und Kieferheilkunde der Albert-Ludwigs

Aus der Universitätsklinik für Zahn-, Mund- und Kieferheilkunde
der Albert-Ludwigs-Universität Freiburg i. Br.
Abteilung für Kieferorthopädie
Age-related Proliferative Behavior of Mandibular Osteoblasts
- An in vitro study -
Inaugural-Dissertation
zur
Erlangung des Zahnmedizinischen Doktorgrades
der Medizinischen Fakultät
der Albert-Ludwigs-Universität
Freiburg i. Br.
vorgelegt 2004
von Mohammad Samer Juma’a
geboren in Damaskus / Syrien
Dekan: Prof. Dr. J. Zentner
1. Gutachter: Prof. Dr. I. E. Jonas
2. Gutachter: Prof. Dr. Dr. J. Düker
Promotionsjahr: 2005
Table of Contents
1. Introduction…………………………………………………………...……1
1.1. Osteoblasts’ Growth Factors and Mediators…………………………………………3
1.2. Characteristics of Growth Factors and Mediators………..…….............................5
1.3. Proliferation Tests……..………………………………………………………………..9
1.3.1. MTT-Test..…………………………………………………………….......................9
1.3.2. Easy For You (EZ4U)-Test..……………………………………………………….10
2. Literature Review…...……………………………………………………11
2.1. Orthodontic Aspects of Ageing…...……...…………………………………………..15
3. Aim of the Study……..…………………………………………………..18
4. Materials and Methods………………………………………………….19
4.1. Samples………...………………………………………………………………………19
4.2. Materials………………………………………………………………........................19
4.3. Preparation Procedures……………………………...……………………………….20
4.4. Culturing of Samples (first passage)……...……..……..….………………………..22
4.5. Feeding of Cultured Biopsies (first passage)………….….…………………..……23
4.6. Transferring of Cells (second passage)……....………...….……………………….23
4.7. Feeding of Osteoblastic Cells (second Passage)…..……….…...........................24
4.8. Cell Preparation for Proliferation Tests……………….....………………………….24
4.9. MTT-Test…………………….…..……………………………………………………..24
4.10. Easy For You-Test (EZ4U)………………………………………………………....25
4.11. Calk-Proof Experiment According to van Kossa….……………………….……..25
4.12. Light Microscopic Examination……………….…….………….............................26
4.13. Scanning Electron-Microscopic (SEM) Examination..…………………....…......28
4.14. Separation – Thin Section – Technology (Hard Cross Section Technology) …28
5. Results……………………………………………………………………..32
5.1. MTT-Test……………………...………………………………………………………..32
5.2. Easy For You (EZ4U)-Test...…………………………………………………………33
5.3. Statistic Analysis……...……………………………………………………………….34
5.3.1. EZ4U-Test..………………………………………………………………………….34
5.3.2. MTT-Test...………...………………………………………………………………...35
5.3.3. Cell-Counting by Using the Separation – Thin Section – Technology (Hard
Cross Section Technology)………..………………………………………………36
5.3.4. Measurement of Cell Surface……………...………………………………………37
6. Discussion………………………………………………………………...38
6.1. EZ4U (easy for you)-Test…………………………………………………………….38
6.2. MTT-Test……...………………………………………………………………………..39
6.3. Comparison…………………………………………………………………………….39
6.4. Cell Number and Surface…………………………………………………………….40
6.5. Orthodontic Aspects of Morphological and Histological Osteoblast-Ageing…….43
7. Summary…………………………………………………………………..45
8. Zusammenfassung………………………………………………………46
9. Appendix….……………………………………………………………….47
9.1. EZ4U-Test...……………………………………………………………………………47
9.2. MTT-Test.………………………………………………………………………………49
9.3. Number of Cells per View-Field…….………………………………………………..51
9.4. Surface of the Cells per View-Field………………………………………………….52
10. References…..…………………………………………………………..54
11. Curriculum Vitae………………………………………………………..61
1. Introduction
Successful orthodontic treatment is dependent upon the adaptive potential of
human hard and soft tissues. At a cellular level, bone remodeling is related to the
proliferation capacity of osteoblasts and osteoclasts beside the muscular tissues
and vascular system. It has been thought that the proliferation capacity of bone
cells decreases with age due to cellular and extracellular factors.
At a microscopic level, even normal action of daily activities can induce cracks in
bone. With age these microscopic cracks appear to accumulate exponentially and
are associated also with an increase in bony porosity (Frank et al. 2002). It has yet
to be fully elucidated whether this is largely due to a reduction in the process of
repair. After using histomorphometrical methods, the examination of the incidence
and the localization of microcracks in human bone specimens have proven that
the amount of microdamages increases dramatically with advancing age (Schaffler
et al. 1995).
It has been observed that at the same time as bone loss occurs on the endosteal
surface, bone is being added to the periosteal surface, but much more slowly than
during growth (Parfitt 1984). Even the fracture healing in young patients is faster
than in the elderly, but it is unknown whether these changes are attributed to the
number of osteogenic precursor cells of bone or whether they are connected with
a change in the proliferative cellular kinetics (Shigeno & Ashton 1995).
There are several possible mechanisms to explain the age-related reduction in
bone formation. It could be related to the availability of fewer stem cells or the
changing biological activy of local regulatory factors. The responsiveness of the
osteoblast lineage may also decrease (King & Keeling 1994).
It has been suggested that the number of proliferative precursor cells on trabecular
bone surfaces is higher in younger subjects (Shigeno & Ashton 1995), and the
decrease in bone amount is accompanied with a decrease in bone formation rate,
which can be due to the reduction in the number and activity of osteoprogenitor
cells, or the osteoblasts (Kabasawa et al. 1996).
A human osteoblast is similar to other diploid cell types. It has a limited
proliferative capacity and undergoes ageing and senesence (Kassem et al. 1997).
Therefore age-related bone loss is partially accounted for by an increased
1
osteoblastic maturation and a decrease in osteoblastic proliferation (Martinez et al.
1999).
Enlow (1982) has reported that bone tissue itself does not have the capacity to
increase in size with senesence because the cellular components do not have any
growth activity after maturation. However, there remains proliferative potential
processes in the soft tissues of the periosteum, endosteum and the surrounding
muscular, vascular, and nerval tissue.
2
1.1. Osteoblasts’ Growth Factors and Mediators
The interaction of the human osteoblast with its surrounding local and systemic
environment is complex. It has influences from the extracellular matrix, cytokines
and systemically derived hormones. Some of these interactions have been clearly
established in the recent literature, others remain to be defined.
The extracellular matrix is composed of various products which are from local or
exogenous resources. Many of these proteins are known to be growth factors like;
transforming growth factor-ß (TGF-ß), insulin-like growth factors (IGFS) and
fibroblast growth factors (FGF). These concentrate in mineralized bone and
probably play a role in bone regeneration after injury (Termine 1990). Growth
factors and cytokines are important mediators of communication between cells
and also between the cell and its extracellular matrix. Furthermore they mediate
locally the effects of several hormones on bone cells (Rifas 1999).
An example of these communication factors can be seen in the osteoblast which
synthesizes four attachment proteins and molecules to mediate the interaction of a
connective tissue cell with its extracellular environment. These are; fibronectin
(FN), thrombospondin (TSP), osteopontin (OP), and bone sialoprotein (BSP). The
later three all bind ionic calcium and exist in the bone matrix to influence the
processes of cellular development and differentiation. With advancing age they
degrade to lower molecular weight fragments, probably this degradation is
associated with deactivation of their bioactivities. At present it is unknown if this
alteration leads to a decrease in cell function in adult individuals (Termine 1990).
Other systemic factors such as parathyroid hormone, vitamin D metabolites, and
calcitonin mediate the process of bone remodeling (Saito et al. 1994). Moreover it
is well-known that osteoblasts exhibit estrogen- and parathyroid hormone- (PTH)
receptors, and produce cAMP following PTH-stimulation and synthesis of type-Icollagen, and osteocalcin (Simmons et al. 1994).
The developing bone matrix also contains two different small proteoglycans,
Prostaglandins: PG-I and PG-II. While mature bone matrix contains primarily PGII, the fetal connective tissue contains more abundant concentration of PG-I
(Termine 1990).
In addition to the above-mentioned growth factors, one of the most distinct
characteristics of osteoblasts is the synthesis of alkaline phosphatase and other
3
secretory proteins like osteonectin, osteopontin, and fibronectin (Pavlin et al.
1994).
