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THE USE OF PLATELET-RICH FIBRIN MATRICES TO
ENHANCE GROWTH FACTOR DELIVERY FOR
CONNECTIVE TISSUE HEALING

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LANCE CHARLES VISSER

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THE USE OF PLATELET-RICH FIBRIN MATRICES TO ENHANCE GROWTH
FACTOR DELIVERY FOR CONNECTIVE TISSUE HEALING

By

Lance Charles Visser

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Comparative Medicine and Integrative Biology

2010

ABSTRACT

THE USE OF PLATELET-RICH FIBRIN MATRICES TO ENHANCE GROWTH
FACTOR DELIVERY FOR CONNECTIVE TISSUE HEALING

By
Lance Charles Visser

Growth factors are known to play a crucial role in the repair and regeneration of
connective tissues. Research unraveling the basic biologic mechanisms of wound healing
in a variety of tissues has led to the development of numerous exogenous and autologous
growth factor products designed to enhance connective tissue repair. However, the
optimal growth factor delivery method for tissue repair and regeneration remains
unsettled. Autologous platelet-rich fibrin matrices (PRFM) may represent a promising
method to deliver locally increased concentrations of platelet-derived grth factors and
other bioactive molecules for a prolonged period of time. The goal of this thesis is to
examine the role of a PRFM in enhancing growth factor delivery for connective tissue
healing. In addition to providing a thorough review of the literature relevant to the
PRFM, the growth factor elution kinetics and mitogenic capacity of PRFMs in vitro is
examined. The role of a PRFM in enhancing and accelerating tendon healing in a canine
model in vivo is presented. The use of PRFM-related technology to create a bioactive
scaffold for tissue engineering applications is also presented. The results of this thesis
supports the use of PRFMs to enhance growth factor delivery for connective tissue
healing, particularly in biologically compromised or chronically injured tissues where a

prolonged increase in grth factors may be particularly desired.

Copyright by
LANCE CHARLES VISSER
2010

This work is dedicated to the two most important women in my life.

To my wife Blair:
Thank you for your continuous love, support, patience, and understanding.

To my Mom:
Thank you for teaching and showing me the true meaning of hard work.

iv

 

ACKNOWLEDGEMENTS

I would like to acknowledge a number of individuals, as the work of this thesis
would not have been possible without their unique contribution and support along the
way.

First and foremost, I must express my deepest gratitude to my mentor, Dr. Steven
Amoczky, for teaching me countless lessons in science and life. I am a better person for
having trained under you and I could not have asked for a better mentor.

I must also thank the other members of my graduate committee Drs. Bryden
Stanley, Kurt Williams, and Vilma Yuzbasiyan-Gurkan for their expertise and guidance.

I am indebted to the members of the Laboratory for Comparative Orthopaedic
Research, especially Oscar Caballero and Keri Gardner. I am also grateful to Dr. Michael
Lavagnino, Dr. Monika Egerbacher, Jeremy Gingrich, and Brian Cummings for their help
along the way.

I must also thank Dr. Mike Scott for helpful discussions regarding platelet
biology, Dr. Joe Hauptman for help with statistical analysis, Sharon Steck for technical
assistance in surgery, the MSU Investigative Histopathology Laboratory, and the MSU
Center for advanced Microscopy.

I am very grateful for my funding sources, the NIH T-32 one-year training grant
for veterinary students and the Wade 0. Brinker Chair in Veterinary Surgery and the
Laboratory for Comparative Orthopaedic Research.

Lastly, I would like to thank my wife and family for all of their love and support.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. viii

LIST OF FIGURES ................................................................................. ix

LIST OF ABBREVIATIONS ..................................................................... xii

GENERAL INTRODUCTION ............................................................................................ 1

CHAPTER 1.

Review of the Literature ............................................................................... 6
Overview of Connective Tissue Healing ................................................................. 7
Platelet Biology ...................................................................................................... 10
Fibrin and Connective Tissue Healing ................................................................... 15
Growth Factor Delivery Methods .......................................................................... 18

Exogenous Growth Factor Delivery .......................................................... 19

Autologous Growth Factor Delivery: Platelet-Rich Plasma ...................... 20

Autologous Growth Factor Delivery: Platelet-Rich Fibrin Matrix ............ 31

Purpose and Hypothesis ......................................................................................... 35

References .............................................................................................................. 38
CHAPTER 2.

Platelet-Rich Fibrin Constructs Elute Higher Concentrations of TGF—Bl and
Increase Tendon Cell Proliferation Over Time When Compared to Blood Clots: A

Comparative In Vitro Analysis ....................................................................................... 49
Abstract .................................................................................................................. 50
Introduction ............................................................................................................ 52
Materials and Methods ........................................................................................... 53
Results .................................................................................................................... 59
Discussion .............................................................................................................. 63
References .............................................................................................................. 67

CHAPTER 3.

The Use of an Autologous Platelet-Rich Fibrin Membrane to Enhance Tendon

Healing: An Experimental Study in Dogs ...................................................................... 70
Abstract .................................................................................................................. 71
Introduction ............................................................................................................ 72
Materials and Methods ........................................................................................... 74
Results .................................................................................................................... 80
Discussion .............................................................................................................. 86
References .............................................................................................................. 91

vi

CHAPTER 4.
Growth Factor-Rich Plasma Increases Tendon Cell Proliferation and Matrix

Synthesis on a Synthetic Scaffold: An In Vitro Study ................................................... 95
Abstract .................................................................................................................. 96
Introduction ............................................................................................................ 97
Materials and Methods ........................................................................................... 98
Results .................................................................................................................. 105
Discussion ............................................................................................................ 1 12
References ............................................................................................................ 1 16

CHAPTER 5.

