development of the discipline, the research
was carried out on the availability of cells for transplantation, and in parallel,
on the best available biomaterials. Both paths were extremely successful thanks
to the extraordinary development in cell biology and cell culture techniques
on the one hand and the amazing progress in material engineering on the other
hand.
Cell-biomaterial interaction became an important issue because in TE
biomaterials must not only play a role of a biocompatible tissue substitute,
but they are also expected to serve as an ideal support for cells in a direct contact
in vitro. This requirement resulted in the more and more sophisticated
investigations of cell-biomaterial interaction - far beyond the requirements
of the routine cytotoxicity analyzes. It turned out that biomaterial may become
a tool to control cell behavior in vitro. I would venture to say that it opens
a new chapter in TE. First of all because it creates new opportunities in cell
engineering but also because it may give rise to the new strategies
in the development of TE products. In order to address the first issue,
in the presentation several examples will be given, illustrating the manner
in which material structure and architecture may dictate cell behavior. Material
stiffness, topography as well as the scaffold architecture will be focused basing
on the data from the literature as well as on our own results. Regarding the new
strategies, the example of a new TE product for bone will be given. It is obtained
in a way that the scaffold degradation occurs under control, during cell culture
in vitro - prior to implantation. The product of the scaffold degradation stimulates
concomitantly the extracellular matrix production by cells. Such cell-biomaterial
interaction results in the gradual replacement of man-made scaffold by a cellmade environment - which is a TE gold standard. The added value of this system
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is the inherent controllability of the process which runs in vitro. It seems
profitable in view of some disappointing reports showing that the presence
of the cells in the implantable systems not always results in a desired clinical
effect. Two examples of more advanced tissue regeneration in response
to the scaffolds without cells as compared to the cells-enriched scaffolds
in animal models will be presented. The ability to take advantage of the proregenerative activity of cells in tissue engineering strategies is crucial
for the further development of the discipline. Cell-biomaterial interaction
is a player in this game.
B.4. Polymers from natural sources for biomedical applications
Prof. Alina Sionkowska PhD, DSc
Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry,
Nicolaus Copernicus University in Torun, Torun, Poland
Polymeric materials are commonly used in everyday life. Especially they
are widely applied in biomedical field. The applications of polymers are essential
in surgery, for prosthetic systems, and in pharmacology, for drug formulation
and controlled drug delivery [1-3]. Although it is much easier to use synthetic
polymers in the biomedical field, natural polymers are also required due to their
biocompatibility and biodegradability [4,5]. New method of preparation
of polymeric materials for biomedical and/or cosmetic applications is blending
two or more polymers. During the last three decades the increasing interest
in new materials based on the blends of two or more polymers has been observed
[6]. To develop modified materials based on the blends of two polymers,
two components of the blend need to be combined into one versatile material
[6,7]. Natural polymers such as collagen, chitosan, elastin, keratin, silk fibroin
can be obtained from nature and very often for such production the waste from
food production is used. The stock of waste materials contains feathers, animal
hairs, animal skin, crustacean shells, fish scales and bones.
The recent advances in studies of the interactions between natural polymers
and application of polymers from natural sources as components of the blends
for biomedical applications will be presented. The short overview presentation
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will be focused on materials made of collagen, chitosan, silk fibroin and keratin
based on current literature and experimental work conducted in the laboratory.
These bioinspired and biobased materials can be good alternative for materials
based on synthetic polymers.
References
1. M. Vert. Prog. Polym. Sci. 32, 755 (2007).
2. M.W.T. Howard. Polymers for tissue engineering scaffolds. In Polymeric
Biomaterials, Second Edition, p.167. Ed. S. Dumitriu, Marcel Dekker Inc.
New York-Basel (2002).
3. J. Kohn, W.J. Welsh, D. Knight. Biomaterials 28, 4171 (2007).
4. A. Sionkowska, K. Lewandowska, A. Planecka, P. Szarszewska,
K. Krasinska, B. Kaczmarek, J. Kozlowska. Key Engineering Materials 583,
95 (2014).
5. A. Sionkowska. Prog. Polym. Sci. 36, 1254 (2011).
6. A. Sionkowska.
Natural polymers as components of the blends
for biomedical applications. Chapter in Polymeric Biomaterials IV Edition,
p.309. Ed. V. Popa, Taylor & Francis (2013).
7. A. Sionkowska. Eur. Polym. J. 39, 2135 (2003).
B.5. Advance your genomic research
Rafał Kochanowski PhD
Roche Diagnostics Polska Sp. z.o.o.
Headquartered in Basel, Switzerland, Roche is a leader in research-focused
healthcare with combined strengths in pharmaceuticals and diagnostics. Roche
is the world’s largest biotech company, with truly differentiated medicines
in oncology, immunology, infectious diseases, ophthalmology and neuroscience.
Roche is also the world leader in in vitro diagnostics and tissue-based cancer
diagnostics, and a frontrunner in diabetes management. Roche’s personalised
healthcare strategy aims at providing medicines and diagnostics that enable
tangible improvements in the health, quality of life and survival of patients.
Founded in 1896, Roche has been making important contributions to global
health for more than a century. Twenty-four medicines developed by Roche
are included in the World Health Organization Model Lists of Essential
Medicines, among them life-saving antibiotics, antimalarials and chemotherapy.
21
SCIENTIFIC SESSION: REGENERATIVE MEDICINE
Chair: Prof. Maria Z. Siemionow MD, PhD, DSc;
Prof. Zbigniew Włodarczyk MD PhD;
Prof. Marek Harat MD, PhD
RM.1. Cell therapy for cardiovascular diseases - ready for clinical
application?
Prof. Wojciech Wojakowski MD, PhD
Medical University of Silesia, Katowice, Poland
Heart failure (HF) is one of the key challenges of clinical cardiology due to very
high rate of hospitalizations and death. There is a need for new therapies,
including the cell-based approaches which could restore the myocardial structure
and function. Cell therapy under went rapid transclation from preclinical models
to human use and have been used in multiple clinical trials in acute myocardial
infarction (AMI), ischemic cardiomyopathy, refractory angina and critical limb
ischemia. Several types of cells including bone marrow, cardiac and adipose
tissue cells were used. In most cases heterogenous population of mononuclear
cells isolated by Ficoll (MNC) centrifugation were used, and to much smaller
extent the preselected cells such as CD133, mesenchymal cells or CXCR4 cells.
Postulated mechanisms of cell therapy include paracrine effects including
microvessicles, leading to reduced apoptosis, inflammation and increased
angiogenesis. So far tere is no direct head-to-head comparison between various
cell types. In acute MI the initial clinical trials using BM-derived MNC revealed
modest but significant improvement of the left ventricular ejection function. More
recent trials with magnetic resonance imaging and recent metaanalyses showed
that MNC infusion does not lead to a significant improvement of cardiac
function. Ongoing BAMI trial will show the long-term effects on all-cause
mortality. So far it would be premature to use the MNC treatment routinely
22
in patients with AMI. In patients with HF the cell therapy has been shown
to improve LVEF and remodeling as well as better clinical status, howeverit
needs verification in large randomized clinical trials with long-term follow-up.
In recent trias in HF patients new types of cells with improved differentiation
capacity, such as cardiopoiesis-guided MSC or adipose tissue-derived
mesenchymal stem cells are evaluated witin clinical trias. Most promising data
are in patients with refractory angina and no possibility of revascularization
where direct intramyocardial injections of cells tend to improve symptoms
and exercise capa city.
RM.2. Modelling cardiovascular disease: a new approach with pluripotent
stem cells
Gábor Földes MD, PhD
National Heart and Lung Institute, Imperial College London, United Kingdom,
ALAB Sp. z.o.o.
Many human cardiovascular diseases are not properly modelled by animal studies
and classical cell lines due to the differences in species or the lack of appropriate
human cells. Recent advancement in technology of human embryonic stem cells
and induced pluripotent stem cells has enabled us to generate various new cell
types and phenotypic models of cardiovascular diseases. Indeed, these cells
are suggested to have many of the properties of authentic cardiomyocytes
and endothelial cells, and their phenotypes provide validation that characteristics
of the disease can be reproduced in vitro. The development of high-content
imaging assays enhances the workflow for understanding progressions
and identification of key cellular pathways of these diseases and searching
of potential drug candidates. The high-throughput capabilities of the hESC/hiPSC
systems can be ideally placed to dissect these pathway interactions by systems
approaches and then to translate into a drug discovery platform. Observations
using newly designed assays on a number of automated platforms and show how
these approaches can identify new targets will be presented. Data will be shown
on our studies that hiPSC diverge systematically from hESC systems
and investigate the reason for the difference at multiple levels from receptor
expression to downstream effector pathways.
23
RM.3. Management of corneal limbal stem cell deficiency with cultivated
epithelial cells
Dariusz Dobrowolski MD, PhD, prof. Edward Wylęgała MD, PhD
Ophthalmology Clinic, II School of Medicine with the Divison of Dentistry
in Zabrze, Silesian Medical University, Katowice, Poland
Aim of the study. Presentation of corneal surface reconstruction techniques
with application cultivated corneal or oral mucosa epithelial cells in acquired
and congenital corneal diseases.
Materials and methods. Surgeries were performed between September 2009
and December 2013. 48 eyes underwent autologous transplantation of cultivated
corneal epithelium performed for partial or total limbal stem cell deficiency
(LSCD) caused by chemical or thermal injury. 14 aniridia patients (17 eyes)
and 16 patients with bilateral chemical burns were qualified to autologous
transplantation of cultivated oral mucosa epithelium. For the procedure corneal
epithelium was taken from superior limbus; oral mucosa epithelial cells were
obtained from buccal mucosa. Suspension of single cells was seeded
on previously prepared denuded amniotic membrane. Cultures were carried
on culture dishes’ inserts in the presence of the inactivated with Mitomycin C
monolayer of 3T3 fibroblasts. Stratified epithelia with their amniotic membrane
carrier were transplanted on the surgically denuded corneal surfaces of patients
with total or subtotal limbal stem cell deficiency. Corneal surface restoration,
epithelial regularity and visual acuity improvement were evaluated. Kaplan
Mayer curves were applied in statistical analysis. 60,4 % of eyes with cultivated
corneal epithelium has not developed recurrent conjuntival neovascularization.
In majority of patients limbal area was irregular with number of vessels
in the limbal region. We qualified eyes as graft failure (39,5%) in case
of superficial revascularization of the corneal centre or stromal scaring with new
stromal vessels, in 4 eyes total conjuctival pannus developed again. Visual acuity
ranged from counting fingers to 0,4. In aniridia group 76,4% of eyes has regular
transparent central epithelium, 23,5% of eyes has developed central corneal haze
or neovascularization involving visual axis. In 70,5% of eyes treatment improved
visual quality from mean 0.02 to 0.12. In bilateral injuries success rate of oral
mucosa epithelial transplantation was 68,7%.
24
Conclusions. Autologous transplantation of cultivated corneal or oral mucosa
epithelia has many benefits for the patient. The procedure stabilizes ocular
surface with satisfactory improvement of visual acuity and reduction of ocular
discomfort.
RM.4. Medical applications of epidermal progenitor cells
Michał Pikuła MSc, PhD1, Paulina Langa1, Anna Wardowska1, Jacek Zieliński2,
Karolina Kondej3, Alicja Renkielska3, Piotr Trzonkowski1
1
Department of Clinical Immunology and Transplantology, Medical University
of Gdańsk, Poland
2
Department of Surgical Oncology, Medical University of Gdańsk, Poland
3
Department of Plastic Surgery, Medical University of Gdańsk, Poland
The skin is the largest organ of the human body, primarily fulfilling a protective
role, but also thermoregulatory, endocrine, immune and receptor functions.
Thus, the correct functioning of the skin and especially its integrity determines
the proper functioning of the whole human body. Skin is one of the organs
with outstanding regenerative properties, enabling continuous self-renewal
of the epidermis, as well as allow for effective wound healing. These properties
are mainly due to the presence of epidermal stem cells, which are located
in the follicle bulge, dermal papillae and basal layer of the epidermis. Isolated
stem cells in cell culture give rise to actively proliferating progenitor cells, which
then differentiate into mature keratinocytes. Progenitor cells expansion is targeted
at stimulation of their proliferation with the smallest possible cell differentiation.
In order to achieve that, media containing appropriate growth factors, peptides,
hormones, and the ionic composition are used. Cultured autologous epidermal
cells have been successfully applied to chronic wounds, serious trauma and burns.
Autologous transplantations of cultured cells have many advantages, including
no immune response, minimal risk of disease transmission, small piece of skin
as a source of cell culture. Transplanted progenitor cells directly accelerate
wound healing through rebuilding the epidermis, producing growth factors
and creating a natural protective layer of the skin. Cultured cells may
be administered to patients in various forms, including: cell suspensions
(collagen, fibrin gels), cultured epidermis or living skin equivalent. The results
obtained in our laboratory confirm that fibrin gel provides high adherence
of transplanted cells to the wound surface, allowing easy application of these cells
25
and facilitating wound healing. Epidermal progenitor cell cultures can also
be used in gene therapy and cellular reprogramming. They might also prove
useful in testing new drugs and cosmetics. The clinical application of cultured
epidermal cells still poses many technical, regulatory and economic challenges.
Therefore, it seems vital to develop effective and safe techniques of stem cells
isolation, cells expansion based on chemically defined, xeno-free media
and forms of clinical administration.
Acknowledgements. This work was supported by the funds of Polish National
Science Centre based on the decision no. Dec-2011/03/D/N25/00555.
RM.5. Regenerative medicine in urogynecology: are we ready?
Klaudia Stangel-Wójcikiewicz MD, PhD
Gynecology and Oncology Department, Jagiellonian University Collegium
Medicum, Krakow, Poland
Urinary incontinence (UI) is a problem in over two hundred million people
in the world [1]. Despite the introduction of minimally invasive surgery
techniques erosion of sling in 3 to 10% cases, resulting in the so-called “rigid
urethra.” For this reason, the possibility of repair with the functioning host tissue,
without the risk of denervation or revascularisation appears to be an almostperfect solution [2]. The research on application of MDC in treating SUI
(stress urinary incontinence) is being conducted in several research centres
in the world. The results presented by them allow assessment of prospective
effectiveness and safety of the cell therapy [3,4,5]. Another aspect
of urogynecology is the pelvis organ prolapse (POP), in which factors of risk
are similar or the same as with SUI. The use of “prosthesising” POP with
synthetic materials, including polypropylene (PPL). Mechanical properties
of mesh (low flexibility) are rather unsuitable for the “mobile” vaginal
environment. Further research is addressing combination of biodegradable frames
for setting autologous cells [6]. Certain aspects of regenerative medicine will
soon be used in urogynecology. However, their introduction into daily standards
of medical practice requires further multi-centre clinical trials.
26
References
1. Ebbesen MH, Hunskaar S, Rortveit G, Hannestad YS. Prevalence, incidence
and remission of urinary incontinence in women: longitudinal data from the
Norwegian HUNT study (EPINCONT). BMC Urol. 2013 May 30;13:27.
2. Stangel-Wójcikiewicz K, Stec M, Nikolavsky D, Chancellor MB. Cellular
Therapy for Treatment of Sterss Urinary Incontinence Current Stem Cell
Research&Therapy 2010, 5, 57-62
3. Peters KM, Dmochowski RR, Carr LK, Robert M, Kaufman MR, Sirls LT,
Herschorn S, Birch C, Kultgen PL, Chancellor MB. Autologous muscle
derived cells for treatment of stress urinary incontinence in women. J Urol.
2014 Aug;192(2):469-76
4. Blaganje M, Lukanović A. Ultrasound-guided autologous myoblast injections
into the extrinsic urethral sphincter: tissue engineering for the treatment
of stress urinary incontinence. Int Urogynecol J. 2013 Apr;24(4):533-5
5. Stangel-Wojcikiewicz K, Jarocha D, Piwowar M, Jach R, Uhl T, Basta A,
Majka M. Autologous muscle-derived cells for the treatment of female stress
urinary incontinence: a 2-year follow-up of a Polish investigation.- Neurourol
Urodyn. 2014 33(3), 324-330
6. Mangera A, Bullock AJ, Roman S, et al. Comparison of candidate scaffolds
for tissue engineering for stress urinary incontinence and pelvic organ
prolapse repair. BJU Int 2013 Sep;112(5):674-85.
RM.6. Treatment of knee osteoarthritis by microfragmented adipose tissue
Konrad Słynarski, Anna Krześniak MD, Carlo Tremolada, Arnold Caplan
Lekmed Hospital, Warsaw, Poland
Aim of the study. The aim of this study was to evaluate the efficacy
of an innovative cell therapy system (Lipogems) for the treatment of knee
osteoarthritis. Lipogems is an apparatus for obtaining non-expanded and ready-touse cell preparation from fat tissue harboring Mesenchymal Stem Cells (MSCs)
and pericytes within a preserved stromal vascular niche. Fat processing involves
mild mechanical forces and does not require any enzymatic or chemical treatment
start from a lipoaspirate.
27
Materials and methods.The study included 40 patients aged from 18 to 83
with osteoarthritis in the knee. In all the patients, the presence of osteoarthritis
symptoms was confirmed by clinical examination,
X-ray and MRI. Patients underwent a three-step procedure of lipoaspiration,
adipose tissue processing using the Lipogems device and injection the preparation
into the knee. Results were assessed using KOOS, KSS and VAS pain scale
before the procedure, at 1 week, 1, 3 and 6 months follow-up.
Results. The improvement of the symptoms occurred few days after treatment
and increased steadily throughout the whole period of our study in KOOS, KSS
and VAS pain scale. The results of all KOOS subscales showed gradual
statistically significant improvement of an average of 21.8 points for each
subscale.
Conclusion. Lipogems system for the treatment of osteoarthritis of the knee
is an easy, safe and effective intraoperative procedure to obtain minimally
manipulated cell therapy product that retains the intact microenvironment
in which MSCs thrive and is amenable for the use in different clinical settings.
RM.7. Cell technologies for treatment of the consequences of civil
and combat bone lesions
V.M. Oksymets1,3, R.G. Vasyliev2,3, A.T. Rodnichenko2,3, A.V. Zlatska2,3,
D.A. Zubov2,3
1
State Institute of Urgent and Recovery Surgery named after V.K. Gusak,
National Academy of Medical Sciences of Ukraine, Kiev, Ukraine
2
State Institute of Genetic and Regenerative Medicine, National Academy
of Medical Sciences of Ukraine, Kiev, Ukraine
3
Biotechnology laboratory ilaya regeneration, Medical company ilaya®, Kiev,
Ukraine
Introduction. Long-term fracture non-unions are observed in 5-6% of the injured
patients. Unsatisfactory outcomes of bone defect treatment are greater than 10%.
The reason is an alteration of osteoreparation mechanisms. Our studies have
shown that resolution of the problem lies in use of cultured multipotent stromal
cells (MSCs).
28
Aim of the study. Aim of study - to determine the effectiveness of cultured
autologous MSCs for treatment of fracture non-unions and limb bone defects.