Alterations of the concentration of any of these factors, as well as the
responsiveness of bone cell to these factors, might lead to a decrease in
osteoblasts’ generation and consequently to an incomplete refilling of the
resorption lacunae (Pfeilschifter et al. 1993)
The cytokines IGF-I, IGF-II, TGF-ß and PGE-2 control the proliferative rate of the
osteoblasts and their capacity to generate various components of bone
extracellular matrix, but osteoblasts also produce constitutively osteoclastogenic
IL- 6 and CSFs as well as osteoclastic inhibitory factors TGF-ß (Simmons et al.
1994).
The bone formation process starts with the recruitment of osteoblasts in the
vicinity of the resorption site as an initial step. This process takes place as a result
of the proliferation of osteoblastic precursor cells. It has been proven that this
proliferation is induced initially through local growth factors, such as TGF-ß,
platelet- derived growth factor (PDGF) and IGFs, which may be released from
bone during resorption (Pfeilschifter et al. 1993)
The bone formative properties of the human osteoblast reduce with increasing
age; this reduction is more obvious by comparing osteoblasts obtained from
fetuses with others obtained from adult donors (Termine 1990).
4
1.2. Characteristics of Growth Factors and Mediators
a) Alkaline phosphatase
Alkaline phosphatase exists in high concentrations in cells which initiate the
mineralized nodule formation in vitro. Furthermore it is well known that periodontal
ligament (PDL) cells containing high levels of alkaline phosphatase show more
rapid or increased nodule formation when compared with PDL-cells with lower
alkaline phosphatase concentrations (Areco et al. 1991).
b) Osteonectin
Osteonectin is a bone glycoprotein, which has its highest concentration levels
between the non-collagenous proteins in the developing bone; moreover it has a
high affinity to bind calcium-hydroxyapatite, collagen, and thrombospondin.
Osteonectin has been found in various non-bone cells, but only in the phase when
the individual undergoes rapid growth and proliferation. Osteonectin production is
highest in bone when compared with the other connective tissues in the human
body.
There is a relation between the existence of osteonectin in high levels in
osteoblasts and its proliferative potential. This is increased in cells with greater
content of osteonectin. In bone matrix this protein seems to be more structural and
may act through its mineral and protein-binding properties (Termine 1990).
c) Type-I-collagen
Type-I-collagen is the prototype of the collagen family, and is known to be the
most abundant component in the extracellular matrix. This is responsible for
several significant biomechanical properties of the connective tissue (D’Souza &
Litz 1994).
Type-I-collagen protein is encoded by two genes, COLIA1 and COLIA2 which are
expressed in all collagen-producing cells in a coordinated manner (Pavlin et al.
1994).
5
Osteoblasts synthesize relatively more type-I-collagen when compared with other
collagen-producing cells. This can be considered as one of the distinctive
characteristics of the osteoblastic phenotype. It has been suggested that this
production of type-I-collagen is modulated by; 1,25-dihydroxyvitamin-D3 (vitamin
D), hormones like (PTH), cytokines, and growth factors such as insulin and (IGF-I).
There are all mechanisms that are partly mediated at the transcriptional level
(Pavlin et al. 1994, D’Souza & Litz 1994).
After synthesizing high levels of type-I-collagen, the osteoblasts deposit it to create
the osteoid layer. This process is followed by a mineralization phase to form the
bone matrix (Pavlin et al. 1994).
d) Vitamin D
Vitamin D is an important calcium-regulating hormone. It has several effects on the
bone cells, including the stimulation of osteopontin and osteocalcin synthesis. It
also inhibits the synthesis of type-I-collagen in osteoblastic cell lines (Pavlin et al.
1994)
An adequate response to several hormones such as 1,25 dihydroxyvitamin D3
(calcitriol) is considered to be a normal function of osteoblast cells (Kveiborg et al.
2001).
e) Parathyroid hormone (PTH)
Several tasks of PTH have been reported, such as the stimulation of cell
proliferation, DNA-synthesis in osteoblast cells in vitro, and bone apposition when
combined with vitamin D.
According to its concentration, it can also have bone resorbing effects by
interacting with the regulation of calcium-homeostasis; PTH may cause inhibition
of cell proliferation when it is applied in high doses (Carvalho et al. 1994).
It has been proven that PTH-inducible cAMP formation increases with ageing,
which seems to be more associated with an increase in PTH-receptors than with
their decrease (Pfeilschifter et al. 1993).
6
f) Interleukin-6 (IL-6)
Interleukin-6 is a cytokine produced by many cell types including the stromal cells
and the osteoblasts which both contain large amounts of it. Some believe that IL-6
is not a regulator of osteoblast function (Saito et al. 1994, De Vernejoul et. al.
1993). Others however despute this and it has been reported that IL-6 has an
inhibitory effect on bone formation through the stimulation of osteoclastic activity
and subsequently bone resorption (Saito et al. 1994)
The high production of interleukin-6 by osteoblasts is associated with high bone
resorption rate in mice after ovariectomy (De Vernejoul et al. 1993).
g) Interleukin-1 (IL-1)
Saito et al. (1994) have defined the cytokine (IL-1) as a potent biological response
modifier, which has a wide range of biological activities, and plays a central role in
the inflammatory process. It stimulates bone resorption and induces osteoblast
proliferation, but shows an inhibitory effect on bone formation in vitro.
h) Transforming growth factor-beta 1 (TGF-ß1)
The cytokine (TGF-ß1) has many effects on osteoblast activities. For instance, its
influence on the net-structure accumulation of the cellular matrix causing either
more degradation or in contrast increase of its synthesis. Moreover, it could act as
an activator of type-I-collagen gen transcription (D’Souza & Litz 1994).
i) Prostaglandin E (PGE)
It has been proven that Prostaglandin E stimulates bone resorption in vitro and
causes more calcium release when applied in high doses (Saito et al. 1994).
7
j) Insulin-like growth factor-I (IGF-I)
This is a polypeptide that exhibits a pro-insulin-like chemical structure with insulinlike and cell growth- promoting properties.
As reported insulin-like growth factor-I stimulates osteoblastic proliferation which is
considered as the most important influence on osteoblasts (D’Avis et al. 1997).
8
1.3. Proliferation Tests
There are many methods to evaluate the proliferation rate and the survival of the
different tissue types of the mammalian creatures. Methods include; the counting
of dye in included or excluded cells, measurement of released
51
Cr-labeled protein
after cell lysis, measurement of the incorporation of radioactive nucleotides such
as [3H] thymidine or [125I] iododeoxyuridine during cell proliferation, radiolabeled
glucose utilization, and the automated image analysis of crystal violet-stained cells
(Mosmann 1983, Carmichael et al. 1987).
Some of these methods are inefficient, either because of the low ability of plating
or because of technical problems or needing a lot of time.
1.3.1. MTT-Test
The MTT-test has been developed by Mosmann (1983) to solve time-problems in
examinations containing a big amount of samples during cell refeeding in cases
with increased incubation time. On the other hand cell death and the loss of
dehydrogenase activity must be allowed to have enough time to take place, thus
the recommended incubation time is between four and seven days.
In regard to these considerations, this proliferation test is accounted to be a semiautomated one, because it can be performed in 96-well plates and by using a
scanning multiwell spectrometer (Carmichael et al. 1987).
The principle of MTT-test depends on the cleavage of the tetrazolium salt MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) into a blue colored
product (formazan) which can be measured spectrophotometrically by the
mitochondrial enzyme succinate dehydrogenase (Denizot & Lang 1986). The
tetrazolium ring is cleaved in active mitochondria and therefore it can be only
applied by intact mitochondria of living cells. A distinct signal by reading the color
reaction using the spectrometer is revealed form cells with a high rate of
metabolism. It can also help by the evaluation of the cell proliferation or activation
as well as by the evaluation of the cytotoxicity, namely by a wide spectrum of cell
kinds.
9
The MTT-test has been found to be capable of detecting cell activities regardless
of their amounts, and the spectrometric absorbance increases proportionally by
increased cell number. However, in order to avoid the protein preciptations an
organic solvent can be advantageous (Mosmann 1983).
As a difference between MTT-test and the test of the incorporation of [3H]
thymidine has to be mentioned that MTT-test measures the cell proliferation at the
end of the test procedure, while [3H] thymidine incorporation evaluates the cell
activity at the last few hours of the test.
1.3.2. Easy For You (EZ4U)-Test
The EZ4U-test is usually used to assess both osteoblasts and keratin cells. This
procedure has been accomplished in our study as a second experimental process
for the assessment of the proliferation potential of sheep-derived osteoblasts.
