Platelets

Larry D. Galuppo
William R. Pritchard
Veterinary Medical Teaching Hospital
University of California , Davis
https://vmacs.vmth.ucdavis.edu/userpages/ldgaluppo/
Tahoe2009
Novel Autogenous Therapeutics
!! PRP
– Platelet Rich Plasma
!! BMAC – Bone Marrow Aspirate
Concentrate
!! IRAP – Interleukin-1 Receptor Antagonist
Protein
!! Stem Cells
Introduction
!! Orthopedic
Disorders
"! Tendon & ligament injuries
"! Meniscal injuries
"! Fracture repair
"! Traumatic osteochondral fragmentation
"! Traumatic arthritis
"! Degenerative joint disease
"! OCD
Platelet Rich Plasma
!! The
Platelet
Platelet Function
!! Clot
formation
"! Activation
!! Collagen
!! Thrombin
!! Thromboxane A2
!! ADP
Adhesion & Degranulation
!!
Growth Factors
"! PDGF
"! TGF-"
"! VEGF
"! FGF
"! PDEGF
"! PDAF
"! IGF-1
"! PF4
!!
These factors signal the local
mesenchymal, epithelial and
endothelial cells to migrate,
divide, and increase collagen
and matrix synthesis
PRP Components
!! Platelets
"! Growth factors
!! Hematocrit
!! Granulocyte
recovery
!! Mononuclear cell recovery
!! Volume
Platelet Concentration
!!
Percentage Recovery vs Concentration Times Baseline
"! Percentage recovery – VOLUME INCLUDED
Total platelet number after concentration
Total platelet number before concentration
1400 x 103/µl x 6 ml X 100 = 76% Total = 8,400,000
185 x 103/µl x 60 ml
"! Increase time baseline – VOLUME NOT INCLUDED
Platelet count/µl after concentration
Platelet count/µl before concentration
140 x 103/µl = 7.6 x baseline Total = 140,000 x 6 ml = 840,000
18.5 x 103/µl
TOTAL NUMBER OF PLATELETS IS IMPORTANT
PRP- Veterinary Market
!!
Vantus Laboratories
!!
"! Res-Q
!!
Harvest
"! Genesis
!!
"! Smart Prep-2
!!
VetCell
"! AcelereTM-fPRP
!!
Milbourne
"! Magellan
VetStem
Arthrex
"! ACP
!!
MWI
"! Fibrivet
Product Performance
Final Product
(ml)
Platelet recovery
Times Baseline
Hematocrit
(%)
Smart Prep-2
(Human data)
6
7x
13
Res-Q
6
6x
2
Acelere TM
7
7x
?
GenesisCS
(Human data)
4
7x
32
ACP
(Human data)
2-3
2-3x
1
Magellan®
(Human data)
6
7x
7.5
Fibrivet
(Human data)
?
?
?
Device
Bone Marrow Aspirate Concentrate
!! Platelets
(growth factors) + cells
!! 50-60 ml of bone marrow
!! Concentrated total nucleated cells
"! Mononuclear
!! Macrophages
!! Lymphocytes
!! Stem cells
"! Granulocytes
BMAC-Veterinary Market
!! Harvest
"! R&D
!! Vantus
Laboratories
"! Vantus Xpress®
"! Res-Q under R&D
!! VetCell
"! R&D
!! Vet-Stem
"! R&D
BMAC Components
!! MNC
recovery
!! Platelet recovery
"! Growth factors
!! Hematocrit
!! Cell
viability
!! CFU recovery
!! Volume
!! Granulocyte recovery
Product Performance
Device
Final
Product
volume
TNC
recovery
MNC
recovery
(%)
(%)
Platelet
recovery
Hematocrit
(%)
(ml)
Harvest
SmartPrep
6
58
60
NA
13
Biomet GPS
6
69
66
NA
15
Emcyte
GenesisCS
4
76
NA
NA
32
Vantus
Express
21
95
96
4-6x
7
Hypothetical Data - PRP/BMAC
Hematocrit
WBC
MNC
Neutrophil
Platelets
(%)
(%)
(%)
(%)
(%)
I
5
30
65-80
20
65
II
10
50
76-90
45
75
III
20
85-95
85-95
60
85
Processing
Technique
Hypothetical Data - PRP/BMAC
Hematocrit
WBC
MNC
Neutrophil
Platelets
(%)
(%)
(%)
(%)
(%)
I
5
30
65-80
20
65
II
10
50
76-90
45
75
III
20
85-95
85-95
60
85
Processing
Technique
Res-Q 60v PRP
Equine Whole Blood- PRP( n=8)
Device
Res-Q
60v
Final
Product Vol.