Concluding Discussion ................................................................................................... 120

vii

LIST OF TABLES

Table 1.1 Bioactive molecules found within platelet tut-granules and their functions ...... 14
Table 1.2 Growth factors identified within PRP and their role in tissue repair ............ 24
Table 3.1 Semiquantitative histologic scoring system .................................................... 79

Table 3.2 Results of the semiquantitative histologic scoring system used to assess tendon
healing at 4 (A) and 8 (B) weeks post-operation. The ordinal data is displayed as mean
score 2t SD (n = 4). An unoperated (“normal”) tendon would have a perfect score of 0.
Repair tissue (RT) refers to the regenerated healing tissue within the central-third patellar
tendon defect and tendon tissue (TT) refers to the native patellar tendon tissue. GAGs =
glycosaminoglycans, PRFMem = platelet-rich fibrin membrane ...................................... 85

viii

LIST OF FIGURES

Figure 1.1 Model of a Megakaryoctye (MK) depicting transport of a-granules during
platelet formation. Granules are transported along microtubules from the MK cell body
through extensions termed proplatelets. Platelets form as bulges at the distal end of these

extensions. Contents of a-granules are shown in various colors, green = fibrinogen, red =
von Willebrand factor... .................................................................................................... 11

Figure 1.2 Transmission election micrograph of a human platelet depicting numerous
granules, including the a-granules indicated by the white arrows. Scale bar = 500
nm ..................................................................................................... 13

Figure 1.3 Summary of the coagulation cascade and fibrin formation ............................ 17

Figure 1.4 Schematic of the creation of a platelet-rich fibrin matrix (PRF Matrix) and
platelet-rich fibrin membrane (PRF Membrane) ................................................................ 33

Figure 2.1 Schematic flow chart illustrating the processing steps for the platelet-rich
fibrin matrix (PRF Matrix), platelet-rich fibrin membrane (PRFMembrane) and whole
blood clot (BC) ...................................................................................... 55

Figure 2.2 Mean concentrations of TGF -[31 + SE (n = 4) eluted from a blood clot (BC),
platelet-rich fibrin matrix (PRFMatrix), and platelet-rich fibrin membrane
(PRFMembrane). Statistical significance was not reached (p > 0.05) where p-values for
comparisons are not shown ........................................................................ 59

Figure 2.3 Photomicroscopic live-cell images of representative tendon cell proliferation
results after 48 hours of exposure to day l eluents from the respective constructs (Phase
contrast, 100x original magnification) ............................................................ 60

Figure 2.4 Mean optical density values displayed as n-fold difference over control i SE
(n = 4) of tendon cells subjected to eluent from the blood clot (BC), platelet-rich fibrin
matrix (PRFMatrix), or platelet-rich fibrin membrane (PRFMembrane) from the various
time periods. Statistical significance was not reached (p > 0.05) where p-values for
comparisons are not shown ......................................................................... 61

Figure 2.5 Photomicroscopic images of a representative blood clot (A), platelet-rich
fibrin matrix (B), and platelet-rich fibrin membrane (C) stained for fibrin with a
phosphotungstic acid hematoxylin (PTAH) stain. Fibrin strands are not visible among the
red blood cells in the whole blood clot (A). “Nests” of platelets (arrows) can be seen
among the dense fibrin network of the platelet-rich fibrin matrix (B). The dense
compaction of the fibrin in the platelet-rich fibrin membrane is obvious (C). Scale bar =
50 um and applies to each 1mage62

Figure 2.6 Photomicroscopic images of a representative platelet-rich fibrin matrix (A),
and platelet-rich fibrin membrane (B) immuno-stained for TGF-Bl. In both constructs
there is positive staining of the fibrin strands and localized areas of increased staining
intensity which likely represents “nests” of platelets trapped within the fibrin network.
Scale bar = 50 um and applies to both images. . . . . . .. ........................................................ 62

Figure 3.1 Summary of the creation of the platelet-rich fibrin membrane
(PRFMembrane) used in this study ............................................................... 75

Figure 3.2 Photographs of the surgical procedure. The patellar tendon was isolated and
exposed (A). A central-third patellar tendon defect was created (B). A bed of sutures was
created in the defect to act as caudal support (C). The autologous platelet-rich fibrin
membrane was rolled onto itself and sutured to the defect (D). The contralateral limb
consisted of only steps A and B ................................................................... 76

Figure 3.3 Photograph of a patellar tendon after histologic processing mounted in cross-
section representing an example of how the cross-sectional area (area within the dotted
line) data were gathered in this study. The number of pixels within the dotted line was
converted to area measurements using imaging software ...................................... 78

Figure 3.4 Representative photographs of cross-sectional tissue sections of a sham and
PRFMembrane-treated patellar tendons at 4 (A) and 8 (B) weeks after surgery. Note the
difference in size of the PRFMembrane-treated tendons compared to its surgical control
(sham) ................................................................................................ 81

Figure 3.5 Mean cross-sectional area of the sham and PRFMembrane-treated patellar
tendons 4 and 8 weeks post-operation i SD (n = 4) ............................................ 82

Figure 3.6 Photomicrographs of representative H&E-stained sections of the repair tissue
(RT) within the central-third patellar tendon defect of the sham and PRFMembrane-
treated patellar tendons at each time point. The cellularity, vascularity, collagen
organization, and glycosaminoglycan content (images not shown) of the PRFMembrane-
treated tendons were similar when compared to the sham tendons at each time point. The
white arrows denote blood vessels. Scale bar = 100 um ....................................... 84

Figure 3.7 Representative photographs of the sham (A) and PRFMembrane-treated (B)
patellar tendons 4 weeks after surgery mounted in cross-section. Note that the origin of
the repair tissue was primarily from a proliferative response arising from the fat pad and
paratenon (asterisks) in both groups, albeit more abundant in the PRFMembrane
group .................................................................................................. 86

Figure 4.] Photographs of the synthetic biodegradable polymeric scaffold used in this
study showing an 8-mm-diameter scaffold-disk (A) isolated from a larger sample of the

scaffold (B). Scanning electron photomicrographs of the scaffold at 20x (C) and 100x (D)
magnification ......................................................................................... 99

Figure 4.2 Scanning electron photomicrographs of a GFRP-enriched scaffold after
combining with cell culture media and prior to cell-seeding at 20x (A), 50x (B), 80x (C),
and 3500x (D) magnification. Notice the thin fibrin-matrix coating throughout the
scaffold surface that results from fibrin polymerizing around the scaffold when combined
with culture media containing calcium ......................................................... 101