The achievements of this test do not affect the vitality of the examined cells. It
depends on the active cell ability to reduce the tetrazolium salts into formazan
derivatives. This reduction process is supported from the cellular mitochondria
(Wiedmann-Al-Ahmad et al. 2002)
10
2. Literature Review
Pfeilschifter et al. (1993) taking cultures from the femoral head trabecular bone of
60-90 year-old women have shown that mitogenic responsiveness to plateletderived growth factors and growth hormones is related inversely with the donor
age. This study established that age-related bone loss may be due to some extent
to the decreased proliferative capacity of the osteoblasts influenced by several
systemically and locally secreted osteotropic factors. In comparison to human
bone formation in vivo it has been reported that human bone formation in vitro has
similar biological properties and cellular proliferative rates.
Another study has confirmed that human osteoblast cellular activity is dependent
on the potential of extracellular matrix synthesis and its responsiveness to
systemic and local growth factors. Both of these are contingent on increasing age
of the donors (D’Avis et al. 1997). Findings after examination of cortical bone from
dogs of various ages have suggested that the decrease in both the proliferation
rate and cellular size are associated with progressing age (Williams et al. 1987).
The osteoblast-like cells exhibit more capacity of collagenase inhabitation; this
was five times more in young donors in comparison to the elderly. It could be
possible that the elder osteoblasts produce more collagen than the younger cells if
the collagen degradation has been inhibited (Koshihara & Honda 1994). After
culturing of osteoblast cells from 168 patients (50-70 years of age and older than
70 years) undergoing knee or hip arthroplasty, the results revealed higher
proliferation rates and more osteocalcine secretion in young donors when
compared with the older. In contrast the alkaline posphatase secretion was
decreased. There was no difference in the C-terminal type-I-collagen secretion
between the two generations (Martinez et al. 1999).
Comparing young cells (less than 20 % life span completed) to old cells (more
than 90 % life span completed) indicated a progressive increase in both population
doubling time (PD) and the protein content per cell. Moreover they showed a
decrease in both the attachment frequency, and the rates of DNA, RNA and
protein synthesis. The production of type-I-collagen was significantly decreased in
young donors; the activity of alkaline phosphatase decreased quickly after the first
few passages and became stable during the rest of the proliferative life span.
11
Morphologically young osteoblasts look thinner and spindle-shaped, while the
older cells look larger, more flattened with irregularly shape, and full of intracellular
debris (Kassem et al. 1997).
The in vitro growth of human cells obtained from trabecular bone showed no
relation with progressing age; but the cell number at the confluence has been
decreased with increasing donor age. The results also showed no significant
change in the metabolic efficiency of the bone-derived cells, including the total
protein, osteocalcin, and alkaline phosphatase synthesis in relation with age. In
other words these results could lead to the suggestion that while the ability of
individual cells to divide and to perform specific synthetic activities remains
unimpaired with ageing, there are other fine changes which can cause the
decrease in the osteogenic capacity (Evans et al. 1990).
In an experiment the osteocalcin concentration in bone cells was investigated. The
authors describe that bone formation and turnover rate seemed to decrease with
increasing age, and it has also been reported that osteoblastic activity and bone
turnover are stimulated at the menopause (Vanderscheuren et al. 1990).
The outcomes of a comparison of osteocalcin secretion by human osteoblasts
obtained from 38 donors undergoing either knee or hip arthroplasty have indicated
that secreted osteocalcin was higher in human osteoblasts obtained from young
patients. Moreover the response of osteocalcin secretion and its mRNA to
physiological doses of 1,25-(OH)2-D3 decreased with advancing age (Martinez et
al. 2001). These findings were emphasized by another study which has suggested
that decreased osteoblast functioning with in vitro ageing is due to impaired
responsiveness to calcitriol which is requested for the regulation of the biological
activities of osteoblasts (Kveiborg et al. 2001).
Also Martinez et al. (1999) determined that osteoblastic responsiveness to 1,25(OH)2-D3 in vitro represents an important regulator of osteoblastic functions,
however the degree of this regulation is depending on the stage of osteoblastic
maturation.
Yoshida (1995) investigated the mineralization ability and the growth response to
estrogen. He found that these processes decrease with advancing age of the
donor. This indicates that donor age relates with the depression of proliferation,
mineralization and biological responses to estrogen, 1 alpha, 25-(OH)2-D3 and
retinoic acid in osteoblastic cells of mandibular bone.
12
Young rats showed a significantly increased osteoblastic proliferation when treated
with 1,25-(OH)-D3. This response was dose-dependent. This means that the
osteoblastic proliferation was increased after application of high concentrations of
1,25-(OH)-D3 and decreased after application of lower concentrations. In contrast
osteoblasts obtained from old rats did not show any response to the treatment with
1,25-(OH)-D3. The same effect has been noticed by using the transforming growth
factor-beta-1 (TGF-ß1). This indicates that age has a distinct influence on the
ability of osteoblasts to respond to vitamin D and TGF-ß1 which are considered as
two of the most important factors of the development and maturation process
(Shiels et al. 2002).
After examination of osteoblasts harvested from iliac crest of 98 donors, the
results suggested that TGF-ß has a stimulatory effect on the osteoblastic
proliferation. This effect significantly declined with advanced age. At the same time
the decline of TGF-ß effect on proliferation’s accumulation could be generated
from a single human osteoblast progenitor cell (Erdmann et al. 1999). Cell surface
expression of TGF-ß receptors was studied in human osteoblasts derived from
femoral trabecular bone. It has been proven that no significant differences in cell
differentiation or proliferative behavior between the age groups could be
determined.
The TGF-ß receptor number per cell significantly increased with age, while the
receptor affinity tended to decrease (Batge et al. 2000).
In humans, increasing age has been associated with a linear decline in human
skeletal content of IGF-I (D’Avis et al. 1997).
Another study examined the in vitro age-related changes in osteoblast functions
comparing the constitutive mRNA levels of osteoblast- specific genes in early
passage cells (< 50 % lifespan completed) with those of late passages (> 90 %
life-span completed). The outcomes showed a significant reduction in mRNA
levels of alkaline phosphatase, osteocalcin, and collagen-type-I in in vitro
senescent late passage cells compared to early passage cells, suggesting an in
vitro age-related impairment of osteoblast functions. In addition there was no
change in the amount of the vitamin D receptors (VDR) with in vitro increasing
osteoblast age.
Examination of VDR localization, nuclear translocation and DNA binding activity
revealed no in vitro age-related changes.
13
The treatment of early passage osteoblastic cells inhibited their proliferation and
stimulated steady state mRNA levels of alkaline phosphatase and osteocalcin.
Similarly, calcitriol treatment increased mRNA levels of alkaline phosphatase and
osteocalcin in late passage osteoblastic cells. Thus, in vitro senescent osteoblastic
cells maintain their responsiveness to calcitriol. An in vitro age- related decrease in
biological markers of osteoblast function can be reverted by calcitriol treatment
(Kveiborg et al. 2001).
14
2.1. Orthodontic Aspects of Ageing
Ageing has a distinct negative influence on bone modulation, on its
responsiveness to other factors, and on its metabolism. These changes could
develop if bone underwent inappropriate stimuli which can cause different
responses in young and adult individuals. Ren et al. (2003) found that orthodontic
procedures seem to be more time-consuming in adults than in juveniles. This may
be caused through the delay in response of bone tissue to the orthodontic load
and the consequent turnover processes in bone and the periodontal ligament as
well.
It has been reported that some differences exist between osteoblastic cells of the
mandibular and maxillary alveolar processes and those obtained from bone of
other parts of the skeleton. These differences start to develop in the embryonic
stage and continue to be clearer during the maturation and the ageing process of
these cells (Zernik et al. 1997).
Osteoblasts and/ or osteoclasts from aged rat alveolar bone showed lower activity
in the physiologic state. These cells could be highly activated in response to an
orthodontic force (Kabasawa et al. 1996). In contrast to the ageing effect,
osteoblast cells and their progenitors have the capacity to respond to an external
stimulus and can produce new bone during remodeling as well as during fracture
healing (Termine 1990). Clinically both the young patients and the adults have the
same response potential to mechanical stimuli once tooth movement has started.
Treatment time in older patients could be extended in comparison to younger
individuals because of the delay in the initial response of aged cells (Ren et al.
2003). Furthermore bone stimulation caused by application of an orthodontic force
play a great role by promoting bone resorption through production and induction of
certain factors such as prostaglandins or interleukins, at the same time it
stimulates DNA-synthesis in bone cells (Kabasawa et al. 1996).