(ml)
WBC
Recovery
(%)
MNC
Recovery
(%)
Granulocyte
Depletion
(%)
Platelet
Recovery
(%)
Hct.
(%)
6
37±0.2
79± 1.5
93±2.0
60±6
(6x)
2±0.2
Growth Factors
6
Res Q: TGF ! 1 Release in
Activated Platelets
Fold Increase
Fold Increase
Res Q: PDGF-BB Release in
Activated Platelets
4
2
0
Plasma
PRP-Thrombin
Activated
7
6
5
4
3
2
1
0
Plasma
PRP-Thrombin
Activated
Interleukin -1 (IL-1)
Pro-inflammatory cytokine
!! Major mediator of joint
disease
!! Produced by
!!
"!Synoviocytes
"!Chondrocytes
"!WBCs
!!
Stimulates neutral
proteinase production
IL-1
Synovium
Chondrocytes
Macrophages
Neutral Proteinases
Synovial
Thickening
Tissue destruction
Cartilage
Degradation
IL-1 Ra (IRAP)
Naturally occurring
!! Produced by
!!
"!Synoviocytes
"!Chondrocytes
"!WBCs
The Balance
IL-1
IL-1Ra
Osteoarthritis
IL-1Ra
IL-1
Catabolic effects
The IRAP Theory
Inflammation – IL1
IRAP
Antagonized
24h Incubation of Whole Blood
(Human Data)
!!
Upregulation
"! Anti-inflammatory
!! IL-1ra
"! Pro-inflamatory
!! IL-1"
!! TNF-#
Medical grade 2.5mm diameter boro silicate beads
Incubated in 50% Chromium sulphate stimulates WBCs to
produce cytokines
IRAP II vs IRAP (Human Data)
IRAP II vs IRAP (Human Data)
Growth Factor Data
Device
FGF
Increase
PDGF
Increase
TGF-!
Increase
VEGF
Increase
IRAP II
8.1x
5.0x
3.9x
7.1x
IRAP
7.3x
4.3x
3.1x
5.7x
Arthrex Vet Systems
Indications
Synovitis
!! Capsulitis
!! Arthritis
!! Bursitis?
!! Tenosynovitis?
!!
ACS - Administration Protocol
!! 3
treatments 7 days apart
!! Volume dependent on joint involved
"! 4 - 6 ml
!! Therapeutic effect
"! Within days
"! After third treatment
!! Fourth
series
treatment - 4-6 months after initial
Conclusions
!!Novel Autogenous
"! PRP
"! BMAC
"! IRAP
"! Stem Cells
Therapeutics
Future Research and Applications
!! Treatment
timing
!! Number of Treatments
!! Treatment frequency
!! Combination therapies
"! PRP or BMAC
"! PRP/BMAC + MSCs
PRP
!!
!!
Platelet-rich plasma (PRP) has attracted attention as a safe and cost-effective
source of growth factors that stimulate cells to regenerate tissue. Bone marrow
aspirate was processed with the same protocol to obtain PRP from peripheral
blood. This concentrate contained condensed nucleated bone marrow cells,
which are useful for regenerative medicine, as well as condensed platelets. In
PRP derived from bone marrow aspirate, the density of platelets and levels of
growth factors (platelet-derived growth factor and transforming growth factorbeta) were the same as in PRP derived from peripheral blood. Condensation of
nucleated cells, especially small-sized cells, was confirmed. With a simple and
cost-effective technique, source cells and growth factors can be obtained at the
same time. This simultaneous concentration of platelets and bone marrow cells
has great potential as a source of materials for regenerative medicine
Nishimoto S, Oyama T, Matsuda K. Wound Repair Regen. 2007 Jan-Feb;15(1):
156-62
PRP
!!