Figure 4.3 Representative photomicrographs of the cell-seeded surface of a control (A)
serum-enriched (B) and GFRP-enriched (C) scaffold stained rhodamine phalloidin and
DAPI after 24 hours in culture. Note the dramatic increase in cell density of the GFRP-
enriched scaffold compared to the other groups ............................................... 106

Figure 4.4 Results of the MTT assays performed on the scaffolds after 48 and 72 hours
in culture. Optical density values indicate relative cell proliferation. Bars represent mean
optical density value i SE (n = 5) ................................................................ 107

Figure 4.5 Photomicrographs of representative transected scaffolds stained with
hematoxylin and eosin (H&E) from each group after 14, 21, and 28 days in culture. Note
the relative differences in the surface tissue thickness at each time point. Scale bar = 100
um ................................................................................................... 108

Figure 4.6 Photomicrographs of representative transected scaffolds from each group
after 21 days in culture. Tissue neogenesis on the surface of the scaffolds appeared most
abundant on the GFRP-enriched scaffolds. Hematoxylin & Eosin (H&E) -staining (top
row) showed that cells were able to invade into the scaffold equally well in all three
groups. Picrosirius red (PSR) -staining (middle row) suggested that the majority of tissue
generated was collagen. The bottom row shows scanning electron micrographs of the
scaffolds in each group. Scale bar = 100 um ................................................... 109

Figure 4.7 Cross-sectional tissue thickness measurements on the surface of the scaffolds
after 14, 21, and 28 days in culture. Bars represent the mean surface tissue thickness i SE
(n = 4). Statistical significance was not reached (p _>_ 0.05) where p-values for
comparisons are not shown ....................................................................... 110

Figure 4.8 Total collagen (A) and GAG (B) deposition by tendon cells seeded onto plain
scaffolds (control), serum-enriched scaffolds, or GFRP-enriched scaffolds after 14, 21,
and 28 days in culture. Bars represent the mean total collagen (A) or GAG (B) content
among the scaffolds :1: SE (n = 4). Statistical significance was not reached (p Z 0.05)
where p-values for comparisons are not shown ................................................ 11 1

xi

bFGF

BC

BMP

DMEM

ECGF

ELISA

EGF

FBS

FGF

GAG

GFRP

H&E

HGF

IGF

MMP

MK

MTT

PT

PD-EGF

PDGF

PRFM

LIST OF ABBREVIATIONS

Basic fibroblast growth factor

Blood clot

Bone morphogenetic protein

Dulbecco’s modified Eagle’s medium
Endothelial cell grth factor
Enzyme-linked immosorbent assay
Epidermal growth factor

Fetal bovine serum

Fibroblast growth factor
Glycosaminoglycan

Growth factor-rich plasma

Hematoxylin and eosin

Hepatocyte growth factor

Insulin-like growth factor

Matrix metalloproteinase
Megakaryocyte
3-(4,5-dimethylthiazol-2-yl)-2,5-diphynyltetrazolium bromide
Patellar tendon

Platelet-derived epidermal growth factor
Platelet-derived growth factor

Platelet-rich fibrin matrix/matrices

xii

PRFMatrix
PRFMembrane
PRP

PSR

RT

SEM

TGF-B

VEGF

Platelet-rich fibrin matrix
Platelet-rich fibrin membrane
Platelet-rich plasma

Picrosirius red

Repair tissue

Scanning electron microscopy
Tendon tissue

Transforming growth factor-beta

Vascular endothelial grth factor

xiii

GENERAL INTRODUCTION

Growth factors play a vital role in connective tissue repair and regeneration.
These polypeptides transmit signals that orchestrate the tissue repair process through a
variety of mechanisms including either stimulating or inhibiting cell proliferation,
migration, differentiation, or gene expression.(l) The use of autologous and recombinant
growth factors to improve tissue repair and regeneration is rapidly growing and appears
to hold great promise in a variety of fields in medicine. However, the optimal delivery
method for making these factors available at a desired site remains unresolved. Bolus
delivery of exogenous growth factors has been investigated as a potential method, yet
questions regarding the proper selection and dosage of these recombinant proteins remain
unanswered. The creation of biomaterials, constructs that incorporate growth factors with
polymer or hydrogel scaffolds, designed to mimic the natural release kinetics of growth
factors is an emerging field.(2) Although these technologies appear promising for the
controlled release of grth factors, the optimal grth factor or combination of grth
factors, dosage, timing of application, biosafety, and cost-effectiveness appear to be
significant shortcomings.(3, 4)

Recently, the use of platelet-rich plasma (PRP) has been proposed as a potential
method of delivering locally increased concentrations of a variety of bioactive autologous
growth factors in an effort to optimize connective tissue healing.(5-12) Platelet-rich
plasma has been defined as plasma that contains a platelet concentration above the
“normal” physiologic level found in whole blood.(l3) The increased concentration of
platelets also yields an increase in the concentration of grth factors that are stored in

platelet a-granules.(14, 15) These factors, such as platelet-derived grth factor (PDGF),
1.

transforming growth factor-beta (TGF-B), basic fibroblast growth factors (bFGF),
insulin-like growth factor-1 (IGF-1), and vascular endothelial growth factor (VEGF) have
all been shown to be essential in the healing of connective tissues.(l 6) Because numerous
in vitro studies have shown a direct dose-response influence of many growth factors on
cell migration, proliferation and matrix synthesis,(l7-20) it has been proposed that the
local administration of increased concentrations of these grth factors through the use
of PRP could optimize the local healing environment and thus enhance the ability of
biologically compromised tissues to generate a repair response.(21) While PRP
eliminates questions regarding growth factor selection and dosage, the relatively short
half-life, rapid degradation, and diffusion/wash-out of most growth factors in vivo are
potential significant limitations.(3) Therefore, the local administration of PRP alone may
not be able to provide a prolonged release of chemotactic and mitogenic factors that may
be particularly desired in connective tissue repair and regeneration.(22)

While platelets and their associated growth factors are important for initiating the
healing cascade, of equal importance is the presence of a provisional fibrin scaffold.(23,
24) Fibrin provides a naturally-derived matrix, on which repair cells can adhere, migrate,
proliferate, and deposit a more permanent extracellular matrix.(25, 26) Together with
other plasma-derived proteins such as fibrinogen, fibronectin, and vitronectin, fibrin is
able to bind to many growth factors present within platelet (Jr-granules, thus creating a
reservoir of grth factors(23, 24, 27) able to release growth factors over time.(26)
Therefore, the ability to combine an increased concentration of platelets and their
associated growth factors in plasma (i.e., PRP) within a fibrin scaffold may permit the

sustained availability of increased concentrations of growth factors over time. This, in

2

turn, may provide an optimal environment for engineering biological substitutes for the
body in vitro and/or for orchestrating tissue repair and regeneration in viva.