Materials and methods. MSCs have been used for complex treatment of 43
patients with civil injuries and 9 subjects with combat trauma received during
Eastern Ukraine military operations. The 28 civil patients had fracture non-unions
of femur and tibia (57.6±15.8 weeks) as a result of high-energy trauma: 15 – bone
defects (9 - tibia, 3 – femur, 1 - humerus, 1 - ulna, 1 - calcaneus). One casualty
had a tibia fracture non-union, 8 injured possessed bone defects ranged 3-12 cm
(1 - clavicle, 3 - humerus, 1 - femur, 2 - tibia, 2 - calcaneus). The 6 combat
casualties had blast injuries, 3 – bullet wounds.
MSCs were applied in a form of 3D osteoprogenitor graft (3D-OPG) consisting
of a scaffold (devitalized allogenic bone as blocks or chips in fibrin hydrogel)
and cultured autologous bone marrow MSCs and periosteum cells (3:1). MSCs
were obtained by whole bone marrow seeding and culturing in αMEM with 10%
platelet lysate under 5% O2. Periosteum cells (PCs) were obtained via enzymatic
digestion of fibula periosteum fragments and propagated in DMEM/F12
with 10% human serum and 10 ng/ml bFGF under 5% O 2. Quality assurance
of cell seeded 3D-OPG was done via combined staining with FDA/PI
and histological assay.
Bone formation was assessed by radiography. The first survey was carried out
not earlier than 1.5 months after MSC transplantation.
Results and conclusions. MSC transplantation was done via injection
and surgical methods. The injection method used when inter-fragment diastasis
was less than 5 mm. The surgical method was carried out with use of 3D-OPG.
Bone chips were used in the tangential bone defects, bone blocks - in circular
defects. The injection method was used in 6 civil patients and in one casualty;
the remaining casualties were subjected to surgical methods. Bone fragments
and 3D-OPG were fixed both with Ilizarov device and extramedullary/
intramedullary constructions. The 42 civil patients (97.7%) after a single
transplantation revealed positive treatment results. The average healing time
of fracture non-union in patients composed of 21.6±3.3 weeks and was
comparable with the terms of healing of closed diaphyseal fractures resulting
from low-energy trauma (19.7±2.2 weeks). The results of primary X-rays
of patients with blast injuries have shown that within 1.5-2 months after
transplantation it was an integration of 3D-OPG with recipient bone.
29
ORIGINAL PAPER SESSION
Chair: Prof. Leonora Bużańska PhD, DSc; Prof. Elżbieta Pamuła PhD, DSc;
Prof. Tomasz Drewa, MD, FEBU
O.1. Application of 3D polyethersulfone membranes for repair of articular
cartilage defects
A. Chwojnowski1, M. Płończak2, C. Wojciechowski1, J. Czubak2, T. Jakutowicz2,
B. Kupikowska-Stobba1, E. Łukowska1, D. Lewińska1, J. Nowak1
1
Nałęcz Institute of Biocybernetics and Biomedical Engineering PAS, Warsaw,
Poland
2
Medical Center for Postgraduate Education, Warsaw, Poland
The basic worldwide standard at the present moment, used both in research
and clinical applications, is a variety of collagen scaffolds with various structures,
mainly flat scaffolds. Only a small number of contributions inform
one of attempts at applications of collagen scaffolds with the real 3D structure
in animal models.
Our proposition is about an application of a wide-pore polyethersulfone
membrane (PSE) with the real 3D structure in the role of scaffold. Such
a membrane contains macro-pores which are linked together in a way enabling
an easy colonization of its interior by chondrocytes. Usage of such a structure
minimalizes the occurance of fibroblast-like forms in result of chondrocytes
cultivation, which is common in flat membranes. Chondrocytes in wide-pore
polyethersulfone membranes produce, first of all, type II collagen. In in vitro
cultivations we have confirmed rabbit chondrocytes and human chondrocytes
(White New Zealand) for several times; in 4-6 week old cultivations, and stated
a bigger increase of protein amount in the membrane in comparison with protein
amount in the possessed chondrocytes. The up-to-date results obtained in animal
studies have shown the high efficacy of polyethersulphone (PES) scaffold
30
implants with chondrocytes that have been grown on them (over 90% success
rate). PES membrane is a type of material characterized by considerable
mechanical durability, not changing its properties while in a moist state. These
properties enable moving a membrane colonized by cells during cultivation,
implanting it into the joint and fixing it to the merges of the injury with surgical
stitches as well as with a tissue glue. At the same time durability of a PES
scaffold is definitely bigger than of a collagen one and it is estimated to last
for c.a. 6 months. This allows to protect the just-being-created cartilage
for a significantly longer time and to stabilize it well.
O.2. New approach in evaluation of transplanted cells fate after
transurethral injection in large animal model
A. Burdzińska1, B. Dybowski2, R. Zagozdzon3, M. Godlewski4, Z. Gajewski5,
L. Paczek1
1
Department of Immunology, Transplantology and Internal Diseases, Medical
University of Warsaw, Warsaw, Poland
2
Department of Urology, Medical University of Warsaw, Warsaw, Poland
3
Department of Immunology, Center of Biostructure Research, Medical
4
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw
University of Life Sciences (SGGW), Warsaw, Poland
5
Department of Large Animal Diseases with Clinic, Faculty of Veterinary
Medicine, Warsaw University of Life Sciences (SGGW), Warsaw, Poland
Introduction. Urinary incontinence (UI) is a serious socio-medical problem
which affects 4–14% of younger women and 12–35% of elder women.
Cell transplantation has been proposed as an alternative method for the UI
treatment. Although many results in this field are promising, the recommended
protocol for such a procedure has not been determined. Therefore further preclinical studies are needed, preferentially on large animal models in order
to mimic transurethral injections performed in human patients. One of the most
important determinants of cell therapy efficacy in pre-clinical surveys are:
1) the level of graft survival in the target site, 2) exact localization of injected
cells, 3) the direction of donor cells differentiation and 4) the degree of graft
integration with the host tissue. Traditional methods do not allow for assessment
of all mentioned parameters from one specimen.
31
Aim of the study. The objective was to elaborate a protocol of reliable semiquantitative cell survival assessment and ability of microscopic imaging
of injection site performed in a sample from one animal.
Materials and methods. The study was performed on caprine female urethras
(n=6) collected 28 days after autologous transplantation of muscle derived cells
(MDCs) or after PBS injection. The cells were labeled with DiD fluorochrome
(ex-644; em-665) prior to transplantation. In each animal 8 doses were delivered,
2x10(6) of cells/50 ul per dose. The injections were performed in highly specified
conditions. Collected urethras cut into 5-7 thick cross sections were analyzed with
the use of In Vivo Imaging System (IVIS). Then, the urethral slices were further
cut in cryostat and cross-sections were analyzed either by confocal microscopy
or by slide scanning.
Results and conclusions. Using the IVIS to scan thick cross sections of urethras
injected with DiD stained cells resulted in precise localization of DiD positive
spots both in longitudinal and cross axes of urethra. Donor cells clusters were
identified in all transplanted animals, however in all of them the number of spots
was lower than 8. Moreover, analysis allowed for semi-quantitative assessment
of the donor cells amount in urethras in comparison to the PBS injected control.
Further cross-sectioning and microscopic analysis of histological specimens
confirmed the specificity of IVIS assessment. Examination of immunostained
sections revealed that at least part of transplanted MDCs differentiated into
muscle structures and integrated with host external urethral sphincter.
In conclusion, proposed herein protocol of collected tissue assessment is very
efficient in transplanted cell fate evaluation.
Acknowledgements. This work was supported by Foundation for Polish Science
(FNP), Parent/Bridge Programme, edition 6/2012; Radosław Zagozdzon’s work
was supported by a Grant from the European Commission 7th Framework
Programme: FP7-REGPOT-2012-CT2012-316254-BASTION.
O.3. Differentiation potential
in regenerating skeletal muscle
of
Pax7-/-
pluripotent
stem
cells
A. Helinska1, M.A. Ciemerych1, I. Grabowska1
1
Department of Cytology, Institute of Zoology, Faculty of Biology, University
of Warsaw, Warsaw, Poland
32
Introduction. Skeletal muscles have remarkable ability to regenerate after injury.
The regeneration process depends on the satellite cells which are located between
myofiber basal lamina and sarcolemma. Pax7 transcription factor regulates
maintenance of satellite cells pool and their self-renewal. During myogenesis
Pax7 plays a crucial role in the specification of skeletal muscle precursor cells.
Moreover Pax7 initiates the expression of MyoD and induction of satellite cells
differentiation into myoblasts. In the case of sarcopenia, muscular dystrophy,
and some other muscle diseases endogenous pool of satellite cells is insufficient
to repair damaged tissue. Transplantation of stem cells that could replenish
satellite cells population and support muscle regeneration could be considered
as possible therapy of such diseases.
Aim of the study. This study investigates the role of Pax7 in myogenic
differentiation of mouse embryonic stem cells (mES) and mouse induced
pluripotent stem cells (miPS).
Materials and methods. Fluorescently marked wild type (Pax7+/+) and knockout (Pax7-/-) mES and miPS were transplanted into cardiotoxin injured mouse
gastrocnemius muscles. After seven days of regeneration muscles were isolated
and histological and molecular analysis were performed.
Results and conclusions. Both types of cells, i.e. Pax7+/+ and Pax7-/-,
transplanted into damaged mouse muscles are able to undergo myogenic
differentiation, but only mES participate in the formation of new muscle fibers.
Pax7-/- mES cells more efficiently colonize regenerating skeletal muscles than
control cells. We conclude that myogenic differentiation of Pax7-/- mES cells
occurs more readily than of Pax7+/+ ones.
O.4. Microparticles with vancomycin immobilized on porous scaffolds
for prevention of implant-related infections in bone tissue
U. Posadowska1, M. Brzychczy-Włoch2, J. Lechowicz1, E. Pamuła1
1
AGH University of Science and Technology, Faculty of Materials Science
and Ceramics, Department of Biomaterials, Kraków, Poland
2
Jagiellonian University, Medical College, Department of Microbiology, Kraków,
Poland
33
Introduction. Surgical interventions performed within large bone defects
are associated with high risk of infections [1]. These infections often result
in tissue devastation, creation of treatment-resistant biofilm, osteomyelitis
development and further expansion of microbes to adjacent soft tissues [2].
To eliminate invading pathogens systemic antibiotherapy is applied, but currently
there is an alternative and more accurate recommendation, i.e. the usage
of implants loaded with antibiotics [3].
Aim of the study. Based on that we propose poly(l-lactide-co-glycolide) (PLGA)
micro-particles (MPs) containing vancomycin, seeded on model implant (PLGA
porous scaffold) for local prevention of bone infections.
Materials and methods. Vancomycin was encapsulated in PLGA (85:15, Mn=80
kDa, d=1.8) microspheres via double-emulsification technique. Encapsulation
and loading efficiencies and drug release kinetics were evaluated by an OPA
assay (fluorescence spectroscopy). PLGA scaffolds obtained by solvent castingNaCl leaching [4] were hydrophilized with ethanol and then MPs suspended
in collagen type I (from rat tail, Bioscience BD) were immobilized.
The effectiveness of immobilization was analyzed by scanning electron
microscopy (SEM). Antimicrobial activity was assessed via Kirby-Bauer method.
In vitro behavior of osteoblast-like MG-63 cells seeded on modified scaffolds
was assayed via AlamarBlue test and morphometric analysis.
Results and conclusions. Drug release and presence within the extracellular
space are advantageously longer for microparticles than for nanoparticles [5].
Thus, our first aim was to produce microsized particles characterized by efficient
vancomycin encapsulation. MPs with the size of 5.5±3.4 μm, encapsulation
efficiency of 42.5±3.3% and drug loading of 8.7±0.5% were obtained. Good
efficiency of MPs immobilization within scaffolds illustrated by SEM
microphotographs can be ascribed to already reported affinity of collagen
to PLGA surfaces [6]. Vancomycin release tests showed short burst release
followed by prolonged release. Antimicrobial activity was demonstrated via
growth inhibition of bone inhabiting pathogens (S. aureus, S. epidermidis
classical strains and clinical isolates). In vitro evaluation with osteoblast-like
MG-63 cells showed good cytocompatibility of the scaffolds prepared. Thus we
may conclude that our modification constitutes a promising solution in prevention
of biomaterials-related bone infections.
34
Acknowledgments. National Science Centre (Grant no: 2012/05/B/ST8/00129)
provided financial support to this project.
References
1. Clauss. M. et al., Acta Biomater 6:3791-97, 2010.
2. Bertazzoni E. et al., Anaerobe 17(6):380-3, 2011.
3. Diefenbeck M. et al., Injury 37(2):95-104, 2006.
4. Pamula E. et al., J Mater Sci 19:425-435, 2008.
5. Kohane D., Biotechnol Bioeng 96(2):203-9, 2007.
6. Wojak-Cwik I. et al., J Biomed Mater Res A101:3109-22, 2013.
O.5. Keratin associeted proteins as a new type of biodressing supported
wound healing
M. Konop1, D. Sulejczak2, A. Szczucińska1, P. Kosson1, A.W. Lipkowski1,
A. Misicka1,3, L. Rudnicka1,4
1
Department of Neuropeptides, Mossakowski Medical Research Centre, Polish
Academy of Sciences, Warsaw, Poland
2
Department of Experimental Pharmacology, Mossakowski Medical Research
Centre, Polish Academy of Sciences, Warsaw, Poland
3
Laboratory Peptides, Faculty of Chemistry, University of Warsaw, Warsaw,
Poland
4
Department of Dermatology, Medical University of Warsaw, Warsaw, Poland
Introduction. Keratin associated proteins (KAPs) are a major protein component
of a hair and skin. KAPs form very unique skeleton constructed by polypeptide
chains connected by multiple disulfide bridges. Such structures are very resistant
to chemical and biological degradations. Skin protein structure reconstruction
is one of the major factors of skin healing when injured by accidents, infection
or disease degeneration (e.g. diabetes). Unfortunately, the healing processes
are slow. To enhance healing processes we developed KAPs preparations, which
added to healing space, could be easily colonized by migrated epithelial cells.
We predicted that KAPs structures should be highly complementally
to proliferated cells in the wound and to their keratins.
35
Aim of the study. The aim of this study was to investigate the effect of KAP
on wound healing in a surgical wound model in diabetic mice.
Materials and methods. Chemical studies. Procedure of preparation of KAP
from mice fur/human hair was carried out according method proposed
by Lipkowski et al. [1] (using different enzyme: pepsin, papain, pankreatyn).
Obteined mice-KAP’s (m-KAP’S) was impregnate by silver nanoparticles
(antimicrobial properties), which was prepared by chemical reduction according
to the description of A. Sileikaite et al. [2]. At the sometime in mice
pharmacologically induced diabetes according method K.K.Wu et al.[3].
In vivo studies: STZ diabetic model and surgical procedure. Protocols involve
i.p. administration of multiple, low dose of streptozotocin (80 mg/kg) to C57BL6
male mice by 5 consecutive days, to cause diabetes. Animal from control group
administrated equal volume of citrate buffor (pH 4.5). Mice were considered
diabetic when 3 consecutive measurements were above 250 mg/dL. When
diabetes were stable two incisions wounds were made, and KAP’s bandage
was tested. The wound area was measured on day 0, 4, 7 and 14 after application
of tested biomaterial. Tissue sample were harvested on day 4, 7 and 14
and processed for histopathological studies.
Results. Developed keratin preparations were characterized by lack
of immunogenicity that is independent from the original source. Keratin dressing
has been adsorbed in regenerating skin during healing process, which eliminates
painful and risky stages of dressing replacement. Our dressing well absorbs
exoduses, while maintaining a moist environment inside the wound area.
The preliminary animal in vivo results confirm that obtained keratin powders
could be used as a part of wound dressing that will be adsorbed in regenerating
skin during healing process.
Conclusions. Our preliminary results obtained in diabetic mouse model indicate
that keratin-associcted protein scaffolds with or without colloidal-AgNP
can be applied in managing wound healing.
References
1. A. Lipkowski, B. Gajkowska, A. Grabowska, K. Kurzepa, Polimery, 2009,
54, nr 5.
2. A. Sileikaite, I. Prosycevas, J. Puiso, A. Juraitis, A. Guobiene, Materials
Science, Vol. 12, No. 4, 2006.
3. K.Wu, Y. Huan, Curr Protoc Pharmacol. 2008 Mar;Chapter 5:Unit 5.47.
36
O.6. Autotransplantation of the adipose tissue derived stem cells for diabetic
foot ulcers healing
L. Masłowski1, M. Paprocka2, W. Witkiewicz1, D. Duś2, R. Grendziak1,
J. Kubiak1, A. Buczyńska1, E. Wojtowicz-Prus1, A. Czarnecka1
1
Regional Specialist Hospital, Research and Development Centre in Wroclaw,
Wroclaw, Poland
2
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of Polish
Academy of Science, Wroclaw, Poland
WROVASC Integrated Center of Cardiovascular Medicine
Aim of the study. Adipose tissue could be one of the most suitable cell sources
for cell therapy. The report presents the preliminary results of the use of adipose
tissue stem cells in the treatment of chronic diebetic foot ulcers.
Materials and methods. Adipose tissue was harvested by aspiration after
infiltration by local anesthetic and tumescend isotonic
solution. Stem
and regenerative cells were separated using closed and fully automated system
CELLUTION 800 from Cytori Therapeutics USA. Cell suspension concentrate
volume of 5 ml was implanted to subcutaneous tissue around the wound
and the wound bed. The phenotype of the cells obtained was determined
immediately after separation and after 7 days of culture on RPMI with 10% fetal
calf serum.
Patients: 22 patients, 14 men and 7 women, mean age 60,2 +/- 5,64y,
with diabetic foot syndrome. After autotransplantation all patients were 6 months
of follow-up and received standard local and general treatment.
Results. The preparation obtained from the separator contained an average of 5,6
x 106 +/- 4 x 106 cells. The phenotype of the cells was determined after
separation (CD45 25,52% +/- 7,94; CD31 22,83% +/- 13,47; CD34 59,33% +/28,45; CD133 0,5% +/- 0,84; CD146 18,83% +/- 16,66; CD105 28,5% +/25,99; CD90 55% +/- 27,84) and after 7 days of culture (CD45 7,4% +/- 10,59;
CD31 8,4% +/- 11,84; CD34 34,8% +/- 27,69; CD133 0,0%; CD146 16,8% +/2,17; CD105 81,25% +/- 34,18; CD90 93,8% +/- 9,55).
Clinical results: In patients with diabetic foot improvement was observed
in 77,3% : 8 completely healed and 9 with >50% reduction of ulcer area.
37
5 patients there was no improvement and 2 presented progression of necrotic
changes resulting in amputation. In patients responding to the regenerative
therapy healing acceleration were observed to 10-12 weeks after
autotransplantation.
Conclusions. Autotransplantation of adipose tissue stem cells is a promising
treatment for diabetic foot ulcers, however with transient effect after the single
autotransplantation. The use of cultured stem cells seems to be a good
continuation of the current clinical study.
Acknowledgments. This publication is part of project „Wrovasc – Integrated
Cardiovascular Centre”, co-financed by the European Regional Development
Fund, within Innovative Economy Operational Program, 2007-2013.