Osteogenic cellular synthesis of bone proteins appears to be activated by
intermittent rather than by continuous strain. The dentoalveolar osteoblasts
respond to mechanical strain initially through increase in levels of inositol 1, 4, 5triphosphate (IP3), protein kinase C (PKC) activity, and later of cAMP; however
this response was potentiated when parathyroid hormone (PTH) was applied
together with mechanical strain (Carvalho et al. 1994).
15
Statistically Göz (1990) found that in adults the biological possibilities of tooth
movement are decreased to about one-third of those reported in children.
This decrease in the biological activity of bone cells and their capability to respond
to mechanical strain have been suggested to be reverted to several causes such
as a limited ability to engender the strain necessary to reach the bone remodeling
threshold due to decreased muscle mass and strength, and/ or a decline in certain
hormones or growth factors that may interact with mechanical signals to change
the sensitivity of bone cells to strain (Kohrt 2001).
After applying 40 g tipping forces to maxillary first molars on two groups of rats
(adult: 89-94 days and young: 30-35 days old), the results showed that young rats
have more rapid orthodontic tooth movement accompanied by a faster recovery of
the associated transient alveolar osteopenia. On the tension side of the tooth there
were differences across time in osteoblast surface percent in young animals but
not in adults (King and Keeling 1994).
A comparison of tooth movement cycles and changes in mineral densities of the
alveolar tissue was studied between young (21 to 28 days old) and adult rats (90
to 100 days old) by an application of 60 g mesial tipping force to the maxillary first
molars. A characteristic three-part tooth movement cycle was found in both animal
groups. This cycle consisted of an early “instantaneous” movement that was a
function of the viscoelastic properties of the tissues, a delay period when only little
tooth movement occurred due to tissue hyalinization and undermining resorption
and a late period when bone remodeling and tooth movement occurred.
Compared with the adult group, the amount of “instantaneous” movement in the
young rats was greater, the delay period was shorter, and the rate of secondary
tooth movement was increased. The young rats had significantly lower mineral
densities before orthodontic treatment. Both groups experienced a slight but
insignificant increase in density followed by a rapid decline, which however, did not
reveal any intergroup differences. The young rats reached this point on the fifth
and the adult on the seventh day (Bridges et al. 1988).
Mechanical stress was applied by means of orthodontic elastics inserted between
the first and the second maxillary molar of rats from different ages for seven days.
Results showed that bone formative activity of osteoblasts and bone resorptive
activity of osteoclasts around the maxillary molar section of physiologic rat alveolar
bone declined with age. However, results of mechanically loaded rat alveolar bone
16
revealed no evidence of numerical differences in number, size, and activity of
osteoclasts and osteoblasts among the ages, suggesting that mechanical stress
provokes an increased activity of osteoblasts and osteoclasts similarly in both
adults and younger rat alveolar bone (Kabasawa et al. 1996).
17
3. Aim of the Study
The proliferative behavior of osteoblasts with increasing age is of great interest to the
orthodontist. An understanding of osteoblast behavior increases our understanding of
remodeling response of the bone tissue from individuals of different age-groups to
active external stimuli. The goal of the present study was to establish the proliferative
capacity of osteoblasts with increasing age.
18
4. Materials and Methods
4.1. Samples
The osteoblastic cells used in this study were obtained from sheep of three different
age groups. Five animals were taken from each age group to undergo the
experimental process.
Group (1)
Consisted of five young sheep (of about six months of age).
Group (2)
Consisted of five middle-aged sheep (about two years old).
Group (3)
Consisted of five senescent sheep (ten years of age).
4.2. Materials
As a nutrient for the osteoblast cells, BGJ-B medium (Gibco, Eggenstein, Germany)
was used. This medium had to be supplemented with 2 % Hepes puffer, 5 % fetal
calf serum (FCS), and additionally 1 % penicillin (all products of Gibco, Eggenstein,
Germany).
The utilized materials
•
BGJ-B.
•
Hepes Puffer.
•
Penicillin/ Streptomycin.
•
Fetal Calf Serum (FCS).
•
Trypsin.
19
•
Calcium and magnesium free phosphate buffer (PBS).
•
Osteoblasts were cultured in prefabricated culture bottles (75 cm2), (tissue
Culture Flasks TC-75, Greiner, Frickhausen, Germany).
•
Trypan blue (L 6323; Biochrome KG, Berlin, Germany).
•
For cell cultivation, 96-well micro titer plates (No. 3696; Costar, Tübingen,
Germany) with a volume of 200 µl/ well were used.
•
Dimethylsulfoxid (DMSO) (Baker, Deventer, Netherlands).
•
☯3(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Sigma,
Deisenhofen, Germany).
•
In order to remove crystalline structures, trypan blue and MTT were sterilized
and filtered (filter 0.22 µl, Milli pore, Eschborn, Germany) before the usage.
•
Easy For You EZ4U (assay kit, Biomedica Gruppe, Vienna, Austria).
4.3. Preparation Procedures
•
The preparation procedures were started relatively shortly after the animals
were sacrificed. The time interval ranged between 4 to 6 hours.
•
To remove the biopsy from the bone, the oral mucosa and muscles were taken
off and the bone laid open (Fig. 1). These biopsies have been burred from the
thinnest section distally to the mental foramen of the mandible (Fig. 2).
•
The samples were obtained from the mandibular bone of the sheep by using a
trepan bur with slow speed and under cooling with Ringer’s solution.
20
Figure 1:
The mucosa and
the
periosteal
layers
were
separated
from
the bone behind
the
mental
foramen,
where
the
biopsy
was
taken.
Figure 2:
The
harvested
biopsy
obtained
from the mandible
bone
culturing.
21
before
4.4. Culturing of Samples (first passage)
Each biopsy was disinfected with ethanol 90 % for 10 seconds (Fig. 3) and thereafter
it was washed three times with PBS (each time for 10 seconds).
By using tweezers and forceps the biopsies were cut into small pieces. The samples
obtained from one sheep were cultured in two 75 cm2 Tissue-Culture- Flasks with
approximately 4 to 5 small pieces of bone in each flask (Fig. 4). Each flask contained
1.5 ml of BGJ-B medium. The samples were kept in an incubator for 2 to 3 weeks at
a temperature of about 37° C with 5 % CO2 concentration and before starting the
second passage.
Figure 3:
The bone pieces
were placed for 10
seconds in ethanol
to be disinfected.
Figure 4:
The biopsies were
cultured in 75 cm2
Tissue-CultureFlasks after being
divided
smaller
The
into
4-5
pieces.
flasks
contained 1.5 ml of
BGJ-medium.
22
4.5. Feeding of Cultured Biopsies (first passage)
After insertion into the flasks which already contained 1.5 ml BGJ-B medium the
biopsies were fed for 7 days. 1 ml, 1 ml, 1.5 ml from the same medium were
sequently added to the flasks every 3 days. At the end of the first 2 weeks the flasks
contained about 5 ml BGJ-B medium.
4.6. Transferring of Cells (second passage)
As mentioned above, after 2-3 weeks the cells had grown densely and been ready
for the transfer process to get the second generation of these osteoblasts.
The transferring process was done as follows:
•
The medium was pulled out from the flask.
•
10 ml PBS (not on the flask’s ground where the osteoblasts are glued, but
rather on the flask’s border) was added and pivoted to dissolve the FCS, and
once again pulled out.
•
At the same time trypsin was diluted with PBS (1 ml Tryp + 2 ml PBS).
•
This time 0.5 ml from the diluted trypsin was added directly on the cells on the
flask’s ground, and pivoted a little bit to be distributed on the whole surface.
•
The flask was placed in the incubator for 8 minutes.
•
A 50 ml-tube with a cell-strainer was prepared.
•
The cells were washed with 10 ml PBS, therewith it would be a solution of
PBS and the osteoblasts, which had to be pulled out and poured in the 50 ml
tube through the cell-strainer to avoid the bone pieces to be also poured in it.
•
The last step was repeated for 3 times to ensure that all cells had been
transferred into the tube.
•
After taking off the cell-strainer the solution was centrifuged.
•
PBS was pulled out letting the cells in the tube.
•
1 ml of the medium was added to the cells in the tube and mixed very well.
•
0.5 ml from the cells-medium solution was poured in a new flask which already
contained 5 ml BGJ-B medium and was put in the incubator.
23
4.7. Feeding of Osteoblastic Cells (second Passage)
This has been achieved by means of changing the 5 ml medium in a time interval of 7
days.
After 2 to 3 weeks of incubating the secondary cell culture, it was the time for the
proliferation average of each sample to be analyzed by means of two separate
proliferation tests.
4.8. Cell Preparation for Proliferation Tests
•
The cells were isolated accurately by cells’ transferring to the second passage.