Autologous platelet-rich fibrin matrices (PRFMs) represent a promising method
to improve growth factor delivery by providing a locally available source of prolonged
increased concentrations of growth factors in “normal” physiologic ratios within a dense
provisional fibrin scaffold. However, there have been no published studies investigating
the basic biologic mechanisms behind its proposed benefits. Therefore, the general
purpose of this thesis was to determine if/how a PRFM is able to enhance growth factor
delivery and, consequently, augment connective tissue healing. This was accomplished in
three separate but related studies. First, however, a thorough review of the literature was
conducted in order to become familiar with what is known regarding the topics relevant
to the contents of this thesis and is detailed in chapter 1. Chapter 2 describes our first
study where we sought to determine the elution kinetics and mitogenic activity of the
growth factors eluted from a PRF M compared to a whole blood clot (BC) in vitro. In the
second study detailed in chapter 3 we test the purported benefits of the PRFM-in vivo
using a canine tendon healing model. Chapter 4 contains our third and final study in
which we attempt to create a bioactive scaffold using PRFM technology to enhance tissue
regeneration in vitro for tissue engineering applications. Chapter 5 contains a concluding
discussion and mentions future directions for further work related to the findings of this

thesis.

References

l. Nimni ME. Polypeptide growth factors: targeted delivery systems.
Biomaterials.l8:1201-25. 1997.

2. Lee KY, Peters MC, Anderson KW, Mooney DJ. Controlled growth factor release
from synthetic extracellular matrices. Nature.408:998-]000. 2000.

3. Chen R, Mooney DJ. Polymeric growth factor delivery strategies for tissue
engineering. Pharm Res.20:1103-12. 2003.

4. Tessmar JK, Gopferich AM. Matrices and scaffolds for protein delivery in tissue
engineering. Adv Drug Deliv Rev.59:274-9l. 2007.

5. Alsousou J, Thompson M, Hulley P, Noble A, Willett K. The biology of platelet-
rich plasma and its application in trauma and orthopaedic surgery: a review of the
literature. J Bone Joint Surg Br.9l :987-96. 2009.

6. Aspenberg P. Stimulation of tendon repair: mechanical loading, GDFs and
platelets. A mini-review. Int Orthop.3 1 :783-9. 2007.

7. Creaney L, Hamilton B. Growth factor delivery methods in the management of
sports injuries: the state of play. Br J Sports Med.42:314-20. 2008.

8. Foster TE, Puskas BL, Mandelbaum BR, Gerhardt MB, Rodeo SA. Platelet-rich
plasma: from basic science to clinical applications. Am J Sports Med.37:2259-72. 2009.

9. Gamradt SC, Rodeo SA, Warren RF. Platelet rich plasma in rotator cuff repair.
Techniques in Orthopaedics.22:26-33. 2007.

10. Mishra A, Woodall J, Jr., Vieira A. Treatment of tendon and muscle using
platelet-rich plasma. Clin Sports Med.28:l 13-25. 2009.

11. Nikolidakis D, Jansen JA. The biology of platelet-rich plasma and its application
in oral surgery: literature review. Tissue Eng Part B Rev.l4:249-58. 2008.

12. Sampson S, Gerhardt M, Mandelbaum B. Platelet rich plasma injection grafts for
musculoskeletal injuries: a review. Curr Rev Musculoskelet Med.1:165—74. 2008.

13. Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant
Dent.10:225-8. 2001.

14. Anitua E, Andia I, Ardanza B, Nurden P, Nurden AT. Autologous platelets as a
source of proteins for healing and tissue regeneration. Thromb Haemost.91:4-15. 2004.

15. Nurden AT, Nurden P, Sanchez M, Andia I, Anitua E. Platelets and wound
healing. Front Biosci.13:3532-48. 2008.

l6. Werner S, Grose R. Regulation of wound healing by growth factors and
cytokines. Physiol Rev.83:835-70. 2003.

17. Abrahamsson SO. Similar effects of recombinant human insulin-like growth
factor-1 and II on cellular activities in flexor tendons of young rabbits: experimental
studies in vitro. J Orthop Res.15:256-62. 1997.

18. Costa MA, Wu C, Pham BV, Chong AK, Pham HM, Chang J. Tissue engineering
of flexor tendons: optimization of tenocyte proliferation using growth factor
supplementation. Tissue Eng. 12: 1937-43. 2006.

19. Haupt JL, Donnelly BP, Nixon AJ. Effects of platelet-derived growth factor-BB
on the metabolic function and morphologic features of equine tendon in explant culture.
Am J Vet Res.67:1595-600. 2006.

20. Thomopoulos S, Harwood FL, Silva MJ, Amiel D, Gelberman RH. Effect of
several growth factors on canine flexor tendon fibroblast proliferation and collagen
synthesis in vitro. J Hand Surg [Am].30:441-7. 2005.

21. Marx RE. Platelet—rich plasma: evidence to support its use. J Oral Maxillofac
Surg.62:489-96. 2004.

22. O'Connell SM, Impeduglia T, Hessler K, Wang XJ, Carroll RJ, Dardik H.
Autologous platelet-rich fibrin matrix as cell therapy in the healing of chronic lower-
extremity ulcers. Wound Repair Regen.16:749-56. 2008.