O.7. Under the surface: from hair to digit regeneration unveiling
regenerative potential of distinct skin stem cells
K. Kobielak1
1
Eli and Edythe Broad Center for Regenerative Medicine & Stem Cell Research;
University of Southern California, Los Angeles, USA
Hair follicles (HFs) in adult skin contain a reservoir of stem cells (SCs) which
reside in a niche that provides a specialized extrinsic environment to regulate
their proliferation and differentiation. One of the most important issues
is to understand a precise molecular mechanism how signaling pathways
are integrated with different activators and inhibitors to achieve a network
capable of cyclic activation of SCs. I will present our recent data, which revealed
a new mechanism of hair follicle stem cells (HFSCs) regulation
with a competitive balance between BMP and WNT signaling, which occurs to be
surprisingly intrinsic to SC population and where Bmp inhibition regulates
ligand-receptor dependent canonical Wnt activation in SCs. Thus, this new
intrinsic mechanism added one more hierarchical layer of SC regulation.
Although, hair follicles contain one of the best characterized SCs, much less
is known about existence of SCs in other skin appendages. Interestingly during
development, different skin appendages like hair, sweat gland and nail
are derived from a common ectodermal origin and share similar characteristics
but ultimately adopt alternative, individual homeostatic mechanisms to fulfill
38
the different functions of each distinct mini-organ. Therefore, further I will
discuss our recent discoveries of different skin stem cell populations and their
roles in epidermis, hair follicle, sweat gland and nail regeneration during
homeostasis and injury. Dissecting the underlying similarities and differences
which occur during distinct skin appendage renewal may prove highly beneficial
in unraveling the fundamental principles which govern ectodermal organ
regeneration in vivo.
39
YOUNG SCIENTIST’S SESSION
Sunday, 15 March 2015
Chair: Prof. Dr. rer. nat. Michael Gelinsky; Prof. Samy L. Habib MS, PhD;
Jarosław Maciaczyk MD, PhD
Y.1. In vitro model for probiotics selection based on immune-related gene
expression in chickens
A. Płowiec1, M. Zawadzka2, M. Siwek1, A. Sławińska1, H.A.R. Bluyssen2
1
Animal Biochemistry and Biotechnology Department, UTP University of Science
and Technology, Bydgoszcz, Poland
2
Department of Human Molecular Genetics, Institute of Molecular Biology
and Biotechnology, Adam Mickiewicz University, Poznań, Poland
Introduction. The proper composition of the intestinal microflora is an essential
element for development of the immune and metabolic functions in the body.
Probiotics, the bacterial strains with beneficial properties for microbiome
development, can also express immunomodulatory properties, mediated
by the membrane receptors, such as TLRs (Toll-like receptors). Recognition
of the bacteria by the lymphoid cells is followed by the immune-related gene
expression modulation. In order to select probiotics with immunoregulatory
properties, in vitro models can be used, based on immune cell lines.
Aim of the study. The aim of the study was the selection of the bacterial strain
showing the strongest immunomodulatory properties based on in vitro model.
Materials and methods. Experiment was performed on DT40 cell line derived
from chicken bursal lymphoma, containing immortalized B cells. It was used
for in vitro stimulation with three different strains of the bacterium Lactococcus
lactis subsp. lactis (IBB2955, IBB477, IBB51). Stimulation of DT40 cell line was
carried out for 4, 6 and 8 hours in two variants of media, with and without
gentamicin antibiotic. The suspension of live probiotic bacteria was added
40
to DT40 cells at to meet 10:1 ratio of bacteria to the B cells . At every time point,
the stimulation was terminated by RNA extraction.The downstream analysis
of the immune-related gene expression was based on RT-qPCR. The gene panel
consisted of interferon β (IFN-β), interleukin 1β (IL-1β) and interleukin 8 (IL-8).
The genes were selected in the previous experiment using whole-genome
transcriptome analysis with NimbleGen Gene Expression Microarray (135K).
Results and conclusions. In general, gene expression was higher when B cells
where incubated with probiotic bacteria alone (without antibiotic). Which
indicates the suppression of immunological responses caused by gentamicin
Effect of probiotics stimulation increased with the duration of the experiment.
For IFN-β fold induction parameter was respectively 1,05 at 4h 1,09 at 6h
and 1.59 at 8h, in the medium without antibiotic for Lactococcus lactis subsp.
lactis IBB477. The strongest immunomodulatory effect was observed
for Lactococcus lactis subsp. lactis IBB477.
The results differentiate
the bacterial strains used due to its ability to stimulate an immune response,
which allows the use of a simplified in vitro model for pre-selection of prebiotics
before testing in vivo.
Acknowledgments. The research was supported by the NCN (UMO2013/11/B/NZ9/00783) and by the project of Regional Centre of Innovation
co-financed by the European Regional Development Fund. We would like
to acknowledge Institute of Biochemistry and Biophysics for bacterial strains
used in this experiment.
Y.2. DT40 cell line as a tool for functional analyses of chicken immune
response
A. Dunisławska1, A. Sławińska1, M. Siwek1
1
Animal Biochemistry and Biotechnology Department, UTP University of Science
and Technology, Bydgoszcz, Poland
Introduction. DT40 is commercial cell line of chicken B lymphocytes
established from bursal lymphoma infected with avian retrowirus RAV-1.
The primary cell cultures have the limited ability of continuous multiplication
in vitro. To adapt their lifespan to a long-term cell culture and to delay the aging
process, the cells are immortalized by transfection with viral genes. The derived
cell lines are characterized by a constant proliferation of identical cells. Cell line
41
can be considered as a very simplified model of the living organism in vitro.
In our research DT40 is used for in vitro stimulation of Toll-like receptors (TLR)
by specific ligands, in order to regulate gene expression of the chicken immune
system. Three antigens were used: nonpathogenic keyhole lympet haemocyanin
(KLH) a glycoprotein derived from the marine mollusk, environmental antigens –
lipoteichoic acid (LTA) and lipopolysaccharide (LPS) which account
for the pathogen-associated molecular patterns (PAMPs) from Gram-positive
(LTA) and –negative (LPS) bacteria. KLH and LTA are Th2 type antigens
and stimulate humoral immune responses, whereas LPS is a Th1 type antigen that
stimulates cellular responses.
Aim of the study. The main aim of study was to determine the molecular
mechanisms of the immune response against KLH, LPS and LTA antigens, based
on functional analysis of expression at mRNA and protein levels.
Materials and methods. B cell line was grown in RPMI1640 medium,
supplemented with FBS. Cell culture was incubated in suspension at 37°C
and with 5%CO2. Consecutively it was used for in vitro stimulation with KLH,
LPS and LTA antigens. Candidate genes were selected based on association
between SNP markers and immune responses – adaptive against KLH and innate
against LTA and LPS. Stimulation of B cells was carried out for 3, 6, 9 and 24h.
LPS was added to cells at dose 5µl/ml. Treatment with LTA and KLH
was performed in two doses– 5 and 50µl/ml. In every time point stimulation
was terminated by RNA and protein extraction. The gene expression analysis
at mRNA level was based on RT-qPCR, while analysis at protein level
was performed with Western Blot.
Results and conclusions. Th2 type antigens stimulation resulted in a strong
activation of FOXJ1, ITGB4 and MAPK8IP3 genes. The highest expression
was observed after 24h, triggered by lower dose of LTA antigen and higher dose
of KLH. Selected genes are not activated by Th1 antigen. Obtained results
confirmed that the gene panel is involve in metabolic pathways of immune
responses against KLH and LTA antigens.
Acknowledgments. The research was supported by the NCN (NN311558640)
and by the project of Regional Centre of Innovation co-financed by the European
Regional Development Fund.
42
Y.3. Influence of cross-linking on the amniotic membrane
M. Julkowska1, J. Skopinska-Wisniewska1, K.Wegrzynowska1, J. Tworkiewicz2,
D. Tyloch2, A. Sionkowska1
1
Faculty of Chemistry, Nicolaus Copernicus University in Torun, Torun, Poland
Chair of Regenerative Medicine, Tissue Engineering Department, Ludwik
Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University
in Torun, Poland
2
Introduction. Amniotic membrane is the most inner layer of the fetal membrane.
This material is a thin and semi-permeable tissue. It consists of three layers:
epithelium, basement membrane and avascular matrix. It`s received from
the placenta during cesarean section. It is important that after obtaining
the material must be stored in appropriate conditions. The specific properties
of the membrane cause that it is a good surgical material and it would be very
useful for tissue engineering.
Aim of the study. The aim of our study was to investigate the influence
of various cross-linking agents for the properties of amniotic membrane.
Materials and methods. The amniotic membrane obtained from patients were
stored at -80°C, then defrosted and dried at 8°C before use. The material
was cross-linked by using 0,5 % and 1% solutions of PEG-dialdehyde,
O,O´-Bis[2-(N-Succinimidyl-succinylamino) thyl]- poly ethylene glycol
(PEG-NHS), 1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride/
N-hydroxysuccinimide (EDC/NHS), dialdehyde starch and squaric acid (SQ AC)
for 24 h. After cross-linking the specimens were rinsed with water. Obtained
materials were prepared to various analysis.
The mechanical properties of unmodified and cross-linked amniotic membrane
were tested. After the cross-linking process the amniotic membrane gained better
mechanical endurance and became more rigid. However, the values of tensile
strength noted for amniotic membrane with addition of PEG-dialdehyde,
PEG-NHS and dialdehyde starch are higher than observed for the materials
with EDC/NHS and SQ AC.
Results and conclusions. The mechanical properties of amniotic membrane
are improved by cross-linking process. Our results let us to include that
43
macromolecular cross-linking agents such as PEG-aldehyde, improve these
parameters much more than small molecular reagents EDC/NHS or SQ AC.
Acknowledgments. The authors would like to thank the National Science Centre
(NCN, Poland, Grant no: UMO-2011/03/D/ST8/04600) for providing financial
support to this project.
Y.4. Collagen and elastin hydrolysates hydrogels cross-linked by dialdehyde
starch and pectin
K. Wegrzynowska1 J. Skopinska-Wisniewska1, M. Julkowska1, A. Bajek2,
M. Maj2, A. Sionkowska1
1
in Torun, Poland
2
Introduction. Collagen is the main structural protein of the various connective
tissues in mammals. Collagen, in the form of elongated fibrils, is mostly found
in fibrous tissues such as tendons, ligaments and skin. The amino acid
composition of collagen is unusual for proteins, mostly because of its high
hydroxyproline content. Elastin is a protein which can be mostly found
in connective tissues. It is elastic and allows many tissues in the body to resume
their shape after stretching or contracting. The chemical composition of elastin
is also unusual for proteins, because of desmosine and isodesmosine content.
Cross-linking process of the materials was designed to improve its properties,
like mechanical strength, porosity and susceptibility to degradation.
Aim of the study. The aim of our study was to investigate the influence
of dialdehyde starch (DS) and pectin (P) on the properties of collagen/elastin
hydrogels. DS is a polysaccharide derived by chemical modification from natural
starch. It is prepared by periodate oxidation of starch. Pectin is a complex
polysaccharide consisting mainly of esterified D-galacturonic acid resides
in an alpha-(1-4) chain.
Materials and methods. Collagen was obtained from rat tail tendons. Elastin
hydrolysates were isolated from porcine aorta. 1% solution of collagen in 0.1 M
44
acetic acid and 1% solution of elastin hydrolysates in water were prepared.
Mixtures of the proteins were prepared in different volume ratios (Coll 100 %;
Coll 95%-El 5%; Coll 90 %-El 10%) and were then cross-linked with DS and P.
After that, a dialysis against deionised water was performed. Obtained gels were
then analysed.
Results and conclusions. The FTIR spectra show that the collagen and elastin
structure was not changed by cross-linking with dialdehyde starch or pectin.
The mechanical properties of the collagen and elastin material cross-linked by DS
were improved, while the use of pectin causes deterioration of these properties.
The lyophilized gels exhibit porous structure. The various size is observed.
The in vitro study demonstrates that the materials are attractive for cells.
The addition of dialdehyde starch and pectin causes formation of cross-linking
bonds in the collagen and elastin materials and the transparent, hydrogels
are obtained. However, the gels containing DS are much stiffer than materials
with P. The results show that DS is better cross-linking agent than P. Dialdehyde
starch is a suitable cross-linking agent for protein materials for medicine
and tissue engineering applications.
Acknowledgments. The authors would like to thank the National Science Centre
(NCN, Poland, Grant no: UMO-2011/03/D/ST8/04600) for providing financial
support to this project.
Y.5. Interactions of modified with ascorbic acid poly(ester urethane)s
with phosphate buffered saline (PBS) and their hemocompatibility
I. Gubańska1, J. Kucińska-Lipka1, H. Janik1
1
Department of Polymer Technology, Faculty of Chemisty, Gdansk University
of Technology, Gdansk, Poland
It is commonly known that properly designed poly(ester urethane)s (PEUs)
are well degrading type of polyurethanes. That makes them suitable candidates
for biodegradable biomedical devices. In this study we determined
the physicochemical interactions of obtained unmodified and modified
with 2 wt% of ascorbic acid (AA) PEUs with PBS and their hemocompatibility.
To synthesize unmodified PEUs we used oligomeric α,ω-dihydroxy(ethylenebutylene adipate) (dHEBA), 1,6-hexamethylene diisocyanate (HDI)
45
and 1,4-butandiol (BDO). To improve biological properties of obtained PEUs
we modified them with 2 wt% of ascorbic acid. The results showed that both
obtained types of PEUs were stable during 3 months of incubation in PBS.
Modified PEUs revealed lower mass increase (1%) at the period of the PBS
study. After 3 months of incubation the final mass of ascorbic acid modified
PEUs was almost equal to their initial weight. Both types of polyurethanes
showed good biocompatibility with blood. Modified with 2 wt% of AA PEUs
caused the decrease of blood parameters which were initially elevated (but not
below reference range). In summary obtained polyurethanes may be a promising
candidate for use in biomedical field.
Y.6. Scanning Electron Microscopy of microporous poly(ester urethane)s
modified with and ascorbic acid for tissue scaffolds
J. Kucińska-Lipka1, I. Gubańska1, H. Janik1, A. Klein1, P. Kosiorek1
1
Aliphatic poly(ester urethane)s (PEUs) are well known materials in biomedical
field. They are applied in many areas such as heart valves, vascular grafts,
artificial skin, wound dressings, drug delivery systems as well as soft and hard
tissue scaffolds. Recently, the most popular tissue scaffolds fabrication
techniques are electrospinning, rapid prototyping and salt leaching/solvent casting
connected within phase separation. In this study we obtained microporous
poly(ester urethane)s with the use of salt leaching /solvent casting technique.
PEUs used for the scaffolds fabrication were obtained from oligomeric
α,ω-dihydroxy(ethylene-butylene
adipate)
(dHEBA)
soft
segments
and 1,6-hexamethylene diisocyanate (HDI) reacted with 1,4-butandiol (BDO)
hard segments. Moreover, we modified obtained PEUs with 2 wt% of ascorbic
acid to improve the biomaterial biocompatibility as an antioxidative property
of AA and its large influence on tissue regeneration. Unmodified and ascorbic
acid modified poly(ester urethane)s were dissolved, at concentration of 20 wt%,
in dimethylsulfoxide (DMSO) (wt/wt) or mixture of solvents N,Ndimethylformamide (DMF) and N-methyl-2-pirolidone (NMP) at ratio
of 1:1 wt/wt. Used pore forming agent was salt. Obtained microporous PEUs
were characterized by Scanning Electron Microscopy (SEM). The pore size
of unmodified polyurethanes was in the range of 9-10 µm and for modified PEUs
was in the range of 10-20 µm for both types of used solvents. The porosity
46
of obtained microporous materials was of 88% for unmodified and 87%
for modified PEU scaffolds prepared with the use of DMF/NMP solvents mixtue
and 91% for unmodified and 92% for modified PEU scaffolds prepared
with the use of DMSO.
Y.7. Ti/TiO2 nanotube platform for the electrochemical detection of tumor
necrosis factor (TNFα)
K. Arkusz1, E. Krasicka-Cydzik1
1
Biomedical Engineering Department, University of Zielona Gora, Zielona Gora,
Poland
Corresponding authors: [email protected],
[email protected]
Electrochemical method to detect the inflammatory cytokine tumor necrosis
factor alpha (TNFα) on a new platform has been presented. The selected cytokine
may serve as an early indicator of tumor metastasis, especially to detect bone
metastasis [1]. The novelty of the developed methods consists in using thermally
modified nanotubular layer of titanium dioxide TiO2 on titanium (Ti/TNT –
Titanium/Titania NanoTubes) as biosensor platform for direct immobilization
of TNFα antibodies used as biological elements of biosensors to detect TNFα
antigens electrochemically in 0.01M PBS (Phosphate Buffered Solution) solution.
Due to easy adsorption and high electrical conductance of the modified TNT
layer [2], which allow the effective and direct immobilization of biological
receptors, give the possibility to use the amperometric technique
for electrochemical detection [3]. The calibration curves indicate the possibility
to detect TNFα antibodies within concentrations of 5÷2500 pg/ml in the 0.01 M
PBS (pH 7.4) solution corresponding to clinically important range. The cut-off
limit for the detection of TNFα (5 pg/ml) matches the physiological level
of cytokine, whereas its higher concentrations correspond to inflammation
and metastatic conditions.
References
1. Balkwill F., Nature Reviews Cancer 2009; 9; 361-371.
2. Krasicka-Cydzik E., InTech 2012; ISBN: 978-953-51-0354-7; 175-200.
3. Gongadze E., Kabaso D., Bauer S., Park J, Schmucki P, Iglic A, Mini Rev
Med. Chem 2013; 13(2); 194-200.
47
Y.8. Factors affecting status of isolated human hepatocytes
K.E. Zakrzewska1, A. Samluk1, K. Dudek2, A. Kowalska1, D.G. Pijanowska1,
K.D. Pluta1
1
Nałęcz Institute of Biocybernetics and Biomedical Engineering PAS, Warsaw,
Poland
2
Chair and Department of General Transplant and Liver Surgery, Medical
Currently, the only available treatment for the end-stage liver diseases is liver
transplantation. Unfortunately, this procedure is limited by organ shortage.
The lifetime of the patients could be prolonged through hepatocytes
transplantation or use of the BioArtificial Liver systems (BALs). Such liver
supporting devices, consisting of bioreactor with embedded hepatic cells, have
entered clinical trials phase II/III carried out worldwide. In both cases the basic
requirement is the highest possible quality of the hepatocytes. There are many
reports describing isolation procedures, culturing methods or cryopreservation
protocols. Our study was focused on the factors that might affect status
of the freshly isolated human hepatocytes, especially in the context of their
metabolic capacity. We isolated cells from 33 resected human liver fragments
using isolation method that is based on maceration and analyzed them with flow
cytometry. Obtained results indicate that parameters of the isolated hepatocytes
depend on the patient’s age, sex, and chemotherapy. For example,
sex of the patients had an influence on the level of the albumin production –
we detected higher percentage of albumin positive cells in male than in female
samples (P=0.031). Interestingly, chemotherapy had an impact on albumin
production by hepatocytes of male vs. female patients, but in livers of non-treated
patients we did not observe this effect (P=0.01 and P=0.982, respectively).
We found no such correlations in the case of α-1-antitrypsin synthesis. In turn,
we observed lowered production of this protein in men aged over 60 compared
with men under 60. The collected data and presented conclusions can help
researchers in making decisions about the source of hepatocytes
for transplantation or BALs construction.