Guided bone regeneration is an accepted surgical method employed in implant
dentistry to increase the quantity and quality of the host bone in areas of
localized alveolar defects. The lack of predictability in osseous regenerative
procedures with various grafting materials suggests that improvement in the
osteoinductive properties of these materials is highly desirable. Platelet-rich
plasma (PRP), a modification of fibrin glue made from autologous blood, is being
used to deliver growth factors in high concentration to sites requiring osseous
grafting. Growth factors released from the platelets include platelet-derived
growth factor, transforming growth factor beta, platelet-derived epidermal growth
factor, platelet-derived angiogenesis factor, insulin-like growth factor 1, and
platelet factor 4. These factors signal the local mesenchymal and epithelial cells
to migrate, divide, and increase collagen and matrix synthesis. PRP has been
suggested for use to increase the rate of bone deposition and quality of bone
regeneration when augmenting sites prior to or in conjunction with dental implant
placement Only 6 human studies using PRP have been found in the dental
implant literature and 5 were case series or reports. Thus, there is clearly a lack
of scientific evidence to support the use of PRP in combination with bone grafts
during augmentation procedures. This novel and potentially promising technique
requires well-designed, controlled studies to provide evidence of efficacy.
!!
Sánchez AR, Sheridan PJ, Kupp LI. Int J Oral Maxillofac Implants. 2003 Jan-Feb;
18(1):93-103
BMAC
!!
!!
OBJECTIVE: To analyze a centrifugation-based, point-of-care device that concentrates canine
platelets and bone marrow-derived cells. ANIMALS: 19 adult sexually intact dogs.
PROCEDURES: Anticoagulated peripheral blood (60 mL) and 60 mL of anticoagulated bone
marrow aspirate (BMA) were concentrated by centrifugation with the centrifugation-based,
point-of-care device to form a platelet and a bone marrow concentrate (BMC) from 11 dogs.
Blood samples were analyzed on the basis of hemograms, platelet count, and PCV. The BMA
and BMC were analyzed to determine PCV, total nucleated cell count, RBC count, and
differential cell counts. The BMC stromal cells were cultured in an osteoinductive medium.
Eight additional dogs were used to compare the BMC yield with that in which heparin was
infused into the bone marrow before aspiration. RESULTS: The centrifugation-based, point-ofcare device concentrated platelets by 6-fold over baseline (median recovery, 63.1%) with a
median of 1,336 x 10(3) platelets/microL in the 7-mL concentrate. The nucleated cells in
BMCs increased 7-fold (median recovery, 42.9%) with a median of 720 x 10(3) cells/microL in
the 4-mL concentrate. The myeloid nucleated cells and mononuclear cells increased
significantly in BMCs with a significant decrease in PCV, compared with that of BMAs. Stromal
cell cultures expressed an osteoblastic phenotype in culture. Infusion of heparin into the bone
marrow eliminated clot formation and created less variation in the yield (median recovery,
61.9%). CONCLUSIONS AND CLINICAL RELEVANCE: Bone marrow-derived cell and
platelet-rich concentrates may form bone if delivered in an engineered graft, thus decreasing
the need for cancellous bone grafts.
Thoesen MS, Berg-Foels WS, Stokol T, Rassnick KM, Jacobson MS, Kevy SV,
Todhunter RJ. Am J Vet Res. 2006 Oct;67(10):1655-61.
BMAC
!!
Stem and progenitor cell therapy is a novel strategy to enhance cardiovascular
regeneration. Cell isolation procedures are crucial for the functional activity of the
administered cellular product. Therefore, new isolation techniques have to be
evaluated in comparison to the Ficoll isolation procedure as the current gold
standard. Here we prospectively evaluated a novel point-of-care device (Harvest
BMAC System) for the concentration of bone marrow total nucleated cells (TNC) in
comparison to the Ficoll isolation procedure for bone marrow mononucleated cells
(MNC). The yield in total numbers of TNC was 2.4-fold higher for Harvest
compared to Ficoll. Despite significant differences in their cellular compositions, the
colony-forming capacity was similar for both products. Intriguingly, the migratory
capacity was significantly higher for the Harvest TNC (164 +/- 66%; p = 0.007). In a
mouse model of hind limb ischemia, the increase in blood flow recovery was similar
between Harvest BM-TNC and Ficoll BM-MNC (0.53 +/- 0.20 vs. 0.46 +/- 0.15; p =
0.88). However, adjustment of the injected cell number based on the higher yield of
Harvest TNC resulted in a significant better recovery (0.64 +/- 0.16 vs. 0.46 +/0.15; p = 0.003). Cells concentrated by the Harvest point-of-care device show
similar or greater functional activity compared to Ficoll isolation. However, the
greater yield of cells and the wider range of cell types for the Harvest device may
translate into an even greater therapeutic effect.