23. Laurens N, Koolwijk P, de Maat MP. Fibrin structure and wound healing. J
Thromb Haemost.4:932-9. 2006.

24. Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb
Haemost.3: 1 894-904. 2005.

25. Ahmed TA, Dare EV, Hincke M. Fibrin: a versatile scaffold for tissue
engineering applications. Tissue Eng Part B Rev.l4:199-215. 2008.

26. Breen A, O'Brien T, Pandit A. Fibrin as a Delivery System for Therapeutic Drugs
and Biomolecules. Tissue Eng Part B Rev.in press. 2009.

27. Maori L, Silverstein D, Clark RA. Growth factor binding to the pericellular matrix
and its importance in tissue engineering. Adv Drug Deliv Rev.59:1366-8l. 2007.

CHAPTER 1.

Review of the Literature

OVERVIEW OF CON NECTIVE TISSUE HEALING

Connective tissue healing is an exquisitely designed set of events involving three
overlapping phases commonly referred to as the inflammatory, proliferative, and
remodeling phases. Growth factors and cytokines play a vital role throughout all of the
phases of wound healing. These bioactive molecules bind to transmembrane receptors on
local and circulating cells which, initiates intracellular signaling that ultimately affects
gene expression. Gene expression results in the production of additional growth factors as
well as cytokines that regulate cell proliferation, cell migration, angiogenesis, cellular
differentiation, further grth factor/cytokine expression, and extracellular matrix

production.

Inflammatory Phase

The inflammatory phase of tissue repair last for approximately 24 hours; it can be
further divided into an early vascular (hemostatic) phase and a later cellular
(inflammatory) phase.(1) The vascular phase begins immediately with vasoconstriction of
the arterioles and is the first attempt to limit blood loss at the site of injury.
Vasoconstriction is a transient process lasting for only a few minutes. It leads to platelet
aggregation and to the formation of an unstable clot (platelet plug), which leads to the
cessation of bleeding. Following hemostasis, vasodilation occurs, largely due to factors
released from platelets and damaged endothelium. Factors such as kinins, histamine,
prostaglandins, and leukotrienes increase vascular permeability leading to the leakage of
blood components into the tissues surrounding the injury.(2) Meanwhile, platelets are

exposed to the subendothelial layer of damaged blood vessels (primarily type IV

7

collagen), which causes them to adhere and aggregate. The coagulation cascade is
simultaneously activated in response to the injured vessels via factors from the blood and
the surrounding damaged tissues. Platelet aggregation and the polymerization of fibrin
form what is known as the stable hemostatic clot.(3, 4) The formation of a clot serves
many purposes. In addition to hemostasis, the clot acts as scaffold for many invading
cells and as a bioactive reservoir of growth factors required throughout the healing
process.(3-5) Platelets play a crucial role in the early phase of inflammation via release of
the first wave of growth factors and cytokines from their a-granules.(3, 6) These grth
factors and cytokines are known to play a role in signaling inflammatory cells,
particularly macrophages, and later fibroblasts, to the injury site initiating the cellular
phase of the inflammatory reaction. The cellular phase of inflammation typically begins
first, by the influx of neutrophils and later, by the arrival of monocytes and macrophages.
These inflammatory cells play a crucial role in phagocytosis of necrotic debris.
Additionally, macrophages are particularly important for remodeling the previously
fonned clot, initiating the second wave of growth factors to recruit resident fibroblasts,

and for initiating angiogenesis.(4, 5)

Proliferative Phase

The proliferative phase usually begins within a few days post-injury.(l) It is
characterized by fibroblast and endothelial cell proliferation and migration into the
wound. This is due in large part to growth factors and cytokines secreted mainly from
platelets and macrOphages.(3, 4) It is important that fibroblasts in the surrounding tissues

become activated by growth factors in order to “awaken” from their dormant non-

8

replicative state.(4) Once activated, these fibroblasts secrete their own growth factors and
migrate into the wound from the surrounding tissues with new capillaries lagging behind.
The new capillaries arise from nearby intact vessels in response to angiogenic growth
factors such as VEGF and PDGF.(4) Once the fibroblasts have entered the wound bed
they begin to synthesize various components of the extracellular matrix, dominated by
type III collagen which, at this point, remains disorganized and random. Furthermore,
wound fibroblasts begin to acquire a contractile phenotype and become known as the
myofibroblast, a cell type that plays a major role in wound contraction. The resulting
wound connective tissue is more commonly referred to as granulation tissue due to the

granular appearance of the many new capillary beds.

Remodeling Phase

The remodeling phase is characterized by the repair tissue slowly changing from
cellular to fibrous. In the remodeling phase, continued collagen synthesis and
reabsorption ensues. This phase is classically characterized by extracellular matrix
remodeling, regression of blood vessels, and by the tissue regaining tensile strength.
Overtime, type III collagen is replaced with the stronger type I collagen. As the tissue
matures some mechanical strength is restored as type I collagen fibers are increasingly
orientated along the direction of force through the tissue.(7) Applying controlled
physiologic loads to the healing connective tissue stimulates cross-linking of type I
collagen, granting additional strength to the tissue while allowing further orientation of
type I collagen along the axis of tension.(8, 9) From a period of about ten weeks to a year

the healing tissue continues to mature from a fibrous tissue to a scar beginning to

9

resemble the native tissue. During this time period the metabolism of the resident cells

decreases and blood vessels within the scar gradually regress.

PLATELET BIOLOGY

Traditionally known for their role in hemostasis, platelets are the first cells to
respond to an injury through the process of adherence, aggregation, and degranulation.
Platelets also play a crucial role in inflammation and tissue repair via cell-to-cell
interactions and the release of soluble mediators liberated from activated platelets via the
process of degranulation. Platelets circulate in the bloodstream for approximately 5 to 10
days where they survey the vasculature and respond to injury.(10, 11) Normal platelet
concentrations in both human and canine blood range from approximately 150,000/uL to
350,000/uL. It is important to note that, although there are subtle differences, the basic

biological properties of platelets are conserved across the mammalian species.(12, 13)

Platelet genesis

Platelets are considered to be small (2 um in diameter) subcellular fragments
derived from megakaryocytes (MKS). MKS represent 0.1 to 0.5% of the nucleated cells in
bone marrow.(l4) Megakaryocytopoesis is stimulated by several cytokines (e.g.,
Interleukin (IL) —3, IL-6, and IL-11) and thrombopoeitin, and results in platelet
formation.(15) MKS are located beneath the capillary sinuses in the bone marrow, where
they follow a maturation program culminating in the conversion of most of their
cytoplasmic material into multiple long processes known as proplatelets (Figure 1.1).