48
Y.9. Growth properties and pluripotency marker expression
of spontaneously formed three-dimensional aggregates of human adiposederived stem cells
A. Bogdanova-Jatniece1, U. Berzins1, T. Kozlovska1
1
Latvian Biomedical Research and Study Centre, Riga, Latvia
Adipose tissue is known to be a readily available source of mesenchymal stem
cells (MSCs). Recent findings suggest that their therapeutic potential could
be increased through aggregation into three-dimensional (3D) bodies,
and different culture methods have been employed to obtain 3D spheroids
of MSCs. In the current study we report accidentally encountered spontaneous
formation of adipose-derived stem cell (ASC) bodies in standard ASC culture
of a single donor.
Human ASCs from passages 1 to 3, cultured in a medium containing
5% autologous serum (AS), spontaneously clustered and formed floating 3D
bodies. In the tissue culture flask single ASC bodies along with large aggregates
of interconnected ASC bodies could be observed simultaneously. After a transfer
of single floating ASC bodies onto new adherent plastic dish, they attached
to the surface and gradual migration of spindle-shaped ASCs out of the bodies
was detected. A substitution of AS with allogeneic sera did not hinder this ability,
but commercial medium containing fetal bovine serum delayed the process.
Substantial part of ASCs surrounding transferred ASC bodies showed alkaline
phosphatase (AP) activity, while ASC aggregates were AP negative. Only few
cells in a monolayer culture of ASCs were AP positive. Similar 3D bodies formed
when ASCs were grown on an uncoated glass surface. These ASC aggregates
as well as clusters of ASCs, where formation of the 3D bodies is initiated,
expressed pluripotency marker NANOG, but the expression of OCT4A was not
detected.
Obtained results suggest that spontaneously formed ASC aggregates may
represent a more primitive cell subpopulation within the individual ASC culture.
The ability to form 3D aggregates, the expression of NANOG, and the lack of the
AP activity may be used to enrich ASC cultures with potentially more primitive
cells that could serve as an excellent basis for therapeutic applications.
49
Y.10. Wound repair with cryopreserved cord blood serum treatment
O.O. Vlasov1, I.O. Ishchenko1, G.A. Kovalov1, O.V. Naumova2, O.S. Prokopyuk1,
B.P. Sandomirskiy1
1
Institute for Problems of Cryobiology and Cryomedicine of the National Academy
of Sciences of Ukraine, Kharkov, Ukraine
2
Kharkiv National Medical University, Ukraine
Introduction. Cryopreserved cord blood serum (CCBS) is a natural source
of biologically active substances, stimulating a tissue repair. Its stocks
can be established due to cryopreservation, which enables complete testing
and timely delivery of the serum to any place of its application. Its use
in regenerative medicine is promising.
Aim of the study. The research aim was to study the influence of CCBS
to morphological characteristics of wound healing.
Materials and methods. Cryoinstrument (t=-195C, 60 sec exposure) simulated
wounds in "Sphinx" rats. Introduction of CCMA in experimental group (EC)
and 0.9% solution of NaCl in the control group (CG) was carried out for 10 days,
by intramuscular injection of 0.1 ml/kg of body weight a day, starting 3 days later
the cryodestruction.
During morphological study to day 7 in the area of cryoeffect there was formed
a necrotic zone of soft tissues, surrounded with a perifocal cell bank in both
groups. In CG in deep compartments it is leukocyte and macrophage one,
in peripheral ones it represents a granulation tissue (GT) of horizontal fibroblasts,
thin collagen fibers, islets of newly formed vessels. In EG a neutrophilic response
decreases and macrophage-fibroblast one strengthens, a continuous wide layer
of GT is observed. Regenerating epidermis with focal hyperplasia
and hypertrophy, cell dystrophy (hyperkeratosis, parakeratosis, vacuolar
dystrophy, spongiosis) are most pronounced in CG, and less in EG, moreover
in the latter these changes occur only in 50% of observations.
To day 14 in CG under necrotic area there was formed a layer of developing FG
in lower compartments with the presence of horizontal fibroblasts, vessels
of different dimensions, collagen fibers. Upper compartments comprise the sites
50
of vertically oriented vessels, fibroblast bands, ingrowing between the tag ends
of collagen fibers and necrotic detritus. In EG the GT completely fills the area
of previous cryoinjury in 80% of observations, being characterized
with a strengthened collagen formation in lower compartments and the formation
of continuous layer of vessels in upper ones. If comparing with CG
the epidermization proceeds more actively in EG, focal proliferative
and dystrophic changes in epidermis have less pronounced character.
To day 21 the wounds are purified from necrotic mass with predomination
of such morphological signs in GT rearrangements as: an augmenting content
of fibrous structures, decreased number of cell elements and vessels. If comparing
the epidermis in EG with CG the pronouncement and prevalence of focal
proliferative and dystrophic processes are reduced.
Results and conclusions. Thus, the CCBS demonstrated a significant stimulating
effect on the reparation of wounds, which was manifested in the improved
morphological imaging of injured tissues. Therapeutic effect of CCBS is apparently
implemented via versatile integrated effect of biologically active substances, being
in the certain physiologically balanced ratio.
Y.11. Poly(ester urethane)s modified with ascorbic acid as useful
biomaterials in electrospinning process
J. Kucińska-Lipka1, I. Gubańska1, H. Janik1, P. Kosiorek1, A. Klein1
1
Poly(ester urethane)s are powerful tool in fabrication of tissue scaffolds
for regenerative medicine. Due to their segmented structure, which consists
of hard and soft segments, their mechanical, physicochemical and biological
properties can be tuned directly for material application. Moreover, PEUs have
great tailoring properties, which make them a perfect material for electrospinning
of tissue scaffolds. In this study we took the attempt to electrospun poly(ester
urethane)s modified with ascorbic acid. PEUs used for the study were
synthesized with the use of oligomeric α,ω-dihydroxy(ethylene-butylene adipate)
(dHEBA) polyol, 4,4’-methylene bis(cyclohexyl diisocyanate) (HMDI)
and 1,4-butandiol (BDO) chain extender. To improve the biocompatbility
of obtained PUEs we modified them with 2 wt% of ascorbic acid, which
51
is antioxidant and posses large influence in tissues regeneration. Unmodified
and ascorbic acid modified PEU solutions of 20wt% in DMSO/DMF (1:1) were
subjected to electrospinning under different conditions of voltage (10-13 kV)
and flow rate (0,2-2ml/min). The working distance between needle
and the collector was of 12 cm. Obtained results showed that both types
of poly(ester urethanes) had good processing properties by the electrospinning
technique and may be suitable for applications in regenerative medicine.
Y.12. Microporous hydroxyapatite material for tissues regeneration
in periodontitis in dogs - a clinical study
I. Polkowska1, M. Gołyńska1
1
Department and Clinic of Animal Surgery, Faculty of Veterinary Medicine,
University of Life Sciences, Lublin, Poland
Introduction. Periodontal diseases are the most common oral cavity diseases
in dogs, which are characterized by the destruction of tissues supporting the teeth
(gingival, periodontal ligament, alveolar bone). An important step
in the treatment procedure is regeneration of damaged tissues and restoration their
normal function. One of the most widely used graft biomaterials in both human
and veterinary dentistry is hydroxyapatite (HAp). Hap has a similar structure
to natural
bone
mineral,
high
biocompatibility,
osteocondactivity
and osteoinductivity. Hydroxyapatite allows for regeneration of tooth support
tissues. Moreover, it accelerates the healing process and limits the development
of inflammation.
Aim of the study. The aim of the study was to evaluate the effectiveness
of the use of hydroxyapatite microporus granules and shaped blocks
for the treatment of periodontal diseases and post-extraction defects.
Material and methods. The study was performed on 20 dogs treated
at the Department and Clinic of Veterinary Surgery at the University of Life
Sciences in Lublin. We identified among them two study group of 5 dogs (S1, S2)
and two control group of 5 dogs (C1, C2). All of dogs were diagnosed
with severe periodontitis. Dental examination was performed under general
anaesthesia. There were evaluated depth of gingival pockets (PPD) and tooth
mobility (TM). All of the dogs have 5-8 mm gingival pockets depths and showed
mobility (class 2 and 3) of mandibular/maxillary incisors. In order to avoid
52
extraction of incisors, in five dogs (S1), hydroxyapatite implantation into
the bone pockets was performed. The HAp was applied in the form of 150-500 μg
granules saturated with gentamicin. In group S2 there was required the extraction
of maxillary canines/premolars followed by hydroxyapatite shaped blocks
implanted into extraction socket. The control groups were respectively reflection
of study groups, without the use of hydroxyapatite The groups were determined
by dogs, in which performed only conventional treatment, without using
hydroxyapatite ceramics.
Results. In study group S1, there was observed significant shortness of gingival
pockets after 4 weeks as compared to the control group. The depth of gingival
pockets was reduced by 1,1-2,9 mm and mobile teeth stabilised, thereby avoiding
their extraction. In control group C1 gingival pockets were only slightly
shallower (0,3-0,8 mm). Mobile teeth in these dogs could not be saved
and the extraction was necessary after 2 weeks after the procedure. In study group
S2, after implantation with hydroxyapatite to post-extraction socket, there was
observed considerable acceleration of bone cavity healing. In control group C2,
where there was not performed the implantation of HAp after tooth extraction,
occurred complications in the form of oronasal fistula (80%).
Conclusions. The study confirmed the usefulness of using microporous
hydroxyapatite granules as filling for bone pockets in dogs with periodontal
diseases as well as using shaped hydroxyapatite blocks as filling for faciomaxillary bone defects.
Y.13. Composites of chitosan/collagen/hyaluronic acid with addition
of nanohydroxyapatite
A. Sionkowska1, B. Kaczmarek1
1
Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry,
Nicolaus Copernicus University in Toruń, Poland
Introduction. Collagen and chitosan are natural polymers widely used in tissue
engineering. They are resorbable with a high biocompatibility and stability
to foster the tissue regeneration. Collagen is widely used to fabricate several
biomaterials. Nevertheless due to its weak mechanical properties, other natural
53
polymers or cross-linking agents have to be added to improve the properties
of collagen materials. Collagen and chitosan are miscible because of the hydrogen
bonds and van der Waals forces presence. Into such materials other additives can
be incorporated, for example hydroxyapatite and/or hyaluronic acid. They
modify the properties of material, what can be observed by different experimental
works. Hyaluronic acid is nowadays very popular natural polysaccharide. It has
good biological properties. Moreover addition of this polymer improves
the elasticity of material because of the ability to water absorption.
Aim of the study. The aim of this study was to obtain composites of chitosan,
collagen and hyaluronic acid in 3D form. Solutions of biopolymers were mixed
and then 10, 20, 50 and 80% of nanohydroxyapatite was added. Scanning electron
microscope images were made and the structure of composites was observed.
Materials and methods. Solutions of chitosan, collagen and hyaluronic acid
were made separately with 1% concentration in 0.1 M acetic acid. They were
mixed using magnetic stirrer in a weight ratios 50/50 for chitosan and collagen.
1,2 and 5% addition of hyaluronic acid was added based on sum of chitosan
and collagen weights. Composites were obtained in 3D form in lyophilisation
process. The addition of 10, 20, 50, 80% of nanohydroxyapatite powder
was added to the solutions during its stirring. The morphology of the samples
was studied using Scanning Electron Microscope (SEM) (LEO Electron
Microscopy Ltd, England). Scaffolds were cut with a razor scalpel after being
frozen in liquid nitrogen for 3 min.
Results and conclusions. Chitosan, collagen and hyaluronic acid composites
were obtained in 3D forms by lyophilization process. Nanohydroxyapatite was
added to the solution of chitosan, collagen and hyaluronic acid mixture. After
lyophilization of the mixtures the presence of inorganic particles was observed by
SEM images. One can conclude that the mixture of chitosan/collagen/hyaluronic
acid can form a matrix for inorganic particles. SEM images show that composites
of chitosan/collagen/hyaluronic acid and nano hydroxyapatite have porous
structure. It suggests that such composites can be applied as scaffolds
in biomedicine. The stiffness of the composite can be modified by the amount
of nanohydroxyapatite.
Acknowledgement. Financial support from the National Science Centre (NCN,
Poland) Grant No UMO-2013/11/B/ST8/04444 is gratefully acknowledged.
54
POSTER SESSION
SUNDAY, 15 March 2015
Chair: Agnieszka Banaś PhD; Marta Pokrywczyńska PhD;
Tomasz Kowalczyk PhD; Maciej Nowacki MSc
P.1. Relationship between neural differentiation, hypoxia and epigenetic
regulation in HUCB-NSC at low and ambient oxygen concentration
M. Podobinska1, I. Szablowska-Gadomska2,3, H. Winiarska1, K. DomanskaJanik1, L. Buzanska1
1
Stem Cell Bioengineering Laboratory, Mossakowski Medical Research Centre,
Polish Academy of Sciences, Warsaw, Poland
2
Department of Applied Pharmacy, Medical University of Warsaw, Warsaw,
Poland
3
Faculty of Pharmacology, Institute of Mother and Child, Warsaw, Poland
According to the literature, oxygen concentration may both inhibit and induce
neural differentiation. In our study, we are trying to elucidate the cause of such
different research outcomes using the model of neural stem cells derived from
human umbilical cord blood (HUCB-NSC). We are investigating whether
and how low oxygen concentration influences the neuronal differentiation
HUCB-NSC, how does it correlate to the hypoxia inducible factors (HIF’s)
expression and what is the impact of epigenetic modifications on this process.
To reveal the possible molecular relationship between neural differentiation,
hypoxia and epigenetic regulation, expression of genes MAP2, NANOG, HDAC1,
HDAC2, DNMT3a, DNMT3b as well as HIF1α, HIF2α and HIF3α were
examined. Changing cellular accumulation of HIF’s by DMOG (prolyl
hydroxylase inhibitor) in cells cultured at different developmental stages (induced
by serum free, low serum and 100 μm dBcAMP conditions) at 21% and 5%
oxygen concentration, we looked at mutual dependence of this pathways.
55
We have shown that 5% O2 concentration increase cell proliferation and influence
differentiation in developmental stage dependent manner. Interestingly cellular
responses to the addition of DMOG were also developmental stage dependent.
DMOG increased significantly the expression of HIF1α and HIFα genes in cells
cultured in low serum (2%) medium, but not serum free conditions in both tested
oxygen concentrations. However, HIF1α and HIF2α accumulation associated
with the DMOG treatment was higher in cells cultured in low oxygen
concentration.
Induced by dBcAMP expression of MAP2 was further increased by the presence
of serum and HIF accumulation (under DMOG), however this was not correlated
to different oxygen conditions. Spectacular increase in the expression of MAP2
was observed only in one combination of tested conditions: low serum, low
oxygen in DMOG treated cells, but not pre-differentiated by dBcAMP.
Expression of genes involved in regulation of epigenetic processes (HDAC1,
HDAC2, DNMT3A, DNMT3B) was higher in HUCB-NSCs growing in low serum
conditions in comparison to serum free culture. Moreover the highest expression
of HDAC1, HDAC2, DNMT3A and DNMT3B genes was observed in cells
cultured in low serum/low oxygen conditions with presence of DMOG.
The studies revealed strong correlation between HIF1α and HIF2α accumulation
and expression of HDAC1, HDAC2, DNMT3A, DNMT3B. Moreover, the impact
of oxygen on proliferation rate and the differentiation process has been
demonstrated. That may indicate that oxygen level affects these processes
by epigenetic modifications.
Acknowledgments.
Sponsored
by
grant
Nr 2011/01/B/N23/05401, MMRC statutory funds.
from
Polish
MSRHE
P.2. Cell-penetration in model microorganisms by selected phthalocyanine
photosensitizers
K. Barchiewicz1, K. Jakubczyk1, E. Boniewska-Bernacka2, G. Dyrda1, R. Słota1
1
Faculty of Chemistry, Opole University, Opole, Poland
Department of Biotechnology and Molecular Biology, Opole University, Opole,
Poland
2
56
Currently used anti-tumor therapies still do not provide adequate efficacy
and selectivity, and are often burdened with severe side effects. Hence, the search
for new strategies for the treatment of cancer has become an important challenge
for biomedical scientists. Photodynamic therapy (PDT) is considered as one
of the most promising methods being verified in clinical tests nowadays. The key
elements in such therapy there are the photosensitizer (drug) and light
of a particular wavelength, which in the presence of oxygen would have initiated
a photochemical reaction. It is important for the photosensitizer to be able
to penetrate deeply inside of the cells and therefore, among others, metal
complexes of the macrocyclic phthalocyanine ligand have been extensively
explored, too.
Our experiment was designed to find out whether selected
metallophthalocyanines (SnPc and YbPc2, where Pc = C32H16N8) could be
directly transported into the interior of living cells. For this purpose, cells
of model microorganisms: Saccharomyces cerevisiae and Escherichia coli
were cultured in the presence of the phthalocyanine complex and then the cells
were lysed. The presence of metallophthalocyanines in the lysates
was determined by UV-Vis spectroscopy.
It was confirmed in our study, that some of the tested phthalocyanines were
implemented into the cells. This is a promising preliminary result, which will be
the basis for our further research.
P.3. Goat animal model - an attractive alternative for testing the effects
of cell transplantation into the urethra
A. Burdzińska1, A. Kulesza1, W. Zarychta1, M. Butrym1, M. Dąbrowski2,
K. Gala1, L. Paczek1
1
Department of Immunology, Transplantology and Internal Diseases, Medical
2
Department of Large Animal Diseases with Clinic, Warsaw University of Life
Sciences (SGGW), Warsaw, Poland
Introduction. Urinary incontinence (UI) is a highly prevalent socio-medical
problem. Cell therapy is emerging as an alternative method for the treatment
of UI, however many aspects of the procedure requires further optimization.
57
A big animal model is needed to reliably test the way of cell delivery
and the transplantation efficiency. In larger mammals cells can be administrated
from the lumen of urethra and full urodynamic measurements can be performed
using the same procedure as for human patients. Although porcine model
is accepted in many applications, the size of mature animals can be
a disadvantage in experiments which require the use of adult individuals.
One of possible alternative are small ruminants like goats or sheep.
Aim of the study. The general objective was to characterize caprine animal
model for testing transurethral cell transplantation. The particular goals were:
1) to describe anatomy of female goat lower urinary tract and its regular
profilometry; 2) to isolate and identify muscle-derived cells (MDCs) and bone
marrow (BM) mesenchymal stem cells (MSCs) from adult goats.
Materials and methods. The experiments were performed in 10 aged female
goats (>6 years old). The evaluation of urethral profile pressure (UPP)
was performed in highly specified conditions. The maximal urethral closure
pressure (MUCP), functional urethral length (FUL) and the localization of MUCP
in the longitudinal axis of urethra was assessed. The localization of urethral
sphincters was defined in cross-sections using histo- and immunohistochemical
stainings. The identity of isolated MDCs and MSCs was confirmed by testing
differentation potential followed by cyto- and immunocytochemical stainings.