•
1 ml from BGJ-B was added to the cells to have a 1 ml substrate from each
sample.
•
1 x 104 per 200 µl substrate was considered as a selected cell percentage in
each well, thus to achieve this the isolated cells were counted by using trypan
blue as a staining agent
•
After calculating the needed amount for each sample, the medium was added
to the substrate.
•
200 µl substrate per well in a 96-well Plate, in addition to one well containing
200 µl medium as a blank-probe.
•
The cells were kept in an incubator for 4 days at 37° C with 5 % CO2.
4.9. MTT-Test
•
MTT was dispersed in PBS with a concentration of 8.28 mg /ml.
•
Subsequently the MTT-solution was filtered to remove unsolved MTT.
•
50 µl MTT per well was added, so that every well contained 250 µl (200 µl cellmedium substrate + 50 µl MTT)
•
The cells were incubated (37 °C, 5 % CO2) for 2 hours.
•
After incubation both the MTT-solution and the medium were sucked off
leaving the osteoblastic cells bonded on the well’s ground.
24
•
Every well was washed by 100 µl PBS, followed by adding of 100 µl DMSO
and dispersing it with the cells till they became homogeneous.
•
The optical density was photometrically measured at a wavelength of 540 nm.
4.10. Easy For You-Test (EZ4U)
•
2.5 ml of the activator solution was dissolved in the lyophilized substrate,
which had to be warmed by hand during its mixing with the activator solution.
•
The resulting solution was pre-heated for 15 minutes up to 37° C in the
incubator.
•
25 µl of this solution was added to each 200 µl cell-medium substrate in the
96-well plate.
•
The 96-well plate was incubated for 5 hours at 37° C.
•
The measurement was achieved by determining light absorbance at 540 nm
wavelength with 690 nm as reference.
4. 11. Calk-Proof Experiment According to van Kossa
With this staining the osteoblasts were washed.
•
1x 105 cells/ ml were seeded into a 24-well plate.
•
After one week the cells were dehumidified.
•
Thereafter they were embedded in 5 % silver nitrate solution for 60 minutes in
the darkness and.
•
Washed with distilled water.
•
1 % pyrogallic acid was added for 3-5 minutes.
•
Rewashed with distilled water.
•
5 % sodiumthiosulfate for other 3-5 minutes.
•
Washed with distilled water repeatedly for approximately 15 minutes.
•
The contrast staining was achieved by utilizing nuclear fast red (Fulka, Bucks,
Switzerland) for 8 minutes.
•
Subsequently the staining was rinsed out with water (Fig. 5).
25
Figure 5: Left and right: Light microscopic photos for the calk-proof according to van Kossa’s silver
stain. The black stained areas are a sign of the existence of calcium-containing osteoid as an indicator
of osteoblastic activities.
4.12. Light Microscopic Examination
Photos of the samples under light microscope were taken for assessment and
comparison of the density and the cell size of the different age groups. Preparation
for the light microscopic examination was as follows:
•
The samples were rinsed with PBS.
•
Embedded for 3 days in formol.
•
10 ml from hemalaum (Merk, Darmstadt, Germany) for 5 minutes.
•
Washed with tap-water for another 5 minutes.
•
10
ml
Giemsas’s-solution
(Azur-Eosin-Methylen
blue-Solution,
Merk,
Darmstadt, Germany) was added
•
It was rinsed off with water and let for one day to be dried.
•
The digitalized photos (Fig. 6) were done by means of a light microscope
(Zeiss, Oberkochen, Germany, Software SIS, Münster, Germany).
26
Figure 6:
Light microscopic photos (x20) of
the osteoblasts from the second
cultures.
Top: Young group.
Middle: Middle-aged group.
Bottom: Old group.
27
4.13. Scanning Electron-Microscopic (SEM) Examination
•
The samples were embedded in 4 % paraformaldehyde at room temperature
for about 2 hours.
•
Thereafter they were put in the incubator containing 8 % formaldehyde for two
days at 4°C.
•
They underwent dehydration by using ascending alcohol concentrations
starting with 50 % up to 100 %, and the last one (alcohol 100 %) was repeated
2 times.
•
The samples were dried to a critical point by using liquid carbon dioxide (CPD
030 Baltec, Wallruf, Germany).
•
They were sputtered with gold palladium (Plano, Wetzlar, Germany) in the
SCD 040 (Balzers Union, Wallruf, Germany).
•
The used scanning electron microscope was Zeiss Leo 32 (Zeiss,
Oberkochen, Germany) (Fig. 7).
4.14. Separation – Thin Section – Technology (Hard Cross Section Technology)
a) Sawing of the bone into small pieces
The fresh bone was fixed in formalin for about 3 days before starting the
dehydration process.
b) Dehydration
With ascending concentrations of with distilled water diluted Ethanol of 50 %
up to 100 % for 18 days.
c) Infiltration with Technovit
The bone pieces were embedded in Technovit 7200 VLC (Heraeus Kulzer
GmbH & Co. KG, Wehrheim, Germany), with the use of ascending
concentrations of with ethanol diluted Technovit from 30 % up to 100 % for
approximately 3 weeks.
d) Embedding of the sample
The sample was put in a container and filled up with Technovit, afterwards
they were posed over night in a polymerization equipment then left in an
incubator for approximately 1 day to dry up.
28
e) Loops of the blocks
Have been achieved manually and mechanically in order to reach the desired
smoothness.
f) Sandwich-bonding under press
The sample (block) was pasted with another slide by means of Technovit 7210
VLC (precision adhesives).
g) Cutting of the blocks
Was performed via a diamond saw under vacuum-cooling followed by
polishing of the resulting preparations.
h) Etching of the preparations
With hydrogen peroxide (H2O2) for 15 minutes and then rinsed off under
running water for 5 minutes.
i) Staining
With toluidine solution for approximately 45 minutes
j) Microscopic evaluation (Fig. 8)
Was accomplished by using the SIS software Analysis 3.5 (Soft Imaging
System, Münster, Germany).
29
Figure 7:
Electron microscope photos (x200)
of the osteoblasts from the second
cultures.
Top: Picture from the young group.
Middle: Picture from the middleaged group.
Bottom: Picture from the old group.
30
Figure 8:
Three view-fields (x40) from the
young group (top), from the middleaged group (middle) and from the
old group (bottom)
31
5. Results
The osteoblasts obtained from the young sheep showed a different proliferation rate,
with different number of measurable medium-cells substrate wells, so while 11
substrate wells could be obtained from some animals, not more than 3 wells could be
got from the others. This difference was found in all animals of the three groups, and
it seemed to be not dependent on the age of the sample or the kind of applied
proliferation test.
5.1. MTT-Test
The photometric measurements of the three age-groups of the examined sheep
revealed the following results (Tab. I):
The middle-aged group presented a relative high proliferation rate with a mean value
of 0.2 nm and a standard deviation of 0.081 nm (lowest value 0.125 nm, highest
value 0.157 nm), followed by the old group which offered a proliferation mean value
of 0.139 nm and a standard deviation of 0.129 nm (with a value’s variance between
0.01 nm and 0.33). The lowest proliferation rate was observed in the youngest group
which showed a lower proliferation capacity with a mean value of 0.129 nm and a
standard deviation of 0.102 nm (lowest value 0.018 nm, highest value 0.28 nm).
Table I: Mean values and standard deviations of Osteoblasts’ proliferation rate in the young, middle
aged and old group (MTT-Test).
YOUNG
MIDDLE-AGED
OLD
MEAN VALUE
0.129
0.2
0.139
STANDARD DEVIATION
0.102
0.081
0.129
32
5.2. Easy For You (EZ4U)-Test
As by the results of the last test (Tab. II) the middle-aged group also showed
the most active proliferative function with a mean photometrical absorbance
rate of 0.578 nm and a standard deviation of 0.196 nm (lowest value 0.462
nm, highest value 0.901 nm).
The difference between the middle-aged group and the young one was not
significant, so that the last group showed a relative lower mean value for 0.52
nm. The standard deviation was 0.206 nm (range: 0.331 nm to 0.791 nm).
In contrast to the MTT-test the lowest proliferation potential was measured for
the oldest group with a mean value of 0.281 nm and a standard deviation of
0.393 nm (range: 0.18 nm to 0.893 nm). Furthermore one of the five samples
obtained from the old group showed negative values.
Table II: Mean values and standard deviations of osteoblasts’ active proliferation function in the
young, middle-aged and old group (EZ4U).