One MK may protrude as many as 10-20 proplatelets.(15) These proplatelets begin as
10

blunt protrusions that are driven out away from the MK cell body by microtubule—based
forces. Platelets are derived from bulges at the distal ends of the proplatelets, and as it
matures, it receives granules and organelles delivered as a stream of individual particles
from the MK cell body (Figure 1.1).(16) Once the platelet is saturated with the
appropriate intracellular content, a single microtubule is rolled into a coil, and the platelet
is then released into the bloodstream where it patrols the vasculature for injury for the

remainder of its lifespan.

    

proplatelets

Figure 1.1 Model of a Megakaryoctye (MK) depicting transport of a-granules during
platelet formation. Granules are transported along microtubules from the MK cell body
through extensions termed proplatelets. Platelets form as bulges at the distal end of these
extensions. Contents of a-granules are shown in various colors, green = fibrinogen, red =
von Willebrand factor. Adapted from ref. (16)

Platelet Granules

Platelets contain three major membrane-bound granules: lysosomal granules,
dense granules, and alpha (or) —granules. Lysosmal granules contain reservoirs of
hydrolases such as cathepsins D and E, elastases and other degradative enzymes.(17) The

dense granules contain several bioactive molecules that play a significant role in tissue
11

repair including adenosine triphosphate (ATP), adenosine diphosphate (ADP), calcium,
magnesium, serotonin, histamine, dopamine, and catecholamines. ADP plays a role in
platelet aggregation, whereas ATP plays a role in the platelet-collagen interaction under
flow.(3) Platelets take up and harbor circulating serotonin in the bloodstream and its
release upon platelet degranulation causes vasoconstriction and increased vascular
permeability. Interestingly, platelet-derived serotonin has recently been shown to play a
vital role in liver regeneration.(18) Calcium has also demonstrated to play a central role
in wound healing, as it is a necessary cofactor for platelet aggregation and fibrin
formation and can modulate cell proliferation and differentiation.(l9) The most abundant
granule in the platelet, and perhaps the most well-known, is the (Jr-granule (Figure
1.2).(16) The a-granule is well-known for harboring numerous grth factors and
cytokines thought to have therapeutic potential for augmenting connective tissue healing,
primarily through the use of platelet-rich plasma constructs.(3, 6) These granules contain
a plethora of bioactive molecules including growth factors, adhesive proteins, coagulation
factors, fibrinolytic factors, proteases and antiproteases, platelet-specific proteins, and
membrane glycoproteins (Table 1.1).(16, 20) In addition to wound healing, many of the
secreted a-granule proteins are thought to play critical roles in coagulation, inflammation,

antimicrobial defense, and angiogenesis.(16)

12

 

Figure 1.2 Transmission election micrograph of a human platelet depicting numerous
granules, including the a-granules indicated by the white arrows. Scale bar = 500 nm.
Adapted from ref. (16)

Table 1.1

Bioactive molecules found within platelet a-granules and their functions.

Adapted from ref. (10)

 

Category

Specific Molecules Biologic Function

 

Growth factors

Adhesive proteins

Clotting factors

_..-....._—_.._..—.-_..-._—.—-.__. ___—- ... “-1... ,—17 -

Fibrinolytic factors

_.-—--

VEGF, ECGF

Cell proliferation,
migration, differentiation,
\ “matrix synthesis __

TGF-B, PDGF, IGF-1&II, FGF, EGF,

Cell contact interactions,

hemostasis, coagulation,
Fibrinogen, fibronectin, vitronectin ECM composition,
chemotaxis, grth
factor binding

Thrombin production &
Factor V, XI, protein S, antithrombin regulation, & eventual
fibrin formation

14...... ...,_H .——.- -..._._—. mfln _.__.._.....__..... e—ww--__._ ”Hm—n .—---._._..

Plasmin production
(fibrinolysis) and
vascular remodeling .. . ..

_..__ _._.. _-..- __ 4‘ _-.. .._.

 

 

Plasminogen, plasminogen activator
inhibitor, a-2 antiplasmin

 

Proteases &
Antiproteases

—‘ w-- 4 .—- --.—-~.;.H~

Anti-microbial
proteins

Basic proteins

Membrane
glycoproteins

gillitrypsivaéPéMT.SJ%_._ ..____..£i.¢ge<ie_t.ion.__

 

....~.. .. . --H—r ._,._ ”—9-“.- wick”. H‘

TIMPT-4, MMP-1,-2,-4,—9, (1:1 Angiogenesis, matrix

Thrombocidins Bactericidal & fungicidal
Platelet factor 4, B-thromboglobulin, Inhibit angiogenesis,
endostatins platelet activation

Platelet aggregation and
adhesion, endocytosis,
inflammation, thrombin
generation, platelet-
leukocyte interaction

CD40 ligand, CD61, p-selectin,
tissue factor

 

Platelet Activation and Degranulation

Resting (inactivated) platelets in circulation are disc-shaped, display a relatively

smooth surface, and do not exhibit filopodia or microspikes typical of leukocytes. In

contrast, platelet activation i.e., response to an in vitro or in vivo agonist(s), leads to a

morphologic transformation into spiny spherical cells with numerous filopodia, in