!!
Hermann PC, Huber SL, Herrler T, von Hesler C, Andrassy J, Kevy SV, Jacobson
MS, Heeschen C. Cell Transplant. 2008;16(10):1059-69.
IRAP
!!
Repair of cartilage defects involves sequential participation of specific hormones and
growth factors with potential impairment by inflammatory cytokines. We explored an in
vivo gene therapy treatment to supply adenoviral vectors carrying the genes of
interleukin-1 receptor antagonist protein (IL-1ra) and insulin like growth factor-1
(IGF-1), hoping to enhance repair of full-thickness equine chondral defects treated with
microfracture. We asked whether our treatment could (1) increase proteoglycan and
Type II collagen content in the repair tissue, (2) improve the macroscopic and
histomorphometric aspect of the repair tissue, and (3) induce prolonged and increased
IL-1ra and IGF-1 production in treated joints. Twelve horses had full-thickness chondral
defects created in their carpus and stifle followed by microfracture. Joints were injected
with either equine IL-1ra/IGF-1 adenoviral preparation or Gey's balanced salt solution.
Sixteen weeks later, defect healing was evaluated macroscopically, histologically,
histochemically, and biochemically. Production of IL-1ra and IGF-1 was measured by
enzyme-linked immunosorbent assay and radioimmunoassay. We found increased
proteoglycan content in treated defects along with augmented Type II collagen
associated with substantial transgene expression of IL-1ra during the first 3 weeks.
These data suggest in vivo gene therapy can improve biologic processes associated
with chondral defect repair.
!!
Morisset S, Frisbie DD, Robbins PD, Nixon AJ, McIlwraith CW. Clin Orthop Relat Res.
2007 Sep;462:221-8.
IRAP
!!
OBJECTIVE: To assess the clinical, biochemical, and histologic effects of intra-articular
administration of autologous conditioned serum (ACS) in the treatment of experimentally
induced osteoarthritis in horses. ANIMALS: 16 horses. PROCEDURES: Osteoarthritis
was induced arthroscopically in 1 middle carpal joint of all horses. In 8 placebo- and 8
ACS-treated horses, 6 mL of PBS solution or 6 mL of ACS was injected into the
osteoarthritis-affected joint on days 14, 21, 28, and 35, respectively; PBS solution was
administered in the other sham-operated joints. Evaluations included clinical assessment
of lameness and synovial fluid analysis (performed biweekly); gross pathologic and
histologic examinations of cartilage and synovial membrane samples were performed at
necropsy. RESULTS: No adverse treatment-related events were detected. Horses that
were treated with ACS had significant clinical improvement in lameness, unlike the
placebo-treated horses. Among the osteoarthritis-affected joints, ACS treatment
significantly decreased synovial membrane hyperplasia, compared with placebo-treated
joints; although not significant, the ACS-treated joints also appeared to have less gross
cartilage fibrillation and synovial membrane hemorrhage. The synovial fluid concentration
of interleukin-1 receptor antagonist (assessed by use of mouse anti-interleukin-1 receptor
antagonist antibody) was increased following treatment with ACS. CONCLUSIONS AND
CLINICAL RELEVANCE: Results of this controlled study indicated that there was
significant clinical and histologic improvement in osteoarthritis-affected joints of horses
following treatment with ACS, compared with placebo treatment. On the basis of these
findings, further controlled clinical trials to assess this treatment are warranted, and
investigation of the mechanisms of action of ACS should be pursued concurrently.
!!
Frisbie DD, Kawcak CE, Werpy NM, Park RD, McIlwraith CW. Am J Vet Res. 2007
Mar;68(3):290-6.