YOUNG
MIDDLE-AGED
OLD
MEAN VALUE
0.52
0.578
0.281
STANDARD DEVIATION
0.206
0.196
0.393
33
5.3. Statistical Analysis
The data of both tests (MTT and EZ4U) were statistically analyzed using the
Friedman test.
5.3.1. EZ4U-Test
In consideration of the mean values of the different groups it was noticeable that the
middle-aged sheep had the highest proliferative potential with a value of 0.578 nm
(Fig. 9).
The difference between the middle-aged animals and the younger group was not
significant.
In comparison to the last two groups, the old sample showed a significant reduction
in cellular proliferation (mean value 0.281 nm).
EZ4U-test
1,2
Photometric Values in (nm)
1
0,8
0,6
0,4
0,2
0
Young
Middle-aged
Old
Age-groups
Figure 9: Photometric absorbance values of the osteoblastic cells obtained from the three different
age-groups.
34
5.3.2. MTT-Test
The results of the photometric evaluation of the proliferative behavior of the
osteoblastic cells after the application of the MTT-test were to certain degree similar
to those of the EZ4U-test in relating to both the young and the middle-aged sheep
(Fig. 10).
The absorbance rate in the middle-aged group was the highest compared to the
other two age-groups with an average value of 0.2 nm, while the registered value of
the young samples was 0.129 nm (= mean value).
In the opposite of EZ4U-test results, old samples offered more proliferative capacity
in comparison to the young one with a mean absorbance value of 0.139 nm.
MTT-test
0,45
0,4
Photometric values in (nm)
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
Young
Middle-aged
Old
Age-groups
Figure 10: Photometric absorbance values of the osteoblasts of the three different ages after
application of MTT-test.
5.3.3. Cell-counting by Using the Separation – Thin Section – Technology (Hard
Cross Section Technology)
35
Cell-counting per view-field in the prepared sections of the different age-groups was
performed with a 40’s objective in a measuring-framework of 760 x 570 Pixel.
From the young group three sections were prepared. The cells in 5 view-fields per
one section were counted. The cell number varied between 35 and 60 with a mean
value of 47 cells per view-field.
From the middle-aged group cells in 15 view-fields from two sections were counted.
The variance of cell number was between 26 and 39 with a mean value of 33 cells
per view-field.
That was the same mean value of cell number in the old group as well, however the
lowest and the highest value were approximately the same for about 26 and 38 cells
per view-field in the three prepared sections.
Osteoblast number by using (Hard Cross Section Technology)
70
Cell number per view-field
60
50
40
30
20
10
0
Young
Middle-aged
Old
Age-groups
Figure 11: Osteoblast number of all of the samples of each age-group counted by using the hard
cross section technology.
5.3.4. Measurement of Cell Surfaces
36
For measuring the cell surfaces we used the sections prepared by the separation-thin
section-technology.
Two sections from each sample group were examined, and from each section the
sum of the existing cells in 20 view-fields for each section were measured.
The data showed that the young samples had the larger amount of cell surfaces on
average. The value was about 30 µm with a variance between 22 µm and 37 µm.
Cell surfaces of the middle-aged were smaller and varied between 19 µm and 31 µm
with a mean value of 25 µm.
Compared with the above-mentioned groups the cell surfaces of the old group was
the smallest with a mean value of 21.48 µm (lowest value 16 µm, highest value 33
µm).
Amount of osteoblast-cell surfaces by using (Hard Cross Section Technology)
40
35
cell surfaces
30
25
20
15
10
5
0
Young
Middle-aged
Old
Age-groups
Figure 12: Osteoblast-cell surfaces measured after using the hard cross section technology.
37
6. Discussion
The purpose of this study was to examine the influence of age-dependent changes of
osteoblast proliferation rate. The amount to which bone growth is affected by age
was assessed by two different histological analysis methods. The examined
specimens were obtained from three age-groups of sheep.
A literature search was performed to investigate if the osteoblast cells obtained from
sheep are comparable to those from human donors. Torricelli et al. (2003) published
two studies; in the first one they compared the biologic synthesis activity of human,
rat and sheep osteoblasts. They established that an osteoblastic culture from sheep
was the most similar to human cells because of cell viability and the production of
osteocalcin, Interleukine-6 and transforming growth factor-beta 1.
The aim of the other study was to know if the cultures of osteoporotic sheep cells
could behave similar to those from human origin; the results confirmed the similarity
between the behavior of the both groups after application of 1,25(OH)2D3
stimulation. A significant difference was registered in the production of transforming
growth factor-beta 1 (TGF-ß1) before and after the stimulation applied in both groups.
All of our investigations including MTT-test, EZ4U-test, cell counting, and measuring
the cellular surface size showed a very large standard deviation.
6.1. EZ4U (Easy For You)-Test
The evaluation of the data of EZ4U-test showed that middle-aged samples had the
highest proliferation capacity in comparison to the two other groups. The old samples
had the lowest mean value, although the difference between the young and the
middle-aged animals was not too significant.
As an explanation of these results, it could be considered, that the osteoblast as a
connective tissue cell has the highest proliferation potential if it reaches a certain age
phase. Possibly the cells from the middle-aged animals were affected by the effects
of certain hormonal and physiological factors accompanied with pubertal changes to
increase the cellular activity.
In each of the experimental groups there was a remarkably large individual variation.
38
6.2. MTT-Test
The high absorption value of the middle-aged animals’ results of 0.2 nm confirms the
relatively large activity of the osteoblastic cells obtained from this sample group.
The young and the old animals had lower mean values after the achievement of
MTT-test; however they have offered approximately the same proliferation rate with
0.129 nm and 0.139 nm respectively, although the mean value of the old group was a
little higher. That means that both the young and the old cells had significantly lower
proliferation capacity in comparison to the middle-aged.
After analysing the individual values of the samples of each age-group, it became
evident that the variance was too large for a valid result. While one sample from the
middle-aged group showed a value of 0.104 nm, another one from the young group
showed a value of 0.347 nm, although the young group had the lowest mean
proliferation potential.
6.3. Comparison
By comparing the results of the MTT-test to those of the EZ4U-test, it is remarkable
that in spite of this high individual variation, the middle-aged group showed the
highest proliferation rate in both tests. The lowest mean value in the first assay was
registered for the young samples, while in the EZ4U-test it was registered for the old
samples.
To discuss this variance it has to be considered that both test methods are different,
while the EZ4U-test is a simplified proliferation assessment method with less
possibility of iatrogenic errors, the MTT-test is a multi-phase procedure.
This difference between outcomes of both tests cannot be regarded as relevant
because of the large variance of the individual values for each sample. In addition the
proliferation rate of the young animals determined by the MTT-test was on average
not significantly lower than that of the old sheep. Also the middle-aged animals,
which were in the puberty phase, always had the highest mean values of all the
samples.
39
6.4. Cell Number and Surface
After using the thin section separation technology (hard cross section technology),
the cell number of the young samples was the highest with 47 cells per view-field in
comparison to 33 cells per view-field for each of the middle-aged and the senescent
samples. This could be considered as normal because of the high frequency of the
cell division by young creatures in general.
The same results were suggested by Williams et al. (1987) after morphometric
assessment of the volume of cortical bone osteocytes obtained from dogs’ canine
regions of various age-groups of dogs in the.
This fact does not correspond with the results of the accomplished proliferation tests,
MTT-test depends on the number of cells present and on the mitochondrial activity
per cell (Denizot and Lang 1986). The test's concept is the cleavage of the
tetrazolium salt MTT into formazan product by a certain concentration of 1 x 104 per
200 µl from cell-medium substrate, The same concentration was also used by the
performance of EZ4U-test. This means that the proliferative activity by the young
group is significantly increased, although the cell number of the middle-aged group is
smaller.
The same result was determined in a study by Evans et al. (1989), which indicated
that the in vitro growth of cells obtained from the femoral head of patients from
various age-groups was unaffected with increasing age. It was also associated with a
decrease in cell number; furthermore the synthesis of DNA, total protein, ALP and
osteocalcine was not shown to be different.
The cell surface of the young samples was the largest followed by the middle-aged.
The smallest cell surface was measured in the old cells. This is a normal effect of the
age-related dehydration of the connective tissue cells in general, and particularly the
increase of age-related calcification process in osteoblast cells.
Our data correlates to findings of other studies such as of Williams et al. (1987) which
indicated that cortical bone cell size taken from dogs decreased with increasing age.
We also found correlation with the work of Kassem et al. (1997) who noted that old
cells became more flattened, irregularly shaped and full of debris.
The study of Bellows et al. (2003) showed a similar proliferative variance by each of
the young and the old female rat groups; however only the decreased response of
40
the aged rats was observed after application of dexamethasone or progesterone to
the osteoprogenitor cells.