14

addition to granule content secretion (degranulation), aggregation, and clot retraction.(20)
Shape change occurs in response to most agonists and chilling via mechanisms regulated
by G proteins, actin polymerization, and an increase in cytosolic calcium, and these
mechanisms are reversible in some but not all cases.(20) Platelet degranulation occurs
through a ligand-receptor interaction leading to an actin-myosin-govemed cytoskeleton
contraction. This calcium-mediated contraction culminates in the centralization of the a,
dense, and lysosomal granules that then fuse with the platelet membrane canalicular
system and release their contents into the extracellular environment. Release of granule
contents further propagates platelet aggregation because many granular contents (i.e.,
ADP, thrombospondin, and fibrinogen) are themselves platelet agonists. It is important to
note that platelet agonists are not all of equal potency and the response to different
agonist may vary among species.(l3, 21) However, across all species thrombin and

collagen have consistently demonstrated to be the most potent platelet agonists.(13)

FIBRIN AND CONNECTIVE TISSUE HEALING
As previously mentioned, platelets are involved in the first step in the process of
hemostasis via formation of the so-called unstable platelet plug. This is usually adequate
for the cessation of bleeding. In parallel with this process, the coagulation cascade is
initiated and culminates with thrombin formation whereby the conversion of soluble
fibrinogen to insoluble fibrin fibers ensues. This process stabilizes the primary platelet

plug by forming a fibrin matrix that incorporates platelets via the binding of fibrin to the

(1111,03 (CD61) receptors exposed on activated platelets.(22) The fibrin matrix not only

helps to provide hemostasis, but also is a vital temporary extracellular matrix providing
15

an excellent provisional scaffold for repair cells such as fibroblasts and endothelial cells

to adhere, migrate, proliferate, and synthesize the permanent extracellular matrix.

Coagulation Cascade and Fibrin Formation

The coagulation cascade is a series of several enzymatic reactions made up of two
converging pathways known as the extrinsic and intrinsic pathways (Figure 1.3). The
extrinsic pathway, probably the most important pathway in vivo, is initiated by exposed
tissue factor on the subendothelial surface of blood vessels following injury.(23) Tissue
factor binds to factor VII and leads to the subsequent activation of factors IX and X. The
intrinsic pathway is not essential for coagulation in vivo and is initiated when factor X11
comes in contact with negatively charged substances (e.g., collagen and glass).(24)
Factor X11, which possesses the unique ability to change Shape in response to negatively
charged surfaces, stimulates the activation of factors XI, IX, VIII, and X.(23, 25) Despite
each pathway’s unique trigger, both converge at the activation of factor X, which
subsequently converts prothrombin (factor II) to thrombin (factor Ila). Thrombin is a
multipotent catalyst in the coagulation cascade as it converts soluble fibrinogen to
insoluble fibrin polymers and initiates the entire cascade of events of platelets activation
including shape change, aggregation, and degranulation, among others.(13, 24) The
insoluble fibrin polymers form a network of fibrin fibers that is stabilized via a cross-
linking mechanism due to a thrombin-activated factor XIII.(26) These cross-links not
only provide stability, but also make the clot more resistant to enzymatic degradation.(27)
It is important to note that most of the enzyme complexes of the coagulation cascade,

including thrombin formation, are mediated through a calcium-phospholipid interaction

16

on the surface of activated platelets.(23) Hence, the chelation of calcium, usually via
ethylenediaminetetra-acetic acid (EDTA) or citrate, is essential for keeping blood

incoagulable prior to laboratory coagulation analysis.

INTRINSIC PATHWAY EXT RINSIC PATHWAY

XII —-
Tissue factor
-—’I_7'_E> 1
”_. ._. 1...“...

 

 

 

 

 

 

 

E
E Prothrornbin 1__. A Thrombin
E (II) (Ila)
é Fibrinogen i. Fibrin
5 (I) (la)
0

Fibrin clot

Figure 1.3 Summary of the coagulation cascade and fibrin formation. Adapted from

ref. (2)

Fibrin Functions

During the wound healing process, fibrin plays two major roles in addition to
hemostasis. First, fibrin acts as a provisional scaffold providing a conducive surface for
cell attachment, adhesion, and migration.(28) Fibrin, in conjunction with to the small
adhesive proteins fibronectin and vitronectin, act as bridging molecules and support the

invasion of key repair cells such as macrophages, endothelial cells, and fibroblasts at the

17

wound site in order to initiate tissue repair. Secondly, fibrin has been shown to directly
and indirectly (via fibrinogen, fibronectin, and vitronectin) bind numerous grth factors
including TGF-B, PDGF, bFGF, IGF, and HGF creating a growth factor reservoir within
this scaffold and thus prolonging the biological activity of these factors.(29) F ibrin(ogen)
plays a pivotal role in angiogenesis through the modulation of various pro-angiogenic
factors such as bFGF and VEGF.(30) Further, both bFGF and VEGF have demonstrated
to retain their activity when bound to the fibrin matrix.(3l, 32) Fibrin and fibrinogen also
act in concert by binding PDGF and TGF-B to stimulate fibroblast migration,
proliferation, and matrix synthesis.(33, 34) Therefore, fibrin is a key director of
connective tissue healing via acting and as a temporary scaffold and acting as a source of
numerous grth factors. Moreover, the accumulating knowledge of fibrin’s beneficial
roles in tissue repair and its versatility as a biopolymer have made this molecular a

popular adjunct for connective tissue engineering applications.(28, 35)

GROWTH FACTOR DELIVERY METHODS

Contemporary research on the biology of connective tissue healing has led to the
development of a variety of products designed to enhance connective tissue healing. The
use of autologous products and exogenous products such as recombinant proteins, gene
therapy and biomaterials for grth factor delivery are all rapidly growing in the
orthopaedic and regenerative medicine —related fields and have shown promise in
augmenting connective tissue healing. However, despite the excitement surrounding
these products and their clinical use, it is clear that further study using more rigorous

scientific standards is warranted.(36-3 8)