Results and conclusions. The mean MUCP was 59.5cm H2O, the mean FUL
achieved 3.8 cm and the mean distance from the profile beginning to the MUCP
was 2.1 cm (56% of FUL). The external urethral orifice is located about 2 cm
proximally to the vaginal orifice and is easily accessible. The external urethral
sphincter is well developed, Ω shape and encompasses distal half of urethra.
Isolated caprine MDCs are able to fuse into multinucleated myotubes
and are positive for myogenic marker - desmin, whereas BM-MSCs are able to
differentiate into adipo-, osteo- and chondrogenic lineages and are able to join to
myotubes in vitro when cocultured with MDCs. In conclusion - goats appear to be
a very useful large animal model for testing the effects of cell transplantation
into the urethra.
Acknowledgements. This work was supported by Foundation for Polish Science
(FNP), Parent/Bridge Programme, edition 6/2012
58
P.4. Surface properties of thin films based on collagen from scales
of northern pike (Esox lucius)
J. Kozłowska1, A. Sionkowska1
1
Introduction. Collagen, a major structural component of extracellular matrices
(ECM), is the most abundant protein found in animal body and widely used for
biomedical and pharmaceutical applications [1]. Collagen is known for its high
cell adherence capacity. Surface physicochemical properties of collagen
biomaterials play a significant role in biocompatibility since they govern cellular
adhesion, proliferation and performances. Collagen for biomedical applications
is mainly isolated from animal tissues (bovine or porcine skin). However, due to
bovine spongiform encephalopathy (BSE) disease, the alternative sources
for collagen extraction are required [2]. Fish processing wastes may be alternative
collagen sources for biomedical application and these underutilized resources
have attracted the increasing attention of scientists all over the world [3].
Aim of the study. The aim of this study was to determine the surface properties
of thin fish collagen matrices - an excellent substrate for the culture of many
different cell types.
Materials and methods. Collagen was isolated from fish scale of northern pike,
through demineralization following acetic acid and acetic acid containing pepsin
treatment. The surface properties of collagen films were investigated using
the technique of Atomic Force Microscopy and by contact angle measurements
using diiodometan and glycerol. The surface free energy was calculated using
Owen Wendt method.
Results and conclusions. For fish scales’ collagen films, the surface properties,
such as roughness and wettability, were measured. The surface morphology
characteristics of acid soluble collagen (ASC) film and pepsin soluble collagen
(PSC) are different. RMS (root-mean-square) surface roughness of PSC film
is much bigger than for ASC film. Using AFM technique we observe the fibrils
formed on the surface of ASC film. Diameter of fibril is 67 nm. In PSC film
we do not observe regular fibril formation. The value of polar component
of surface free energy of ASC film is much bigger than for PSC film. The surface
59
properties of collagen films based on collagen isolated from fish scale by acetic
acid extraction and with the aid of pepsin are different. Surface roughness
of collagen film can be improved by addition of pepsin during the collagen
extraction process.
Acknowledgements. Financial support from the National Science Centre (NCN,
Poland) Grant No. UMO-2012/05/N/ST8/02283 is gratefully acknowledged.
References
1. Lee C.H., Singla A., Lee Y., Int J Pharm 2001; 221: 1-22.
2. Senaratne L.S., Park P.J., Kim S.K., Bioresource Technol 2006; 97: 191–197.
3. Muthukumar T., Prabu P., Ghosh K., Sastry T.P., Colloids Surf B Biointerfaces
2014; 113: 207– 212.
P.5. Polyurethanes as biomaterials for cardiovascular system regeneration
and restoration - recent trends and future directions
J. Kucińska-Lipka1, I. Gubańska1, H. Janik1, A. Araszkiewicz1, K. Błażek1
1
Gdansk University of Technology, Faculty of Chemistry, Department of Polymer
Technology, Gdansk, Poland
Polyurethanes (PU) designed for medical devices have found an application
as wound dressings, drug delivery carriers, artificial nerves and skin. Their
application potential was also noticed in regeneration and restoration of tissues
of cardiovascular system. Polyurethanes are used as part of an artificial heart,
heart valves as well as vascular grafts. Recent developments are mostly
concerned with polyurethanes applications as vascular grafts of small diameter.
In respect of other polymeric materials properly designed polyurethanes
characterize with suitable mechanical properties and also biocompatibility,
hemocompatybility and biodegradability. Due to their segmented structure,
consisting of hard and soft segments, PU properties may be directly adjusted
to the implant application. In this poster we present recent developments, reported
at literature data, focused on raw materials, used for the PU synthesis, as well as
fillers and modifiers of PU structure, which may improve PU biocompatibility
and stimulate controllable degradation of such materials according
to requirements of tissue engineering of cardiovascular tissues.
60
P.6. Polysaccharide-protein hydrogels as scaffolds for corneal epithelial
culture
M. Grolik1,2, B. Wowra1, D. Dobrowolski1, K. Szczubiałka2, B. OrzechowskaWylęgała3, M. Nowakowska2, E. Wylęgała1
1
II Ophthalmology Clinic, Medical University of Silesia, District Railway
Hospital, Katowice, Poland,
2
Faculty of Chemistry, Jagiellonian University, Kraków, Poland,
3
Department of Maxillo-Facial Surgery, Silesian Medical University, Katowice,
Poland
Introduction. A critical element in virtually all tissue engineering approaches
is the scaffold. They play an important role in the creation of the cell
environment, adhesion and proliferation. The supports used in the eye surgery
have sufficient mechanical strength to allow surgical suturing of the supports to
the eye surface, fully biodegradable within a proper time scale and to be
nontoxic. Autologous corneal tissue is usually cultured on human amniotic
membrane (AM) or fibrin-cultured epithelial sheets but materials of this kind
are very expensive and relatively difficult to access.
Aim of the study. The purpose of this study was to develop a polymeric material
that could be used to fabricate culture supports for corneal epithelial cells to be
applied during corneal surgeries.
Materials and methods. The materials used to synthesize these supports are
natural polymers (i.e., chitosan, collagen and keratin [7 and 17% w/w]).
The membranes were crosslinked with a natural crosslinker, genipin.
The physicochemical, mechanical, and biological properties of the membranes
were determined.
Results and conclusions. It was found that the addition of keratin results in the
appearance of antibacterial properties against Escherichia coli and in increased
elasticity of the membranes. The hydrogel scaffold based on chitosan (the main
component) is a promising polymer for tissue engineering and suggests that
it may be a useful potential candidate, as a new corneal epithelial culture bed,
for reconstructive tissue engineering of the ocular surgery.
61
Acknowledgements. Project operated within the Foundation for Polish Science
Team Programme co-financed by the EU European Regional Development Fund,
PolyMed, TEAM/2008-2/6. MG gratefully acknowledges a grant from National
Science Centre 2011/01/N /ST5/05544.
P.7. Lysophosphatidylcholine modulates activity of pancreatic cells
A.K. Drzazga1, E. Gendaszewska-Darmach1
1
Institute of Technical Biochemistry, Lodz University of Technology
Introduction. Diabetes mellitus is one of the most common metabolic diseases
nowadays. The most often it results from insulin deficiency due to destruction
of islet cells in pancreas (type 1 diabetes, T1D) or insulin resistance resulting
from reduced number of insulin receptors and disruption of cellular signaling
pathways (type 2 diabetes, T2D). T2D counts for over 90 % of all cases of the
disease and affects over 340 million people worldwide. This also entails
development of other disorders, like heart diseases, strokes, obesity, diabetic
retinopathy, kidney failure or even poor blood circulation in limbs. According to
recent reports, the main metabolite of lecithin - lysophosphatidylcholine (LPC)
is one of the most efficient agonist of G-protein coupled receptors (GPCRs)
expressed in pancreas and gut, responsible for modulation of insulin secretion,
activation of glucose uptake and effective decrease of blood sugar level.
However, the molecular mechanisms behind these observations have not been
understood yet.
Aim of the study. The following study is devoted to LPC and its analogues
originally synthesized at the Institute of Technical Biochemistry, Lodz University
of Technology (IBT), Poland. The goal is to investigate the manner of interaction
of the compound series with murine pancreatic cell line expressing GPCRs
involved in modulation of insulin secretion and to correlate differences
in biological activity of the compounds with their detailed chemical structure.
Materials and methods. All the experiments were conducted in vitro on a βTC-3
murine line of β pancreatic cells. The investigated modified LPC analogues were
originally synthesized at IBT. Experimental methods involve colorimetric
and fluorimetric analysis of compound cytotoxicity and activation of GPCR
signaling pathways.
62
Results and conclusions. The obtained results clearly show the difference
in biological activity of LPC analogues with respect to their chemical structure.
Variations on acyl residues and/or methoxylation of glycerol backbone lead to
observable diversity in the activated GPCR pathways. This emphasize
the importance of specific design of a particular receptor ligand so as to achieve
desired effects on biological models, both in vitro and in vivo.
P.8. Mechanical properties of collagen/silk fibroin composites
A. Sionkowska1, M. Michalska1
1
Nicolaus Copernicus University in Toruń, Faculty of Chemistry, Department
of Chemistry of Biomaterials and Cosmetics, Toruń, Poland
Introduction. Biomaterials used in tissue engineering should meet
the requirements such as biocompatibility, non-toxicity, suitable mechanical
properties. Additionally 3-D composites should have high porosity to provide
good substrate for cell seeding, growth and proliferation [1,2]. Collagen
is a biopolymer which is widely applied in medicine and pharmacy [3].
As a components of extracellular matrix collagen represents nearly 30% of total
proteins in the animal body [4]. Other polymer used in biomedical applications
is Silk fibroin (SF). It is a fibrous protein produced by a variety of species
including silkworms and spiders [5].
Aim of the study. The aim of this work was to create 3-D composites made
of the blends of collagen and silk fibroin.
The mechanical properties
and microstructure of the blends were tested and compared with pure silk fibroin
scaffolds.
Materials and methods. Collagen (Col) was obtained from rat tail tendon [3].
Silk fibroin (SF) was obtained from Bombyx mori cocoons [5].Collagen and silk
fibroin solutions with concentrations of 1% were mixed in final volume ratio
of 50:50 and dialyzed against distilled water for 2 days. After dialysis the solution
Col:SF were put into a polystyrene container and were then placed in a freezer
at −80 °C. Scaffolds were obtained in lyophilisation process during 2 days.
Mechanical properties were tested by a mechanical testing machine. Young
63
Modulus and tensile strength were measured. The microstructure analysis
was performed by the use of scanning electron microscopy (SEM).
Results and conclusions. The properties of Col:SF scaffolds were compared
with SF scaffolds obtained by lyophilisation of 1% solution. Higher Young
Modulus and two times higher tensile strength were observed for composites
of Col:SF scaffolds than for SF scaffold only. High Young Modulus provides
elasticity of material. It proves, that collagen added to silk fibroin causes
th increase in elasticity. SEM results show a structure of irregular interconnected
pores in scaffolds made by silk fibroin and bigger irregular pores with jagged
edges in Col:SF scaffolds.
References
1. F.J. O’Brien, 2011, Biomaterials & scaffolds for tissue engineering, Mater.
Today, 14, 88-95.
2. X. Liu, P.X. Ma, 2004, Polymeric scaffolds for bone tissue engineering, Ann.
Biomed. Eng. 32, 477-486.
3. A. Sionkowska, B. Kaczmarek, K. Lewandowska, 2014, J. Mol. Liq. 199,
318–323.
4. S.Tamilmozhi , A. Veeruraj, M. Arumugam, 2013, Food Research
International 54, 1499–1505.
5. A.Sionkowska, A. Płanecka, 2013, J. Mol. Liq. 178, 5–14.
P.9. Fabrication techniques of polyurethanes designed for tissue engineering
– recent developments and future prospective
J. Kucińska-Lipka1, I. Gubańska1, H. Janik1, K. Błażek1, A. Araszkiewicz1
1
Gdansk University of Technology, Faculty of Chemistry, Department of Polymer
Technology, Gdansk, Poland
Tissue engineering (TE) is one of the fastest growing fields of science. It is due to
its large potential to heal defected and damaged tissues. Tissue engineering (TE)
is directly connected with cellular scaffolds, which seeded with cells, serve
as implantable structure that will cause suitable cells proliferation and migration
64
after implantation. Tissue scaffolds may be made from natural or synthetic
polymers and their blends. One of such synthetic polymers are polyurethanes,
which are widely used in medical field as wound dressings, artificial nerves
and skin, part of artificial heart, vascular grafts and heart valves. In the literature
data, to obtain suitable polyurethane tissue scaffold different techniques were
proposed. Recently, the most popular techniques are electrospinning, salt leaching
solvent casting connected with phase separation and rapid prototyping. Scaffold
fabricated with the use of mentioned techniques possess suitable porosity
and pore sizes for cells growth as well as mechanical properties to serve as
a reinforcing structure for newly formed tissue. In this presentation we focused on
the most often described techniques for polyurethanes scaffold fabrication.
P.10. Calcite scaffolds for tissue engineering
Z. Jaegermann1, M. Lewandowska-Szumieł2,3, A.Chróścicka2,3, S. Michałowski1
1
Institute of Ceramics and Building Materials, Department of Ceramic
Technology, Warsaw, Poland
2
Department of Histology and Embryology, Centre for Biostructure Research,
Medical University of Warsaw, Poland
3
Centre for Preclinical Research and Technology, Warsaw, Poland
Aim of the study. The aim of the study was to optimize the chemical
composition and sintering conditions of synthetic calcite material to be applied
in bone tissue engineered products. Both the physical properties of the material
and its behavior in cell culture were considered within the research.
Materials and methods. Within the research synthetic calcite materials based on
calcium carbonate containing 0,25÷10wt% of lithium fluoride (LiF) were
evaluated. Solid test samples were formed by uniaxial pressing and sintered
in temperatures of 450, 470, 490, 510 and 530oC. Relative density, total porosity
and compressive strength were determined. The method of mapping the porous
structure of polyurethane sponges was applied to obtain porous samples
characterized by suitable and reproducible porosity. Additionally, human bone
derived cells (HBDCs) were cultured on the surface of the sintered samples.
The morphology and the viability (XTT test) of the HBDCs cultured under static
and dynamic conditions were analyzed.
65
Results and conclusions. The results of our studies show that the materials
containing up to 5wt% of LiF behave in a similar way during sintering. They
achieve the highest density and compressive strength in the temperature of 510 oC.
Physical properties and microstructure of porous materials obtained by sponge
method depend mainly on properties of the ceramic slurry and the technique
of extrusion of the surplus slurry applied. The apparent density of porous
scaffolds measured by geometrical method ranged from about 0,5 g/cm3
to 0,9 g/cm3, total porosity ranged from about 60% to 80% and compressive
strength from about 0,5 MPa to 2,3 MPa. The sizes of pores estimated on the
basis of microstructure images from stereoscopic and scanning microscopes were
about 200÷700 µm. Biological tests show that the viability of the HBDCs
cultured on the calcite scaffolds was significantly higher under dynamic culture
conditions. What is crucial, cell viability strongly depends on the LiF content –
viability increased with a decrease of LiF concentration.
Analysis of the results demonstrated that both chemical composition and thermal
treatment highly influence physical and mechanical properties as well as
microstructure of calcite ceramics. It is possible to form porous calcite scaffolds
by organic foam replica, useful for cell culture. Biological tests show that cell
viability cultured on the surface of calcite biomaterial depends strongly
on lithium fluoride content in the material and is the best for the low LiF content.
Our experiments indicate that porous calcite material containing 1wt% LiF, could
be a good option for the purpose of tissue engineered scaffolds.
P.11. Alumina-polyurethane composite for medical application
Z. Jaegermann1, A. Domańska2, A. Boczkowska2, A. Oziębło1, M. Biernat1
1
Institute of Ceramics and Building Materials, Department of Ceramic
Technology, Warsaw, Poland
2
Warsaw University of Technology, Faculty of Materials Science
and Engineering, Warsaw, Poland
During the past two decades significant advances have been made
in the development of materials for biomedical applications. The paper presents
the results of studies on porous alumina ceramics-biodegradable polymer
biocomposites. The idea of the composite is that the polymer degrades
and in the empty space tissue will grow.
66
Porous alumina material was formed by polymeric sponge method using
structural sponges of different pore per inch density. During forming, a 3D
structure of interconnected posts was shaped. Structures of different size
of spaces between posts and differing total porosity can be obtained by the
selection of the sponge density. The main physical properties of tested alumina
foams are, as follows: pore size - 130÷900 µm, total porosity - 84÷90 %,
compressive strength - up to 5,5 MPa.
Biodegradable polyurethanes based on poly(ε-caprolactone)diol (PCL)
and 4,4`-diisocyanato-methylenedicyclohexane (HMDI) were obtained. As chain
extenders two different agents were used: ethylene glycol and water.
The biocomposites were obtained by infiltration of porous ceramics
with prepolymers, solidified in the final stage due to crystallization [Polish Patent
PL212636, 2012]. The prepolymer was crystalized at various temperatures,
and subsequently chain-extended with water. Such composites have higher
compressive strength in comparison to porous ceramics itself (over 6x), as well as
to composites obtained with the application of polyurethanes in which ethylene
glycol was used as chain extender.
Scanning electron microscopy (SEM) was used to investigate the composites
microstructure and to determine whether or not the pores were fully filled
with polymer. Compressive strength of porous alumina ceramics,
of poly(-caprolactono)diol polyurethanes obtained from crystalline prepolymers
extended by water and of the composites fabricated by infiltration method
was tested.
The presented investigations demonstrated that we could fabricate composites
on the basis of porous alumina ceramic filled with polyurethane obtained from
crystalline prepolymers chain extended with water. The results showed that pores
of ceramics were fully filled with polyurethanes. The alumina ceramicsbiodegradable polymer composites exhibit much higher resistance
for compressive stresses than alumina foam itself and higher rigidity than
polyurethanes. These kind of composites give new perspectives for medical
applications, because it assembles with biocompatible components and have
better mechanical properties than porous alumina foams. After particular
biological tests the material could be used for design and future production
of biomimetic type of small joints prosthesis.
67
Acknowledgments. This work was financed by Polish Ministry of Science
and Higher Education (grant nr 3 T08D 03129).
P.12. Nucleoside 5’-O-thiotriphosphates as promising agents in wound
healing
E. Węgłowska1, E. Gendaszewska-Darmach1
1
Institute of Technical Biochemistry, Lodz University of Technology, Lodz,
Poland
Introduction. Wound healing requires coordination of many cellular processes
such as chemotaxis, angiogenesis, proliferation, migration and phagocytosis
which overlap in time and space. Chronic wounds, formed as a result of improper
healing, are frequently occurring medical problem. Angiogenesis connected
with formation of new blood vessels plays a key role in wound healing process
and protein growth factors are significant molecules involved in this process.
Particularly, VEGF (Vascular Endothelial Growth Factor) released by fibroblasts
and keratinocytes promotes endothelial cells to create new blood vessels
in wound environment.
Aim of the study. The object of our studies was to improve some processes
involved in wound healing.
Materials and methods. We have analysed the effect of nucleoside
5’-O-thiotriphosphates on the viability and proliferation of cells naturally present
in the skin and involved in wound healing in vivo. We have also investigated
the influence of those compounds on production of VEGF (Human VEGF
ELISA) by fibroblasts. Additionally, we have examined antioxidant activity
of tested nucleotide analogues.