Other findings by Sibonga et al. (2000) and Koshihara et al. (1991) revealed the
independent effects of ageing on the capacity of bone formation and the response to
an external stimulus. No age-related differences in cell density per colony were found
by Koshihara et al. (1991). The same findings of a study from Evans et. al. (1990)
indicated that an in vitro cell growth is not affected by ageing, although the cell
number at the confluence showed a decrease.
These results led us to find an explanation for the age-related decline in bone mass,
which could be the influence of the release of several local and systemic factors in
this region of bone (Pfeilschifter et al. 1993).
The osteoblast-like cells obtained from the old donors in an in vitro study by Ergise et
al. (1996) revealed even much more rapid proliferative activity in comparison to the
younger samples. This means that age-increasing bone loss is due to the deficiency
of some local factors and proliferation inhibitors which can be secreted in the human
body and affect the osteoblastic cells.
Edrise et al. (1992) found that the proliferation rate was approximately the same in
mature and aged rats, although the amount of cells which they obtained was
decreased in the old group. The authors registered a significant difference in the
percentage of mineralization between the trabecular surfaces compared with the
endocortical surfaces which were low in the aged animals.
According to Paulsen et al. (1999) the fibrocartilage and the underlying bone have
only slight age-related quantitative differences.
In contrast Martinez et al. (1999) found that age-related bone loss was associated
with a decrease in the proliferation rate of osteoblasts in addition to an increase in
their maturation, and might be reverted to osteoblastic differentiation. On the other
hand their explants were obtained from patients who were unhealthy and underwent
surgical procedures. Our differences to their findings might be caused due to the fact
that they used different skeletal sites.
In other words, bone samples obtained from the human jaws differ from those taken
from other parts of the bony skeleton. The origin of the first one is from the neural
crest-derived ectomesenchyme, while the bone tissue from the rest of the skeleton is
derived from mesenchymal cells. The other characteristic is the difference in their
development time (Zernik et al. 1997).
41
This difference in site of the bone biopsies is the critical point in many studies which
registered a decline in the biological activity of osteoblast cells. This is the case in the
study of Kassem et al. (1997) who reported a decrease in the rates of DNA, RNA,
type-I-collagen and protein synthesis after examining cells obtained from patients
suffering from osteoarthritis. In the same study the researchers examined an
osteoporotic patient who showed a dramatically reduced capacity of cell division and
several features of accelerated senescence.
The results of Shigeno and Ashton (1995) showed that the outgrowths of cultured
bone cells taken from young patients were significantly higher than in older
individuals. The authors interpreted, that the number of the proliferative precursor
cells on the bone surface was higher in the young group. Compared to our study the
latter one has determined the cell proliferation by using a dye-binding assay which
only considers the cell number but not the cell activity as was done in our experiment.
Such a result can be obtained if the examined cells undergo enzymatic treatment
during preparation as in the study of Battmann et al. (1997), who used type IV
collagenase in their work.
Even the synthesis of the extracelluar matrix in bone tissue – like type-I-collagen,
fibronectin, thrombospondin and others – degrade with increasing age (Termine
1990). This decline in cell function could be influenced by several local and systemic
factors which could affect the cells’ differentiation and maturation. In addition Evans
et al. (1990) reported that the metabolic efficiency of an aged donor did not show any
difference when compared with the younger individuals.
Jäger (1996) suggested that aging is correlated with a decrease in the fraction of
osteoid surface covered with active osteoblasts. The samples of his study were
obtained from mandibles of human cadavers. He reported that the cause of the
subject’s death did not have any influence on the skeleton. This statement lacks
credibility as people are often ill before dying, and secondly the study did not mention
the length of the period between the date of death and the time of sample
preparation, which could have had a significant effect on cellular activity.
42
6.5. Orthodontic Aspects of Morphological and Histological Osteoblast-Ageing
Some of the samples of the old group showed a high proliferation rate and activity,
which reveals that bone remodeling in adult patients can be achieved successfully.
Similar suggestions have been reported by Kabasawa et al. (1996), who never found
any significant difference in cellular number, size, and activity with increasing age,
and after a mechanical force application.
It is well-known that orthodontic tooth movement in adult patients is more timeconsuming. Our results confirm that an aged osteoblast could have the same
proliferative capacity and activity. The rapid tooth movement in young people could
be related to a delay caused by age-related changes in the periodontal structures
which take place in the initial response to an external stimulus, while it is similar after
reaching the linear phase. This means that the potency of the osteoblastic cell activity
stay the same independent of donor’s age (Ren et al. 2003).
Our point of view agrees also with those of King & Keeling (1994) who emphasized,
that no difference existed in the amount of tooth movement between various agegroups; however the higher elasticity of young periodontal ligaments allowed an
increased initial tipping of the teeth in the younger age-groups with a more apical
position of their rotation center compared to the aged animals examined in their
study.
These recommendations are outcomes of in vitro studies, which could not be reliable
in clinical orthodontics, because of the age-related changes occurred to the secretion
of local and systemic growth factors, enzymes, proteins, proliferation inhibitors, and
other functional mediators (Yoshida 1995).
For example, high levels of PTH result in more rapid tooth movement caused by an
increased rate of bone metabolism associated with decreased bone density (Midgett
et al. 1981). In another study the high initial response to mechanical stress was
tightly correlated with application of PTH dosage which induces the DNA synthesis in
treated cell cultures (Carvalho et al. 1994).
There are other factors which could play a role in the mechanism of orthodontic tooth
movements such as; the age-related changes in the morphology and the histology of
the periodontal ligament like the decrease in collagen fiber synthesis, the decrease in
the width of the periodontal ligament itself, the increase in the cement thickness
around the roots, and in addition to an increase in the irregularity and complexity of
43
oxytalan fibers which are the only elastic-like elements in the periodontal ligament
(Chantawiboonchai et al. 1999).
Bridges et al. (1988) determined that tooth movements in young rats after mechanical
stress application were more remarkable than in elderly (not to be ignored the high
variance of the data of the examined and the control group as well).
44
7. Summary
Knowledge of age-related changes occurring in the proliferative behavior of
osteoblasts is a necessity for orthodontists because the performance of an
orthodontic tooth movement requires a sufficient remodeling ability of the affected
bony tissue. This could be affected by several local and systemic factors which
influence the proliferative activity of the osteoblast with increasing age.
As the activity of osteoblasts obtained from sheep are similar to that of human cells,
sheep osteoblast cells were obtained to investigate the limit of the dividing function
which could be reached by a senescent osteoblast.
In an in vitro study, the proliferation rate of 15 sheep from 3 different age-groups was
measured using two different proliferation tests (MTT and EZ4U). These depend on
the mitochondrial functional activity of the cells and not only on their staining. The
difference in cell number and size between the three age-groups was also
determined and statistically analyzed using the Friedman-test.
The findings of this study indicated a large variance in the proliferative activity
between young, middle-aged, and old specimens with a significant standard
deviation in each group. However the middle-aged group always showed the
relatively highest mean values, which could be reverted to the maturing phase of the
cells when the animal had reached the age of puberty. This developmental stage is
normally associated with secretion of many hormones and growth factors.
The EZ4U-test proved that some of the individual old cells have shown more
proliferation potential than others from the young cells, and secondly by MTT-test the
proliferation rate on average was higher in the old than in the young group. This
means that ageing may have an influence on the proliferative function of the
osteoblasts.
Further studies on larger samples are needed, in order to verify the frequency of a
high proliferative potential in aged osteoblasts.
45
8. Zusammenfassung
Das Verständnis von altersabhängigen Änderungen im proliferativen Verhalten der
Osteoblasten besitzt für den Kieferorthopäden eine große Bedeutung, da die
Dürchführung
einer
orthodontischen
Zahnbewegung
eine
ausreichende
Remodellierungsfähigkeit des Knochengewebes erfordert. Dies kann durch lokale
und systemische Faktoren beeinflusst werden, die wiederum Einfluss auf die
proliferative Aktivität einer Osteoblastenzelle bei zunehmendem Alter nehmen.
Abhängig von der Tatsache, dass die biologische Aktivität der der aus Schafen
gewonnenen Osteoblasten der menschlicher Zellen ähnlich ist, wurden die in dieser
Studie gewonnenen Osteoblasten auf die Limitierung der Teilungsfunktion der
alternden
Osteoblasten
Proliferationsrate
von
hin
untersucht.