Results and conclusions. According to our results some nucleoside
5’-O-thiotriphosphates affect proliferation and viability of HaCaT keratinocytes.
Less effect was observed in the case of HDFa fibroblasts. Some of tested
compounds also stimulate the release of VEGF from fibroblasts. An added value
of these type of analogues is antioxidant activity, while unmodified nucleotides
are unlikely to have antioxidant properties.
Naturally occurring nucleoside 5’-O-triphosphates are agonists of transmembrane
G-protein coupled receptors. We postulate that nucleoside 5’-O-thiotriphosphates
68
may also bind to these receptors and in this way affect cellular processes. What
else, phosphorothioate analogs are more stable than native nucleotides being
better inducers of angiogenesis compared to their unmodified counterparts.
Acknowledgments. The study was funded by the National Science Centre
in Project No. 2012/05/N/NZ5/02626.
P.13. Biological activity of selected phthalocyanines
G. Dyrda1, R. Gaida1, E. Boniewska2, R. Słota1
1
Faculty of Chemistry, Opole University, Opole, Poland
Institute of Biotechnology and Molecular Biology, Opole University, Opole,
Poland
2
Introduction. Phthalocyanines belong to a class of chemically stable and nontoxic molecular materials, featured by strong absorption of photons from the red
range of the visible light. Since the red light shows good tissue-penetrating
properties, phthalocyanines have been considered important candidates
as photosensitizers in medical treatment, such as photodynamic therapy (PDT)
and photoinactivation of bacteria and viruses (Photodynamic Antimicrobial
Chemotherapy, PACT).
Aim of the study. This studies were designed to determine whether
phthalocyanines show biochemical activity in deactivation of: Saccharomyces
cerevisiae D273 (1), Gram-negative Escherichia coli DH5 (2) and Grampositive Bacillus subtilis (3).
Materials and methods. Microorganisms were treated with phthalocyanines
in DMF and their growth in the liquid and solid phases was observed. Selected
samples were illuminated by UV light (366 nm) for 20 min. Toxicity
of the solvent was also studied. The maximum dosage of DMF was estimated
as 33 % (v/v). Growth Conditions: (1) D273 yeast: YPG medium (1g yeast
extract, 2g bactopeptone, 2g glucose); (2) E.coli DH5: LB medium (1g yeast
extract, 1g bactotryptone, 0,1g glucose, 0,5g NaCl); (3) B.subtilis: broth (0,2 g
yeast extract, 0,2 g beef extract, 0,4 g NaCl).
69
Phthalocyanines: ZnPc, PbPc, SnPc, EuPc2, GdPc2-R8 (Pc = C32H16N8,
phthalocyanine macrocycle; R = O-PhenC15H31) in DMF, c = 1·10-4 M.
Maximum dosage of the solution was 33 % (v/v).
Results. In DMF, inhibition of microbial growth was observed already
at concentrations of 5% (v/v) for yeast, 9% (v/v) for E. coli DH5a and 20% (v/v)
for B.subtilis. SnPc and GdPc2-R8 in DMF 9% (v/v) completely deactivated B.
subtilis after 48 h of incubation, while ZnPc in DMF 7% (v/v) effectively
inhibited the growth of only E. coli DH5 after 20 min of UV irradiation (=366
nm). PbPc and EuPc2 were found inert.
Conclusions. DMF was identified as the principal toxic component in all systems
explored. E. coli DH5 was found more DMF-sensitive than Gram-positive B.
subtilis, whereas the growth of S. cerevisiae had been stopped at considerable
lower DMF concentration than for the bacteria tested. SnPc and GdPc2-R8
in DMF evinced biological activity in deactivation of B. subtilis after 48 h
of incubation and ZnPc in DMF effectively deactivated E. coli DH5 only after
UV irradiation. Other phthalocyanines used in this study did not reveal any
biological activity towards inactivation of the microorganisms examined.
P.14. Synthesis and photodegradation
phenyl]squaraine in organic solvents
of
bis[4-(dimethylamino)-
P. Kuchnicki1, D. Chełminiak1, U. Kiełkowska1, M. Ziegler-Borowska1,
A. Kaczmarek-Kędziera1
1
Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Toruń, Poland
Disubstituted derivates of squaric acid account for a relatively important class
of organic dyes characterized by unique donor-acceptor-donor type structure
and possessing strong absorption in the visible and near-infrared regions
with a strong fluorescence in solution. Because of their properties, these
compounds became attractive in a variety of technological applications
and materials science, such as organic solar cells, non-linear optics, fluorescent
markers or sensitizers for photodynamic therapy [1-2].
70
Fig. 1 Structure of DMASQ
The present work is focused on the study of the photodegradation properties
of bis[4-(dimethylamino)phenyl]squaraine (DMASQ-Fig.1) in solution. DMASQ
was synthesised by dicondensation of squaric acid with two equivalents of N,Ndimethylaniline, performed in (1:1) n-butanol/benzene mixture as medium [3].
The mixtures of this squaraine in organic solvents of various properties
(chloroform, ethanol, THF DMF, DMSO) were subjected to polychromatic UV
irradiation. Changes in the structure of the dye have been controlled by means
of spectroscopic methods. The increase of the absorbance intensity is observed
in the case of DMSO solvent upon irradiation, while the remaining solvents
lead to gradually decreases of absorbance as result of exposure to irradiation.
Acknowledgment. Financial support from Government wherewithal for science
(years 2013-2015) IP2012034472 is gratefully acknowledged.
References:
1. K.Y. Law, Chem Rev., 93 (1993) 449-486.
2. Y. Hyodo, H. Nakazumi, S. Yagi, Dyes and Pigments 54 (2002) 163–171.
3. L. Beverina, P. Salice, Eur. J. Org. Chem. 2010, 1207–1225.
P.15. Modern 3D bioprinting capabilities as the future of regenerative
medicine?
M. Chojnacki1,2
1
Faculty of Health Sciences, Ludwik Rydygier Collegium Medicum in Bydgoszcz,
Bydgoszcz, Poland
2
Students Scientific Group International Health`s Problems and Preventive
Medicine, Faculty of Mechanical Engineering, UTP University of Science
and Technology in Bydgoszcz, Bydgoszcz, Poland
Coresponding author: [email protected]
71
The purpose of the poster is to present the current possibilities of using 3D
printing technology in medicine - defined as bioprint - and the analysis
of the opportunities and risks that are associated with the implementation of these
procedures on a larger scale. The poster will be presented a brief history
of the technology of bioprinting, stages of development, and the first application
in medical science. The poster will include bioprinting comparison to other
previously known or developed methods for tissue engineering.
As part of the poster will be presented the latest developments in the use
of bioprint in different specializations in the field of medicine - including
orthopedics, angiology and aesthetic medicine. Listed are the biggest obstacles to
the further development of this technology, global trends and predictions about
the future role of this technology in clinical use. Will stress the role of the areas
surrounding - such as chemistry, engineering or biomedical engineering materials
in the development of further experience of the bioprint.
Discussion will be given to the use and purpose of use of this technology to other
known methods for tissue engineering. In view of the rapid development
of medical technology will address ethical issues associated with the possibility
of getting a faster regeneration of tissues and organs by using technologies such
as bioprint.
P.16. Effect of C60 fullerene aqueous colloid solution on biochemical
parameters of liver and visco-elastic membrane properties of rats’
erythrocytes
O.O. Vlasov1, S.I. Panov1, G.A. Kovalov1, I.V. Belochkina1, E.E. Nipot1,
N.M. Shpakova1, I.A. Iefimova2, B.P. Sandomirsky1
1
Institute for Problems of Cryobiology and Cryomedicine of National Academy
of Sciences of Ukraine
2
Kharkov Regional Clinical Traumatological Hospital
The data of studies, concerning the biological safety of fullerene C 60 (C60) and the
prospects for its application, including in the regenerative medicine, are quite
contradictory. In vitro and in vivo effects of C60 depend on the modification of its
surface, dose, concentration, type of target cells. C60 therapeutic potential is
mainly associated with its high antioxidant activity. Examples of antitumor
72
activity of C60 are explained by its effects on cell membrane and nuclei. Besides
the target effect when administering C60 the possible side effects should be
considered.
The research aim was to study the effect of C60 fullerene aqueous colloid solution
(C60FAS) on biochemical markers of hepatotoxicity, as well as visco-elastic
properties of the erythrocyte membrane in rats.
C60FAS in a dose of 1 mg/kg was intraperitoneally administered. Stability of the
solution is restricted C60 concentration and medium tonicity. In isotonic solutions
(150 mМ NaCl) the sols with fullerene concentration of not more than 30-35
µМ are stable. There was administered either diluted (34.7 µМ) or concentrated
(173 µМ) C60FAS (the groups 1 and 2 respectively), as the control – there was
corresponded water volume added. The concentrations of aspartate
aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase
(ALP) in blood serum were studied. Erythrocytes’ osmotic fragility was
investigated in NaCl hypotonic solutions (40-100 mM).
In the first group 24 hrs later the AST and ALT activities were increased in 1.9
and 1.8 times correspondingly, the content of ALP did not change and five days
later the activity of all the analytes did not differ from the control. In group 2 all
studied biochemical parameters did not differ from the controls. This is probably
due to lower penetration of C60 from the concentrated solution into the systemic
circulation because of their aggregation and precipitation. It was visually
confirmed when assessing the injection site at autopsy.
When studying the sensitivity of erythrocytes to hypotonic medium in 24 hrs later
C60FAS administering for both groups the hemolysis level increase by 30-60%
was observed. To 5th day in group 1 the hemolysis level remained the same high,
whereas in group 2 this index decrease down to the control level was observed.
C60 in concentration of 10 µM is known to decrease the erythrocyte membrane
elasticity. The penetration time of C60 via lipid membranes is of 1 nanosecond
order. In both groups the necessary concentration of C60 in the blood, which
enabled the reduction in the stability of erythrocytes to hypotension was
apparently achieved.
Thus, when using C60FAS in concentration, ensuring a solution stability under
physiological conditions, the moderate short-term hepatotoxic effect of C60 was
found. Intraperitoneal administration of C60FAS affects the visco-elastic
73
properties of membrane of erythrocytes, by increasing their sensitivity
to hypotonic medium.
P.17. Enzymatically induced mineralization of gellan gum for bone tissue
engineering
applications:
impact
of
manufacturing
conditions
on mineralization effectiveness
K. Pietryga1, K. Reczyńska1, B. Kolecka2, K. Pach2, E. Pamuła1
1
Department of Biomaterials, Faculty of Materials Science and Ceramics, AGH
University of Science and Technology, Krakow, Poland
2
Department of Automatics and Biomedical Engineering, Faculty of Electrical
Engineering, Automatics, Computer Science and Biomedical Engineering, AGH
University of Science and Technology, Krakow, Poland
Introduction. Gellan gum (GG) is a polysaccharide hydrogel produced by the
microbial fermentation (Sphingosomonas elodea) and is widely used in food
industry and medicine (e.g. in drug delivery systems) [1]. GG in presence of Ca2+
ions forms three dimensional network consisting of more than 99% of water [2].
Gellan gum is a promising material for tissue engineering purposes due to its
excellent biocompatibility and similarity to natural extracellular matrix. However,
in terms of bone tissue regeneration, non-modified GG is not the best option (lack
of the capacity to calcify, low stiffness). Mineralization can be used for adjusting
hydrogels for hard tissue engineering purposes. Among many techniques,
enzymatic mineralization is one of the most successful [3]. The enzyme e.g.
alkaline phosphatase (ALP) can be incorporated into GG and it can precipitate
insoluble calcium phosphates (CaP) in presence of organic phosphate groups and
Ca2+ ions [3].
Aim of the study. In the present study, GG hydrogels were subjected to
enzymatic mineralization. Since ALP is not chemically bound to GG network, it
is released to the surrounding medium during the incubation in calcium
glycerophosphate (CaGP). Mineralization effectiveness depends therefore on the
interplay between incoming phosphates and Ca 2+ and outgoing ALP molecules.
Our hypothesis is that more frequent CaGP exchange in the first day of
incubation results in more efficient CaP formation. To this end, samples with
74
several GG and ALP concentrations were tested and two CaGP exchange regimes
were employed.
Materials and methods. GG was dissolved in UHQ-water at 90oC and cooled
down to 50oC. ALP and CaCl2 water solutions were added to obtain hydrogels.
Samples containing 0.5 mg/ml ALP and different GG concentrations (0.4, 0.55
and 0.7% w/v) and samples with 0.7% w/v GG and different ALP concentrations
(0.25, 0.5 and 2.5 mg/ml) were produced. After gelation cylinder samples were
cut with a hole punch and immersed in 0.1 M CaGP. For each sample group two
CaGP exchange regimes were used: A (2, 4, 6 h, 1, 3 and 5 days) and B (1, 3 and
5 days). After 7 days of incubation amount of mineral formed and samples'
mechanical properties (Young’s modulus) were measured.
Results and conclusions. In the case of hydrogels with different concentration of
GG, more frequent CaGP exchange (regime A) caused significant improvement
in mineral formation and increased material stiffness as compared to regime B.
The influence of CaGP exchange regime was not that evident in the case of GG
with different ALP concentrations. Although the amount of mineral content was
higher when more ALP was incorporated into GG, Young's modulus of the
samples was similar. The results confirmed that more frequent CaGP exchange in
the first hours of incubation (regime A) increased mineral formation, which in
consequence led to improvement in mechanical properties of the material. This
confirms that our hypothesis was right – in the first day of incubation, most of
ALP is still entrapped within the sample and can precipitate more CaP in
presence of fresh organic phosphates. Higher ALP concentration did not enhance
minerals formation. Mineralization was the most effective in the case of samples
containing 0.55% w/v GG and 0.5 mg/ml ALP.
Acknowledgements. This study was supported from National Science Centre,
Poland (2012/05/B/ST8/00129).
References
1. Osmałek T. et al., Intern J Pharm 466 (2014) 328-340.
2. Morris E.R. et al., Food Hydrocolloid 28 (2012) 373-411.
3. Douglas T.E.L. et al., J Tissue Eng Regen Med 8 (2014) 906-18.
75
P.18. Characterization of thin chitosan/hyaluronic acid blend films
K. Lewandowska1, A. Sionkowska1, S. Grabska1
1
Faculty of Chemistry, Department of Chemistry of Biomaterials and Cosmetics,
Nicolaus Copernicus University in Toruń, Toruń, Poland
Corresponding authors: [email protected], [email protected]
Introduction. The physico–chemical properties of polymer blends are important
from both scientific and practical point of view. The blends composed of chitosan
(Ch) with hyaluronic acid (HA) at various component ratios have been prepared
as material designed for food and cosmetic industries or biomedical applications.
Aim of the study. The purpose of this study was to evaluate the physico –
chemical properties of chitosan/hyaluronic acid blends. The properties of chitosan
(Ch), hyaluronic acid (HA) and their blends have been investigated
by the tapping-mode atomic force microscopy (AFM) and monitored by contact
angle measurements.
Materials and methods. Hyaluronic acid (HA) is a commercial polymer from
Aldrich Company with a viscosity average molecular weight of 1.8x10 6. Chitosan
(Ch) sample has a degree of deacetylation of 78% with a viscosity average
molecular weight of 0.59 x106. Ch/HA blends were prepared from mixed polymer
solutions in aqueous 0.1M CH3COOH/0.2M NaCl and 0.1M HCl respectively.
The composition of Ch/HA was 80/20, 50/50 and 20/80. Polymer films were
obtained by casting solution onto glass plate at room temperature. The films were
dried in the vacuum oven. Measurements of the contact angle for two different
liquids (diiodomethane and glycerol) on the surface of chitosan, HA and Ch/HA
blend films were made at room temperature by using the DSA10 goniometer
of Krüss GmbH (Germany).
The surface morphology of films were analyzed by using a commercial AFM
a MultiMode Scanning Probe Microscope Nanoscope IIIa (Digital
Instruments/Veeco Metrology Group, Santa Barbara, CA) operating
in the tapping mode in air.
Results and conclusions. The values of polar and dispersive components
of surface free energy show that Ch film is more polar than HA film. In the case
of Ch/HA blends, the values of dispersive component of surface free energy
are much higher than these of polar component which indicate mainly
76
hydrophobic properties of the surface. The surface morphology characteristics
of Ch, HA and their blend films are different. The surface roughness increases
in the Ch/HA blends.
All the above results suggest that modification in properties is a consequence
of the strong interaction between the polymeric components. Summarizing,
the structure and surface properties of chitosan and hyaluronic acid blends depend
on the mixture composition and on the thermodynamic goodness of the solvent.
P.19. Synthesis and properties of magnetic nanoparticles with polymer
coating for bioligands immobilization
D. Chełminiak1, M. Ziegler-Borowska1, T. Siódmiak2, H. Kaczmarek1,
E. Grodzicki1, A. Ilnicka1, J.P. Łukaszewicz1, P. Kuchnicki1, A. KaczmarekKędziera1
1
Faculty of Chemistry, Nicolaus Copernicus University in Torun, Poland
Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus
University in Torun, Bydgoszcz, Poland
2
Recent progress in nanomaterials synthesis has made the possibility to prepare
nanometer-size materials with controlled structure and functionalities. Magnetic
nanoparticles have numerous potential applications in magnetic recording
technology,
catalysis,
photocatalysis,
biotechnology,
medical
uses
and environment processes [1-3]. In biomedical applications, the nanoparticles
have been applied in targeted drug delivery, immunoassay, and hypothermia.
The present work reports on synthesis methods and properties of magnetic
nanoparticles coated with different polymers, such as: poly acrylic acid, chitosan
(pure and modified), and collagen. The chemical structures of the magnetic
nanoparticles have been characterized by the ATR-FTIR spectroscopy. SEM
and TEM images have been registered. The amount of amino groups
on the prepared nanoparticles was estimated. Furthermore, adsorption isotherms
were measured in order to typify the pore structure of magnetic nanoparticles.
The obtained nanomaterials were applied for lipase from Candida rugosa and
human serum albumin immobilization. The degree of immobilization, lipolytic
77
activity and operational stability of the obtained nanomaterials were examined
[4,5].
Acknowledgment. The project was supported by research grant: National
Science Centre 2014/13/B/ST8/04342.
References
1. Fajaroh F, Setyawan H, Nur A, Lenggoro I. W. Adv. Powder Tech. 24 (2013)
507.
2. Chełminiak D, Ziegler-Borowska M, Kaczmarek H. Polimery 60 (2015) 12.
3. Chełminiak D, Ziegler-Borowska M, Kaczmarek H. Polimery 60 (2015) 15.
4. Ziegler-Borowska M, Siódmiak T, Chełminiak D, Cyganiuk A, Marszałł M.P.
J. Mol. Cat. B 288 (2014) 641.
5. Ziegler-Borowska M, Chełminiak D, Siódmiak T, Sikora A, Marszałł M.P,
Kaczmarek H. Matter. Lett. 132 (2014) 53.
P.20. Expansion and directed differentiation of human endometrial stromal
cells under conventional and low-oxygen cell culture conditions
A.V. Zlatska1,2, R.G. Vasyliev1,2, A.E. Rodnichenko 1,2, D.A. Zubov1,2,
S.N. Novikova1.
1
2
Ukraine
Introduction. The endometrium has a great potential for physiological
regeneration (more than 400 cycles during female life course) based on the
presence of epithelial, endothelial and stromal stem/progenitor cells in its basal
layer. The endometrial stromal cells (EndSCs) are of great interest both for basic
research and for practical use in cell therapy of thin endometrium.
Aim of the study. To investigate the potential of EndSCs for proliferation
and differentiation at their expansion under conventional and low-oxygen cell
culture conditions.
78
Materials and methods. Endometrium specimens (n=5) were obtained by
curettage biopsy during first menstrual cycle phase from the patients with focal
endometrial hyperplasia from normal endometrium area. Endometrium fragments
were dissociated by enzyme treatment during 1 h in mixture of 0.05 %
collagenase IA and 0.05 % pronase solution. The obtained cell suspensions were
cultured in the DMEM:F12 medium with 10% FBS, 2 мМ L-glutamine and 1
ng/ml FGF-2 in the multi-gas incubator at 370 С, saturated humidity, 5% СО2 and
5% or atmosphere (21% conventionally) О2. The EndSCs were selected as
adhesive fraction of cells attached to the culture plastic during 24 hours. The
EndSCs were expanded during 5 passages at 5% and 21% О2. Finally the cell
population doubling number (PDN), population doubling time (PDT), colonyforming efficiency (frequency of CFUf), phenotype and capacity for directed
differentiation into adipocytes and osteoblasts were determined.
Results and conclusions. The EndSCs possess fibroblast-like morphology
and have CD73+CD90+CD105+CD34-CD45- phenotype both under conventional
and low-oxygen conditions. The EndSCs expansion was more efficiency under
low-oxygen conditions: 25.5 PDN vs 20.9 PDN; 33 h PDT vs 41 h PDT. Colonyforming efficiency (frequency of CFUf) was higher in cultures expanded under
5% О2: 35.7% vs 23.2%. Noteworthy the EndSCs formed bigger colonies at 5%
О2 at the expense of the greater number of cells within the colony. Further
we observed the formation of 3D spheroids in the majority colonies at 5% О2.
The EndSCs possess capacity for directed adipogenic and osteogenic
differentiation. No qualitative differences in outcome of differentiation when
culturing under 5% or 21% О2 were seen. Thus the EndSCs satisfied minimal
ISCT criteria for mesenchymal stem cells in terms of the phenotype and capacity
for multilineage differentiation. Culturing under 5% О2 has resulted in more
efficient expansion of the EndSCs with an increased content of clonogenic cells
in the culture and with unchanged multilineage differentiation potential.
79
P.21. Neural crest-derived stem cells from bulge region of whisker follicle:
potential for multilineage differentiation and regeneration of injured
peripheral nerve
R.G. Vasyliev1,3, A.E. Rodnichenko1,3, A.S. Demydchuk2, S.M. Shamalo2,
I.F. Labunets1, Y.B. Chaikovsky2, G.M. Butenko1
1
2
O.O.Bogomolets National Medical University, Kiev, Ukraine
3
Ukraine
Introduction. The bulge region (BR) of whisker follicle (WF) contains the neural
crest-derived stem cells (NCSCs) [Sieber-Blum M., 2004]. Study of their
potential for differentiation and possibilities for their use in regenerative medicine
seem interesting.
Aim of the study. To explore differentiation potential of NCSCs at the clonal
level and to assess the possibilities of their use for stimulation of injured
peripheral nerve regeneration.
Materials and methods. NCSCs were obtained from the explants of BR WF.
The cells were cultured on collagen type I in DMEM:F12 medium with 10%
FBS, 5 ng/ml bFGF, 1% B27 under 5% O2 and 5% СО2. The capacity of cells to
clonal growth was assessed by seeding of 100 cells (P1) per 100 mm Petri dish.
To obtain clonal cultures, the colonies have been subcultured with cloning
cylinders. The primary and clonal NCSC cultures were examined using RT-PCR,
flow cytometry and immunocytochemistry analyses. The clonal cultures were
differentiated into neurons, Schwann cells, melanocytes, adipocytes
and osteoblasts. The 12 FVB strain mice aged 4-6 months underwent sciatic
nerve transection (standard sciatic nerve injury). The animals were divided into
two groups: 1) control and 2) with NCSCs transplantation in the fibrin gel (106
cells per 50 µl of gel). Four weeks after the injury the operated (transected) sciatic
nerves were studied by means of neurohistological and morphometric methods.
Results and conclusions. From the explants of BR WF we obtained migration
and proliferation of the nestin+cytokeratin- fibroblast-like cells expressing Sox10
and Sox2. NCSCs possessed high colony-forming efficiency – 72.58±12.35 %
80
at P1. The clonal cultures (n=9) were derived from colonies containing more than
100 cells (500-1000 cells avg). The clonally derived NCSCs had nestin+Sca1+CD44+CD73+CD90+CD117+CD45- phenotype. Clonal cultures also contained
a considerable number of ALDHbright cells (28.7±3.8%). NCSCs were
successfully differentiated into the βIII-tubulin+ neurons, S-100+ Schwann cells,
pigmented melanocytes, adipocytes (Oil Red O stains) and osteoblasts (Alizarin
Red S and alkaline phosphatase stains). Study of injured sciatic nerve has
revealed formation of regenerative neuroma consisting of nerve fibers, blood
vessels, cells (predominantly fibroblasts) and collagen fibers. The 1 st group
of animals displayed less compact placement of regenerative nerve fibers. Many
of these fibers had tangential or transverse orientation along longitudinal nerve
axis. The 2nd group animals had more compact placement of nerves, most of them
being longitudinally oriented. The peripheral segment of the injured nerve
contained myelinated and nonmyelinated nerve fibers. Their total amount in the
2nd group animals was statistically greater (10522.8±1044.0/mm2 vs
8409.5±739.5/mm2). Thus the NCSCs from bulge region of whisker follicle
possess the capacity for directed differentiation into the main cell typesderivatives of neural crest at clonal level. Transplantation of NCSCs leads to
a more successful regeneration of the injured peripheral nerve in mice.
P.22. Biomedical (nano)engineering – nanofibers used in regenerative
medicine
T. Kowalczyk1, T. Kloskowski2, M. Pokrywczynska2, J. Adamowicz2,
A. Jundzill2, B. Noszczyk3, E. Zabost4 , T. Drewa2
1
Department of Theory of Continuous Media, Institute of Fundamental
Technological Research, Polish Academy of Sciences, Warsaw, Poland
2
Chair of Regenerative Medicine, Department of Tissue Engineering, Nicolaus
Copernicus University in Torun, Ludwik Rydygier Medical College in Bydgoszcz,
Bydgoszcz, Poland
3
Department of Plastic Surgery, Medical Centre of Postgraduate Education,
Warsaw, Poland
4
Faculty of Chemistry, University of Warsaw, Warsaw, Poland
Introduction. Nanofibers are very promising material to be used in biomaterial
engineering. Their structure resembles Extracellular Collagen Matrix
and is treated by cells as a native environment. Membranes made of electrospun
nanofibers were used for regeneration of ureter, urinary bladder wall and skin.
81
Their use is limited to flat or tubular tissues. They have been evaluated for tissue
engineering, wound dressing and Drug Delivery Systems. 3D scaffolds for cell
culturing are commercially available and are believed to better mimic cell
surrounding (behavior and signaling) than flat 2D culturing.
Aim of the study. The aim of scaffold made of nanomaterial is to guide the cells
to proper rebuilt the damaged tissue. Its’ structure should completely degrade
in predetermined time span and be replaced by naturally synthesized collagen.
Materials and methods. Nanofibrous membranes cannot perfectly mimic
extracellular collagen matrix, yet they have multiple advantages over
decellularized collagen matrix harvested from natural sources, they are also easier
to produce and handle. Nanofibers are synthesized from biomaterials such
as polyesters and therefore are free from animal pathogens or allergens. Their
manufacturing allows tailoring of shape, thickness and porosity. Fiber dimensions
and material type can be varied to attain different degradation time for
applications in urology and plastic surgery.
Results and conclusions. Cells cultured on biomaterial used for membrane
construction showed affinity depending mainly on the material post-treatment.
The materials were tested on an small animal model. Applications in skin
and ureter regeneration were possible without stem cells seeding – yet necessary
for urinary bladder wall replacement. Additional work is needed to optimize
the nanomaterial to attain its’ best performance.
Acknowledgements. The authors wishes to thank for the cooperation:
T. Chmielewski, P. Nakielski, K. Zembrzycki, G. Mikulowski and prof. T. A.
Kowalewski from IPPT PAN.
82
P.23. What are the potential prospective applications of stem cells in clinical
dermatology and plastic surgery? The scientific report from in vivo studies.
K. Pietkun1,2, M. Nowacki2, M. Pokrywczyńska2, T. Kloskowski2, D. Tyloch2,
K.Warda2, M.Rasmus2, T. Drewa2, R. Czajkowski1
1
Department
of
Dermatology,
Sexually
Transmitted
Diseases
and Immunodermatology. Ludwik Rydygier Collegium Medicum in Bydgoszcz,
Nicolaus Copernicus University in Torun, Poland
2
in Torun, Poland
Introduction. Currently we can observe the interesting development of different
regenerative medicine therapy methods and simultaneously, implementation
of many of novel therapy forms and techniques based on tissue engineering into
standard clinical practice in different medical specialties. But there is still a small
number of publications relating to the possibility of use autologous stem cells
in dermatology and plastic surgery. Currently conducted studies are mainly
related with the pre-clinical in vitro and in vivo experimental projects.
Aim of the study. The aim of this paper was to present the scientific reports from
in-vivo studies performed by the Chair of Regenerative Medicine (Ludwik
Rydygier Collegium Medicum in Bydgoszcz, NCU –Torun) together
with a scientific collaboration with Department of Dermatology, Sexually
Transmitted Diseases and Immunodermatology (Ludwik Rydygier Collegium
Medicum in Bydgoszcz, NCU –Torun).
Materials and methods. In our study we have presented the detailed scientific
reports and results from two in-vivo studies performed by our scientific teams.
In the first project we have analyzed the potential role of stem cells aplicated
as a compound of selected dermal fillers in rat model. In this study we used
Wistar-lab rats. Animals were divided into four different groups. All type
of fillers were injected to the frontal glabelar, the dorsal and chest anatomical
region. Adipose derived stem cells (ADSC) were labeled with PKH-26
fluorochrome. In the second project we have analyzed the usefulness of stem cells
as a main substrate of tissue adhesive substances used currently very often
in plastic surgery. In this study we used 10 Nude RNU (Crl:NIH-Foxn1rnu).
83
The artificial surgical cut was performed in the dorsal anatomical regio. Different
clousere techniques including suture placement and stem cell based adhesive
solution application were performed. In both projects sbeside the clinical,
morphological and topical changes we have evaluated the cell migration
and direct biological influence of used stem cells onto skin milieu.
Results. In the first study project the average of filing effect in all groups were
ADSC were used was significantly better than control. In the cell migration assay
the highest expression of fluorochrome were observed in the ADSC-HA group.
The expression has also been reported in spleen. We haven’t observed any
expression in the brain, kidneys and lungs tissues. In the second project we have
observed different morphological changes in each of study and control groups.
The specific migration level was also observed.
Conclusions. Stem cells can become a potential and future essential component
of dermal fillers and fast adhesive substances. Probably such modifications can
help to improve the clinical effect and reduce the number of possible
complications and failures correlated with many of invasive dermatological
and surgical procedures.
P.24. Is amniotic membrane a promising material for regeneration
of the urinary tract?
D.Tyloch¹, J. Tworkiewicz¹,², M. Nowacki¹, J. Adamowicz¹, T. Kloskowski¹,
M. Pokrywczyńska¹, J. Skopińska-Wiśniewska³, K. Pietkun¹, T. Drewa¹,²
¹Chair of Regenerative Medicine, Department of Tissue Engineering, Collegium
Medicum, Nicolaus Copernicus University in Toruń, Poland
²Department of General and Oncological Urology, Nicolaus Copernicus Hospital
in Toruń, Poland
³Department of Biomaterials and Cosmetics Chemistry, Faculty of Chemistry,
Nicolaus Copernicus University in Toruń, Poland
Introduction. Amniotic membrane is the inner layer of the amniotic sac.
It is composed of epithelium, basement membrane and stroma. Amniotic
membrane is characterized by a low immunogenicity - lack of expression
of HLA-A, B, D, DR, it reduces fibrosis, scaring, angiogenesis and inflammation.
It is obtained during ceasarian section – relatively readily available.
Its extracellular matrix is composed of collagen fibronectin, nidogen, laminin,
84
proteoglycans and hyaluronan. It seems to be a promising material for use
in regenerative medicine.
Materials and methods. In this paper we have presented the review of currently
published literature. We have selected only publications related to the
experimental use of amniotic membrane for the urinary tract regeneration. In our
analysis we have asses such scientific problems like: potential usefulness
of amniotic membrane for regeneration of different parts of urinary tract,
proposed methods and systemic concepts of amniotic membrane implantation,
chemical and physical processes of implant preparation. We have also supported
the presented material with the results and protocols obtained by our scientific
study during performed projects related to regeneration of bladder wall using
amniotic membrane and their modifications (cross linking).
Results. Amniotic membrane was used in regeneration of cornea, skin –
particulary after burns, cartilage and nerve. Its properties and previous
experiences give hope of the possibility of the use of amniotic membrane in the
regeneration of the urinary tract. Our study shows that the amniotic membrane is
a promising matrix for bladder regeneration assessed histologically but more
research on improvement of bladder function after implantation of the graft
is needed.
Conclusions. Amniotic membrane is a promising material in regeneration
of the urinary tract, however more research on improving its functionality
and modifying its physical properties is needed.
P.25. Influence of surface heterogeneities on
and osteoblast-like cells adhesion and proliferation
collagen
adsorption
M. Krok-Borkowicz1, M. Duda1, S. Mauquoy2, C. Dupont-Gillain2, E. Pamula1
1
AGH University of Science and Technology, Faculty of Materials Science
and Ceramics, Department of Biomaterials, Krakow, Poland,
2
Institute of Condensed Matter and Nanoscience, Université catholique
de Louvain, Louvain-la-Neuve, Belgium
85
Introduction. It is known that surface properties, chemistry as well
as topography of polymer substrates, influence organization of adhesive proteins
(fibronectin, laminin, collagen) resulting in controlled cell adhesion, proliferation,
migration and differentiation1. In this study phase separation approach was
explored to produce defined heterogeneous polymer blends 2 with the aim to better
control organization of adsorbed protein layer and thus cell behaviour. In detail
we investigated the effect of surface properties of polymer films produced
by demixing of polystyrene (PS) and polymetylmetacrylate (PMMA)
on the organization of adsorbed collagen type I and on MG-63 osteoblasts-like
cells adhesion and proliferation.
Materials and methods. Polymer thin films were produced by spin-coating
(speed = 5000 rpm, acceleration = 20000 rpm/s, time = 40 s) of PS, PMMA
and PS/PMMA (30/70 w/w) solutions in dioxane or toluene onto silanized glass
coverslips. The total polymer concentration was 10 mg/mL. Collagen type I (from
followed by washing in UHQ water and drying in nitrogen flow. The samples
were characterized by atomic force microscopy (AFM) and water contact angle
measurements. Biological experiments were performed by culturing MG-63
osteoblast-like cells for 4 h, 1, 3 and 6 days on prepared substrates. Cell viability
(Alamar Blue test), morphology and distribution on the polymer substrates after
eosin/DAPI staining was also tested.
Results. AFM pictures show that surface roughness and organization of adsorbed
collagen molecules varied depending on type of polymers and solvent used:
on more hydrophobic PS collagen formed bigger assemblies than on more
hydrophilic PMMA. On heterogeneous PS/PMMA blends organization
of collagen molecules depended on PS domains in accordance with previous
study2. Biological evaluation showed that cell adhesion and proliferation were
enhanced on surfaces with collagen layers. Higher proliferation was observed for
blends dissolved in dioxane than in toluene. Morphology investigation showed
that already after 4 h incubation cells on surfaces with collagen were better spread
and have less spherical shape than cells incubated on films without collagen layer
and on control materials i.e. glass and tissue culture polystyrene (TCPS).
Conclusions. Presented results show that surface properties of polymer substrates
have a major influence on cell behavior. Polymers blends i.e. PS/PMMA
86
and those with adsorbed collagen promote cell adhesion and proliferation
in a higher extent than pure polymers.
References
1. Stevens M. et.al., Science. 310:1135-8, 2005.
2. Zuyderhoff E. et.al, Langmuir. 28:2007-14, 2012.
Acknowledgments. This study was supported from National Science Centre,
Poland (2012/05/B/ST8/00129).
P.26. Autotransplantation of the adipose tissue derived stem cells for venous
stasis ulcers healing
L. Masłowski1, M. Paprocka2, W. Witkiewicz1, D. Duś2, R. Grendziak1,
J. Kubiak1, A. Buczyńska1, E. Wojtowicz-Prus1, A. Czarnecka1
1
Regional Specialist Hospital, Research and Development Centre in Wroclaw,
Poland,
2
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy of Polish
Academy of Science
WROVASC Integrated Center of Cardiovascular Medicin
Aim of the study. Adipose tissue could be one of the most suitable cell sources
for regenerative cell therapy. The report presents the preliminary results
of the use of adipose tissue stem cells in the treatment of chronic venous stasis
ulcers.
Materials and methods. Adipose tissue was harvested by aspiration after
infiltration by local anesthetic and tumescend isotonic
solution. Stem
and regenerative cells were separated using closed and fully automated system
CELLUTION 800 from Cytori Therapeutics USA. Cell suspension concentrate
volume of 5 ml was implanted to subcutaneous tissue around the wound and the
wound bed. The phenotype of the cells obtained was determined immediately
after separation and after 7 days of culture on RPMI with 10% fetal calf serum.
Patients: 14 patients, 11 women and 3 men; mean age 66,6 +/- 9,5y; with chronic
venous stasis ulcers. After autotransplantation all patients were 6 months
of follow-up and received standard local and general treatment.
87
Results. The preparation obtained from the separator contained an average of 5,6
x 106 +/- 4 x 106 cells. The phenotype of the cells was determined after
separation (CD45 23,5% +/- 2,38; CD31 20,75% +/- 5,12; CD34 71,75% +/19,97; CD133 0,0%; CD146 49% +/- 25,25; CD105 41,5% +/- 29,03; CD90 79%
+/- 17,06) and after 7 days of culture (CD45 10% +/- 10,98; CD31 8% +/- 11,31;
CD34 33,75% +/- 34,97; CD133 0,0%; CD146 15,75% +/- 13,37; CD105 82,25%
+/- 32,84; CD90 94,5% +/- 7,05)
Clinical results: In patients with venous ulcers by 10 of 14 (71,4%) improvement
was observed: 3 completely healed and 7 with >50% reduction of ulcer area.
Ulcer progression was observed in 1 patient, in 3 patients no change of ulcers
size. In patients responding to the regenerative therapy healing acceleration were
observed to 10-12 weeks after autotransplantation.
Conclusions. Autotransplantation of adipose tissue stem cells is a promising
treatment for chronic venous ulcers, however with transient effect after the single
autotransplantation. The use of cultured stem cells seems to be a good
continuation of the current clinical study.