In
einer
Unterkieferosteoblasten
In-vitro-Studie
von
15
wurde
Schafen
aus
die
3
unterschiedlichen Altersgruppen mit zwei unterschiedlichen Proliferationstestverfahren gemessen (MTT und EZ4U). Beide Tests lassen zusätzlich zur Anfärbung
eine Untersuchung der mitochondrischen Funktion der Zellen zu. Der Unterschied in
der Zellanzahl und -größe zwischen den drei Altersgruppen wurde statistisch mit
dem Friedman-Test evaluiert.
Die Resultate dieser Arbeit haben eine große Varianz der proliferativen Aktivität
zwischen dem jungen, mittelalten und älteren Tierkollektiv belegt, jeweils mit einer
signifikanten Standardabweichung. Bei der Gruppe mittleren Alters wurden die
höchsten
Mittelwerte
gefunden,
was
auf
die
Reifungsphase
der
Zellen
zurückzuführen zu sein scheint, wenn das Tier das Pubertätsalter erreicht hat. Dieser
Entwicklungsstand steht in Zusammenhang mit der Absonderung verschiedener
Hormone und Wachstumsfaktoren. Das mittels EZ4U-Test nachgewiesene stärkere
Proliferationspotential bei einigen Zellen des älteren Kollektivs im Vergleich zu
einigen Zellen des jungen Kollektivs, und die zweitens beim MTT-Test vorliegende
im Durchschnitt höhere Proliferationsrate des älteren Kollektivs verglichen mit dem
jungen
Kollektiv
lässt
den
Schluss
zu,
das
fortschreitende
Zellalterung
möglicherweise einen Einfluss auf die proliferative Funktion der Osteoblasten hat.
Eine höhere Probenanzahl ist erforderlich, um das Vorhandensein eines hohen
Proliferationspotentials bei älteren Osteoblasten weiter zu verifizieren.
46
9. Appendix
9.1. Data of The EZ4U-Test (in nm)
Young animals
Y1
Y2
Y3
Y4
Y5
0,334
0,324
0,443
0,82
0,724
0,374
0,334
0,441
0,822
0,819
0,348
0,312
0,486
0,733
0,704
0,364
0,326
0,711
0,32
0,306
0,737
0,311
0,349
0,709
0,401
0,691
0,377
0,698
0,359
0,714
0,325
0,249
0,231
0,733
0,34183333 0,33127273 0,45666667 0,79166667 0,68081818 0,52045152 Mean
value
0,20678292 Standard
deviation
47
Middle-aged animals
M1
M2
M3
M4
M5
0,35
0,79
0,595
0,949
0,816
0,593
0,746
0,497
0,78
0,767
0,433
0,835
0,358
0,975
0,704
0,413
0,521
0,462
0,79033333 0,48333333 0,90133333 0,76233333 0,57855556 Mean
value
0,19630484 Standard
deviation
Old animals
O1
O2
O3
O4
O5
0,6
0,151
0,236
-0,22
0,226
0,945
0,196
0,461
-0,15
0,199
1,002
0,185
0,297
0,11
0,21
1,026
0,181
-0,5
0,14
-0,2
0,208
0,521
0,183
0,89325
0,18075
0,33133333 -0,192
0,19433333 0,28153333 Mean
value
0,39327861 Standard
deviation
48
9.2. Data of The MTT-Test (in nm)
Young animals
Y1
Y2
Y3
Y4
Y5
0,049
0,075
0,16
0,027
0,229
0,081
0,095
0,16
0,011
0,305
0,071
0,082
0,158
0,016
0,309
0,12
0,072
0,309
0,089
0,106
0,306
0,102
0,107
0,302
0, 081
0,101
0,29
0, 088
0,093
0,312
0, 111
0,115
0,347
0, 094
0,106
0,298
0, 071
0,081
0,165
0,087
0,09390909 0,15933333 0,018
0,28836364 0,12932121 Mean
value
0,10202051 Standard
deviation
49
Middle-aged animals
M1
M2
M3
M4
M5
0,274
0,096
0,127
0,125
0,135
0,327
0,147
0,23
0,109
0,149
0,347
0,104
0,114
0,166
0,145
0,1255
0,157
0,13333333 0,143
0,326
0,315
0,32
0,20083333 Mean
value
0,08148071 Standard
deviation
Old animals
O1
O2
O3
O4
O5
0,325
0,058
0,251
0,019
0,062
0,334
0,113
0,254
0,001
0,064
1,409
0,142
0,116
0,008
0,057
1,252
0,074
0,017
0,057
0,005
0,053
0,029
0,33
0,09675
0,207
0,01
0,05366667 0,13948333 Mean
value
0,12923977 Standard
deviation
50
9.3. Number of cells per view-field (with 40’s objective)
Young animals
Section
1
Section Section
2
3
45
60
51
44
35
47
51
48
52
52
36
44
46
45
53
47
50
45
47
Mean
Value
Middle-aged animals
Section
1
Section
2
26
30
34
47
33
39
28
30
34
32
28
32
31
33
38
34
33
32
34
33
51
Mean
value
Old animals
Section
1
Section Section
3
2
33
34
32
33
37
38
38
26
31
27
30
34
37
36
31
34
32
34
33
Mean
Value
30
Mean
value
9.4. Surface of the cells per view-field (in µm)
Young animals
Section
1
31
32
34
37
23
35
36
30
24
24
30
Section
2
26
33
22
27
31
27
30
37
30
37
37
32
35
33
29
33
25
33
27
26
26
27
25
31
28
27
32
28
33
24
30
52
Middle-aged animals
Section
1
26
24
25
23
30
22
28
21
27
30
Section
2
23
25
24
22
19
29
24
22
28
31
25
19
25
27
21
29
22
31
24
19
27
29
26
28
28
25
22
25
26
20
27
25
25
Mean
Value
21,5
Mean
value
Old animals
Section
1
29
22
18
28
19
19
25
20
19
18
23
Section
2
33
25
25
23
25
20
19
22
22
28
17
23
19
21
17
31
25
28
19
17
21
19
17
16
26
21
16
22
16
17
20
53
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60
11. Curriculum Vitae
Name:
Juma'a
Forename:
Mohammad Samer
Nationality:
Syrian
Date of birth:
25.09.74
Place of birth: Damascus, Syria
1979-1985:
Ibrahim Hananou-primary School, Damascus, Syria
1986-1988:
Jawdat Alhaschmi-secondary School, Damascus, Syria
1989-1991:
Ibn khaldoun School, University-entrance diploma, Damascus, Syria
1991-1996:
Dental study, Faculty of Dentistry, Tischreen University, Lattakia, Syria
1997-2000:
Clinical practice as a general dentist in the dental office of
Dr. Misbah Diab PHD. (Scientific Director of the Dental Faculty, University
of Damascus), Syria
1999-2000:
German language courses (Deutsch als Fremdsprache) basic level
(Grundstufe) Goethe-Institute, Damascus, Syria
2000-2001:
Clinical experience as a resident in the Department of Conservative
Dentistry and Periodontology, Albert Ludwigs University of Freiburg im
Breisgau, Germany
2002-2004:
Postgraduate education in Orthodontics, Department of Orthodontics (Head:
Prof. Dr. I. Jonas), Albert Ludwigs University of Freiburg im Breisgau,
Germany
10.2004:
Board degree in orthodontics, Stuttgart, Germany
61
12. Acknowledgments
I would like to express my most sincere gratitude to:
Prof. Dr. I. E. Jonas, Chair, Department of Orthodontics, Albert Ludwigs University,
Freiburg, Germany for supervising this dissertation, and the scientific support in the
orthodontic postgraduate program.
Prof. Dr. J. Schulte-Mönting, Institute of Medical Biometry and Medical Information,
Albert Ludwigs University, Freiburg, Germany for his support with the statistical
analysis of the data.
PD. Dr. Dr. E. C. Rose, Department of Orthodontics, Albert Ludwigs University,
Freiburg, Germany for his assistance and advice.
Mrs. U. Hübner, Cell Laboratory, Department of Maxillofacial Surgery, Albert
Ludwigs University, Freiburg, Germany.
Mrs. U. Feger, Library, Dental Clinic, Albert Ludwigs University, Freiburg, Germany,
for her help with searching the scientific literature for this study.
PHD. M. Diab, Scientific Director, Damascus University, Damascus, Syria for the
constant encouragement during my practical 5-years.
Dr. M. Krah, Albert Ludwigs University, Freiburg, Germany, for his efforts in
simplifying of my stay in Germany.
Above all, I would like to thank my family especially my parents for the love,
education and the support they gave me till now, and my brother Dr. Mohammad
Firas Juma’a for giving me the motivation and the light for my whole life.
62