Acknowledgments. This publication is part of project „Wrovasc – Integrated
Cardiovascular Centre”, co-financed by the European Regional Development
Fund, within Innovative Economy Operational Program, 2007-2013.
P.27. Review of 3D printing applications in medicine
M. Cykowska-Błasiak1, E. Chlebus1, B. Dybała1, Ł. Pałka2, M. Dobrzyński3,
O. Parulska4, M. Szymonowicz2, Z. Rybak2
1
The Department of Laser Technology, Automation and Production Organisation,
Wroclaw University of Technology, Wrocław, Poland
2
The Department of Experimental Surgery and Biomaterials Research, Wrocław
Medical University, Poland
3
The Chair and Department of Conservative and Paediatric Dentistry, Wrocław
Medical University, Poland
4
The Chair and Department of Dental Surgery of Wrocław Medical Universit,
Poland
Introduction. 3D printing is a highly developing technology in recent years.
The use of 3D printing technology in medicine does not only help to manufacture
88
custom-designed exoskeletons for patient rehabilitation, but also enables
the production of reconstructed bone implants. Prostheses or implants can be
produced by technology three-dimensional, which occurs by melting layer after
layer of a metal, polymer or resin powder, by a high power laser. Each melted
layer has a cross-sectional contour defined by a 3D model of the object being
produced.
Aim of the sstudy. The aim of the study was to present the results of a literature
review on the implementation of 3D printing technologies in medicine.
Materials and methods. In this paper the literature on medical applications for
3D printing was analyzed. The article presented the possibilities to manufacture
individually fitted patient implants, as well as organs by using additive
technologies.
Results and conclusions. Based on the review it has been shown that the most
3D printing technology is most used for the manufacture of dentures. In addition,
customized implants are made for bone defect. There are works that describe for
the first time the use of living cells to build organs. Aditive technologies offer
a lot of possibilities, because with their help, one can easily create complex
shaped implants. The shape of the implant can be individually adapted to the
patient's anatomy, and one can even fill it with a functional structure (beam,
or a sandwich filled with pores of any shape, etc.). These structures allow bone
ingrowth into the interior of the implant.
P.28. Chemical synthesis and analysis of peptides modified on their
C-terminus with 4-amidinobenzylamine (Amba)
T. Łepek1, I. Małuch1, M. Lewandowska1, A. Prahl1
1
Department of Organic Chemistry, University of Gdańsk, Gdańsk, Poland
Serine proteases are a group of enzymes, which play an important role in many
physiological processes, both normal and disease-related. It has been shown that
the proteolytic activity of these molecules is associated with the development
of many serious dysfunction of the body, such as cancer, heart diseases,
neurodegenerative diseases, bacterial and viral infections. Nowadays, scientists
are seeking relatively specific and potent inhibitors of these enzymes
as a potential drug [1].
89
The L-arginine moiety is involved in series of biochemical transformations, such
as the recognition of specific biomolecules that interact with DNA and RNA.
There is a wide range of well known arginine mimetics, that incorporated into the
peptide chain create strong inhibitors of serine proteses, for instance urokinase
[2]. It turned out, that one of these compounds 4-amidinobenzylamine (Amba)
is a promising idea for the development of effective strategy of blocking the
activity of chosen enzymes. Moreover, the replacement of Arg in position P1
of the inhibitor's peptide chain with Amba has the potential to be less susceptible
to enzymatic degradation and more biostable [3].
In this work, we present different synthesis ways of peptides modified on their Cterminus by introducing of 4-amidinobenzyloamine. According to obtained
results we determined the optimal conditions necessary for the synthesis
of analogues containing mentioned arginine mimetic in P1 position.
References
1. Drag M, Salvesen G.S. ,Nat. Rev. Drug Discov, (2010), 9, 690-701.
2. Balakrishnan S, Scheuermann M.J, Zondlo N.J. ChemBioChem, (2012), 13,
259-270.
3. Meyer D, Sielaff F, Hammami M, Böttcher-Friebertshäuser E, Garten W,
Steinmetzer T. Biochem J, (2013), 452, 331-343.
P.29. Biocompatible poly-L-lactic
composite for bone tissue regeneration
acid/hydroxyapatite
nanofibrous
M. Wojasiński1, J. Bożyk, T. Ciach
1
BioMedical Engineering Laboratory, Department of Biotechnology
and Bioprocess Engineering, Faculty of Chemical and Process Engineering,
Warsaw University of Technology, Warsaw, Poland
Correspodning author: [email protected]
Hydroxyapatite is a synthetic material with crystalline and chemical structure
similar to apatites naturally occurring in vertebrate’s skeletal system. Due to that
fact, it is the first-choice material for bone regeneration applications. In form
of nanoparticles, hydroxyapatite might be synthesized in wet chemical
precipitation techniques in presence of toxic surfactants and/or high temperature.
90
In order to sustain physical loads in bone tissue regeneration, hydroxyapatite
is often mixed with polymers to create composites.
In this paper we propose novel approach to wet chemical precipitation technique
to produce modified by phosphatydylcholines (PC) - added to precipitation
system as a surfactant – nanoparticles of hydroxyapatite (nHAp-PC).
Additionally, we present the way of introducing resulting particles into the
structure of poly-L-lactic acid (PLLA) nanofibers.
Synthesis of nHAp-PC was performed with addition of soybean-derived lecithin
(mixture of phosphatydylcholines, PC) from calcium nitrate and hydrated
diammonium phosphate in molar ratio 1.67. In order to control nHAp-PC
morphology and size, concentration of lecithin solution added to the reaction
system varied in range of 0.3 to 9.0% w/w. Nanofibrous composite was produced
from the mixture of suspension of nHAp-PC in dichloromethane : N,Ndimethylformamide (9:1 v/v) and solution of PLLA in the same solvents mixture.
Characterization of the physical properties was performed: size and size
distribution of particles and fibers – NanoSight measurement, scanning electron
microscopy (SEM); porosity of the nanocomposite – gravimetric method.
For chemical properties evaluation the Fourier Transform Infrared spectroscopy
(FTIR) was performed on nanoparticles and nanocomposite, and X-ray diffraction
(XRD) for crystalline structure of nHAp-PC. The cytotoxicity
of the nanocomposite material was performed in vitro with MG63 osteosarcoma
cell culture. The adhesion and proliferation of cells were analyzed in SEM.
We synthesized nHAp-PC with size in the range of 60 to 90 nm with
phosphatydylcholines bonded to the surface of the particles. We show
the changes of nanoparticles morphology as a function of lecithin concentration
in the reaction system. Subsequently, we produced nanofibrous composite
material PLLA/nHAp-PC with different polymer/bioceramics mass ratios.
Between tested composite materials, nanofibers with 1:1 PLLA/nHAp-PC ratio
exhibit average fiber diameter 215 ± 5 nm and porosity of about 95%.
Additionally, we indicated presence of nHAp-PC in the material by FTIP.
This nanofibrous composite results with 80% surface coverage by MG63 cells.
We described synthesis method for biocompatible hydroxyapatite nanoparticles
in simple reaction system, with addition of lecithin as a surfactant. Additionally,
we presented an application of the particles in the nanofibrous composite material
for bone tissue engineering.
91
P.30. Implantation of xenopericardium and aortic valve leaflets
A.A. Manchenko1, I.P. Mikhailova1, B.P. Sandomirsky1
1
Experimental Cryomedicine Department, Institute for Problems of Cryobiology
and Cryomedicine of National Academy of Sciences of Ukraine, Kharkov,
Ukraine
Introduction. For creation of tissue grafts the porcine pericardium tissue (P)
and aortic valve leaflets (AVL) after low temperature treatment and β-radiation
have been offered. Current method provides a complete destruction of cell
elements and preservation of connective tissue base of devitalized tissue.
Structural matrix proteins maintain the architecture and provide the tissue
scaffold functions.
Aim of the study. The research aim was to study the features of tissue response
to xenotransplantation of different P and AVL groups, as well as the
peculiarities of their resorption and biointegration.
Materials and methods. P and AVL groups have been studied: N – native,
R - irradiated at a dose of 25kGy, F – after freeze (to -196°С)-thawing, FR - after
freezing and irradiation. Samples have been subcutaneously implanted into
the interscapular region of rats. The work has been performed in 80 outbred rats.
Observation time was 7, 14, 30, 90, 180 days after the surgery. The samples were
dissected with the surrounding and underlying tissues and examined
by histological methods with a semi-quantitative assessment of the severity
of inflammatory response. Morphological markers of engraftment or graft
biodegradation were: inflammatory response in surrounding tissue in the graft,
the capsule and its degree of maturity, the graft compactness, presence of various
cell elements in the infiltrate, granulation tissue development.
Results. From day 7 in the group N the lympho-macrophage infiltration begins
to absorb an implanted sample, invading and growing in-between its structures.
Resorption goes from the periphery to the center, to the 3 rd month there were
small fragments of tissue. At the points of R contact with the recipient tissues
an inflammatory reaction with severe neutrophilic infiltration with destruction
of tissue structures develops. As a result of the lysis, the implant is divided into
narrow bands. By the 1st observation month the F implants are represented
by a compact structure, encapsulated by fine connective tissue with small
92
inflammation foci as the clusters of macrophages, lymphocytes and plasma cells.
At the early stages around FP and AVL devitalized grafts (FR group) a capsule
of immature connective tissue begins to form, the fibroblasts forming small bands
start their active migration into the region of transplantation. Graft maintains its
integrity. To the week 2 there is an active angiogenesis, proliferation
and migration of cells of fibroblast line continue. To the month 3 rd the implants
retain their integrity, enclosed in a thin fibrous capsule, well vascularized. Graft
virtually merges with the surrounding tissues, surrounded by a thin fibrous
connective tissue capsule. Proliferating fibroblasts actively synthesize
a connective tissue skeleton around the graft.
Conclusions. The results show that the cryoirradiation modified xenopericardium
and leaflets stimulate formation of fibrous tissue, cause minimal inflammatory
reaction of surrounding tissues and can be used as grafts and base for tissueengineered prostheses.
93
TRICOMED LECTURER
T.L. New generation mesh implants used in urogynaecology
Witold Sujka PhD
TRICOMED S.A.
Aggravating social problems related to stress urinary incontinence as well
as pelvic organ prolapse require the development of ever more modern methods
of treatment. One of such methods involves surgical procedures with the use
of mesh implants. The improvements being implemented include first of all
changes of implant shapes for more atraumatic ones as well as a modification
of their surface in order to achieve a better biocompatibility and thus reduce
the cost of the procedure and shorten the duration of convalescence
of the patients. The goal is to reduce the implant surface mass and thus
the amount of synthetic material implanted in the body. One of the ways to make
a product more biocompatible is to cover its surface with appropriate substances.
Very good results have been achieved with titanium covered surfaces. The lecture
will deal with the titanization process of polypropylene implants. Also, the results
of clinical tests of Dallop NM urological tape as well as titanium covered mesh
implants for pelvic floor repair will be presented.
94
INDEX OF AUTHORS
Ciemerych M.A. ................................ 32
Cykowska-Blasiak M. ....................... 88
Czajkowski R..................................... 83
Czarnecka A. ............................... 37, 87
Czubak J. ........................................... 30
A
Adamowicz J. ............................... 81, 84
Araszkiewicz A. ........................... 60, 64
Arkusz K. ...........................................47
D
B
Dabrowski M. .................................... 57
Demydchuk A.S. ............................... 80
Dobrowolski D. ........................... 24, 61
Dobrzynski M. ................................... 88
Domanska A. ..................................... 66
Domanska-Janik K. ........................... 55
Drewa T. .................................81, 83, 84
Drzazga A.K. ..................................... 62
Duda M. ............................................. 85
Dudek K. ........................................... 48
Dunislawska A. .................................. 41
Dupont-Gillain C. .............................. 85
Dus D........................................... 37, 87
Dybala B. ........................................... 88
Dybowski B. ...................................... 31
Dyrda G. ............................................ 56
Bajek A. ..............................................44
Banas A. .............................................12
Barchiewicz K. ...................................56
Bednarczyk M. ...................................10
Belochkina I.V....................................72
Berzins U. ...........................................49
Bidas J. ...............................................11
Biernat M............................................66
Biskup D.............................................11
Blazek K. ......................................60, 64
Bluyssen H.A.R. .................................40
Boczkowska A. ...................................66
Bogdanova-Jatniece A. .......................49
Boniewska E. ......................................69
Boniewska-Bernacka E. .....................56
Bozyk J. ..............................................90
Brzychczy-Wloch M. .........................33
Buczynska A. ............................... 37, 87
Burdzinska A. ............................... 31, 57
Butenko G.M. .....................................80
Butrym M. ..........................................57
Buzanska L. .................................... 9, 55
F
Foldes G. ........................................... 23
G
Gaida R. ............................................. 69
Gajewski Z. ....................................... 31
Gala K................................................ 57
Gendaszewska-Darmach E. ......... 62, 68
Godlewski M. .................................... 31
Golynska M. ...................................... 52
Grabowska I....................................... 32
Grabska S. ......................................... 76
Grendziak R. ................................ 37, 87
Grodzicki E. ....................................... 77
Grolik M. ........................................... 61
C
Caplan A. ............................................27
Chaikovsky Y.B. ................................80
Chelminiak D. .............................. 70, 77
Chlebus E. ..........................................88
Chojnacki M. ......................................71
Chroscicka A. .....................................65
Chwojnowski A. .................................30
Ciach T. ..............................................90
95
Gubanska I.................. 45, 46, 51, 60, 64
Kubiak J. ...................................... 37, 87
Kuchnicki P. ................................ 70, 77
Kucinska-Lipka J. ....... 45, 46, 51, 60, 64
Kulesza A. ......................................... 57
Kupikowska-Stobba B. ...................... 30
H
Helinska A. .........................................32
I
L
Iefimova I.A. ......................................72
Ilnicka A. ............................................77
Ishchenko I.O.......................................50
Jaegermann Z. .............................. 65, 66
Jakubczyk K. ......................................56
Jakutowicz T. .....................................30
Janik H. ...................... 45, 46, 51, 60, 64
Julkowska M. ............................... 43, 44
Jundzill A. ..........................................81
Labunets I.F. ...................................... 80
Langa P. ............................................. 25
Lechowicz J. ...................................... 33
Lepek T.............................................. 89
Lewandowska K. ............................... 76
Lewandowska M................................ 89
Lewandowska- Szumiel M. ......... 19, 65
Lewinska D. ....................................... 30
Lipkowski A.W. ................................ 35
Lukaszewicz J.P. ............................... 77
Lukowska E. ...................................... 30
K
M
Kaczmarek B. .....................................53
Kaczmarek H. .....................................77
Kaczmarek-Kedziera A. ...............70, 77
Kielkowska U. ....................................70
Klein A. ........................................46, 51
Kloskowski T. ........................ 81, 83, 84
Kobielak K. ........................................38
Kochanowski R. .................................21
Kolecka B. ..........................................74
Kondej K. ...........................................25
Konop M. ...........................................35
Kosiorek P. ...................................46, 51
Kosson P.............................................35
Kovalov G.A. ................................ 50, 72
Kowalczyk T. .....................................81
Kowalska A. .......................................48
Kozlovska T. ......................................49
Kozłowska J. ......................................59
Krasicka-Cydzik E. ............................47
Krok-Borkowicz M. ...........................85
Krzesniak A. .......................................27
Maciaczyk J. ...................................... 13
Maj M. ............................................... 44
Maluch I. ........................................... 89
Manchenko A.A. ............................... 92
Maslowski L. ............................... 37, 87
Mauquoy S. ....................................... 85
Michalowski S. .................................. 65
Michalska M. ..................................... 63
Mikhailova I.P. .................................. 92
Misicka A. ......................................... 35
J
N
Naumova O.V. .................................... 50
Nipot E.E. .......................................... 72
Noszczyk B. ....................................... 81
Novikova S.N. ................................... 78
Nowacki M. ................................. 83, 84
Nowak J. ............................................ 30
Nowakowska M. ................................ 61
96
Sandomirsky B.P. ........................ 72, 92
Sha M. ............................................... 14
Shamalo S.M. .................................... 80
Shpakova N.M. .................................. 72
Siodmiak T. ....................................... 77
Sionkowska A. 20, 43, 44, 53, 59, 63, 76
Siwek M. ..................................... 40, 41
Skopinska-Wisniewska J. .......43, 44, 84
Slawinska A. ................................ 40, 41
Slota R. ........................................ 56, 69
Slynarski K. ....................................... 27
Stangel-Wojcikiewicz K. ................... 26
Sujka W. ............................................ 94
Sulejczak D. ....................................... 35
Szablowska-Gadomska I. .................. 55
Szczubialka K. ................................... 61
Szczucinska A. .................................. 35
Szymonowicz M. ............................... 88
O
Ochiya T. ............................................12
Oksymets V.M....................................28
Orzechowska-Wylegala B. .................61
Ozieblo A. ..........................................66
P
Pach K. ...............................................74
Paczek L. ......................................31, 57
Paczesny Ł..........................................18
Pałka L. ..............................................88
Pamula E. ......................... 16, 33, 74, 85
Panov S.I. ...........................................72
Paprocka M. .................................37, 87
Parulska O. .........................................88
Pietkun K. .....................................83, 84
Pijanowska D.G. .................................48
Pikula M. ............................................25
Plonczak M. ........................................30
Plowiec A. ..........................................40
Pluta K.D. ...........................................48
Podobinska M. ....................................55
Pokrywczynska M. ................. 81, 83, 84
Polkowska I. .......................................52
Posadowska U. ...................................33
Prahl A. ..............................................89
Prokopyuk O.S. ..................................50
T
Tack A. .............................................. 14
Teratani T. ......................................... 12
Tremolada C. ..................................... 27
Trzonkowski P. .................................. 25
Tworkiewicz J. ............................ 43, 84
Tyloch D. ................................43, 83, 84
V
Vasyliev R.G. .........................28, 78, 80
Vlasov O.O. .................................. 50, 72
R
Rasmus M. ..........................................83
Reczynska K. ......................................74
Renkielska A. .....................................25
Rodnichenko A.E. ........................ 78, 80
Rodnichenko A.T. ..............................28
Rudnicka L. ........................................35
Rybak Z. .............................................88
W
Warda K. ........................................... 83
Wardowska A. ................................... 25
Weglowska E. .................................... 68
Wegrzynowska K. ....................... 43, 44
Winiarska H. ...................................... 55
Witkiewicz W. ............................. 37, 87
Wojakowski W. ................................. 22
Wojasinski M..................................... 90
S
Samluk A. ...........................................48
Sandomirskiy B.P. ...............................50
97
Wojciechowski C. ..............................30
Wojtowicz-Prus E......................... 37, 87
Wowra B. ...........................................61
Wylegala E. .................................. 24, 61
Zakrzewska K.E. ............................... 48
Zarychta W. ....................................... 57
Zawadzka M. ..................................... 40
Ziegler-Borowska M.................... 70, 77
Zielinski J. ......................................... 25
Zlatska A.V.................................. 28, 78
Zubov D.A. .................................. 28, 78
Zweigerdt R. ...................................... 14
Z
Zabost E..............................................81
Zagozdzon R. .....................................31
98

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