18

Exogenous Growth Factor Delivery

Although many recombinant grth factors and cytokines are now available for
research purposes and some have also been tested in humans, the clinical experience thus
far has been somewhat disappointing. For example, there has been extensive research
performed on the use of bone morphogenetic proteins (BMPS) as therapeutic tools for the
treatment of various orthopaedic disorders.(39) Despite the numerous BMPS discovered
thus far, only recombinant human BMP-2 and BMP-7 (also Osteogenic protein-l) have
been developed for clinical use. The former has been shown to be effective in fresh
fractures(40) and interbony spinal fusion,(4l) whereas the latter has shown efficacy in the
treatment of non-union fractures.(42) However, the use of these recombinant BMPS has
been associated with complications including heterotropic ossification and significant
swelling.(39, 43) One explanation for the reason that few grth factors have been
approved and commercialized for therapy in humans might lie in the manner in which
these bioactive molecules are delivered: namely, bolus injection into the tissue of interest.
Unfortunately, the rapid degradation and low local availability of growth factors
delivered in a bolus manner likely do not meet the physiologic criteria to augment tissue
repair. Hence, this might explain why several large-scale clinical trials utilizing bolus
delivery methods have yielded unrewarding results.(44-46) Furthermore, it seems that the
use of growth factors should attempt to mimic the natural tissue repair process as much as
possible and thus should not be limited to a single growth factor. It seems that delivering
a combination of key growth factors known to play a role in tissue repair at the proper

physiologic ratio and in the proper spatiotemporal pattern should be the goal.(47)

l9

In the search for better control over growth factor release, several innovative
technologies are currently being explored, and the delivery of growth factors by means of
3D micro- or nanoparticles, injectable gels, composites, polymer scaffolds, or gene
therapy are but a few examples. These so-called biomaterials appear promising for the
controlled release of growth factors and previous studies have incorporated recombinant
growth factors, transfected cells, or plasmid DNA with polymer or hydrogel
scaffolds.(48) Vehicles for growth factor delivery take the physical form of porous
scaffolds, microspheres, and micro- or nano-capsules. The release profile of the desired
bioactive factor can be altered either by manipulating the polymer properties or by
adjusting the physical and chemical properties such as porosity, pore size, degree of
cross-linking, and degradation rate.(48, 49) Consequently, biomaterials can be designed
to produce differential profiles of growth factor release, distinct spatial gradients of
factors, and even the release of grth factors in response to specific cues from the
cellular microenvironment.(50) Although these technologies appear promising for the
controlled release of growth factors, the optimal grth factor or combination of grth
factors, dosage, timing of application, biosafety, and cost-effectiveness appear to be

Significant limitations.(47, 51)

Autologous Growth Factor Delivery: Platelet-Rich Plasma

Platelet-rich plasma (PRP) is an example of an autologous product that has been
used for decades and is currently being studied as a growth factor delivery method for
connective tissue healing. PRP has been used extensively in oral and maxillofacial

surgery to accelerate peri-implant soft tissue and bone healing,(52) and more recently has

20

been investigated in orthopaedics and general surgery.(53-57) PRP is becoming very
popular in Sports Medicine and it is becoming more familiar to the general public,(36) in
large thanks to a recent front page article in The New York Times describing the use of

PRP to treat professional football players prior to the Superbowl.(5 8)

Definition of Platelet-Rich Plasma

Platelet-rich plasma has been defined as plasma that contains a platelet
concentration above the “normal” physiologic levels found in whole blood, and PRP is
considered to be, by definition, autologous.(59) As previously mentioned, normal platelet
counts range from 150,000/uL to 350,000/uL whereas a platelet concentration of at least
1,000,000/uL in PRP has been associated with the enhancement of wound healing.(59)
Lesser concentrations of platelets are thought not to be reliable to enhance wound healing
and greater concentrations have yet to Show further benefit.(59) The increased
concentration of platelets also provides a 3-to 5-fold increase in the aforementioned
platelet-associated grth factors. Throughout the literature several terms have been used
to describe preparations that isolate and concentrate platelets. The term “platelet
concentrate” is frequently used in the literature, but is not the same as PRP because this
implies a solid composition of platelets without plasma, which would therefore not
clot.(59) PRP can be thought of as nothing more than a concentration of platelets and
their associated growth factors delivered in a normal clot. It is important to keep in mind
that the clot also contains several bioactive molecules namely the cell adhesion
molecules: fibronectin, vitronectin, and fibrin itself. As previously mentioned these

molecules play a significant role in would healing, acting in concert both as a provisional

21

scaffold and a growth factor reservoir (due to their ability to bind growth factors). The
term “platelet gel” is also used in the literature and is also incorrect terminology because

the gel does not contain the cell adhesion molecules present in the clot.(59)

Formulation of Platelet-Rich Plasma

The increased use and increasing popularity of PRP has resulted in numerous
proprietary and commercial methods of formulating PRP. However, three main
formulation techniques exist: the gravitational platelet sequestration (GPS) technique
(using centrifugation), standard cell separators, and autologous selective filtration
technology (plateletpheresis).(53) Only the GPS technique will be discussed herein as
this is the method used in the work of this thesis and is considered by many to be
standard.(55, 59) PRP can only be made from anticoagulated blood, as it is impossible to
concentrate platelets in clotted whole blood or serum. The formulation of PRP usually
begins with adding whole blood to a collection tube pre-loaded with citrate.
Anticoagulant citrate dextrose is considered the preferred anticoagulant in order to best
support platelet viability.(59) Citrate reversibly binds to the ionized calcium and therefore
inhibits the coagulation cascade. Next, two centrifugation steps must follow to truly
concentrate the platelets.(59) The first centrifugation step, called the “hard spin” (higher
g-force), separates the red and white blood cells from the plasma containing platelets and
clotting factors. The second spin, called the “soft spin” (lesser g—force) further
concentrates the platelets thus creating a platelet-rich and platelet-poor plasma
component. The PRP must next be clotted in order to activate and degranulate the

platelets.

22

Most PRP formulations use thrombin, a combination of thrombin and calcium, or
collagen to induce clotting. The use of thrombin induces platelets to secrete
approximately 70% of their stored growth factors within 10 minutes and close to 100% is
secreted within the first hour.(59) Thereafter platelets may continue to synthesize small
amounts of grth factors for the remainder of their lifespan (5-10 days) or until they are
depleted.(59) Therefore, PRP should only be clotted when it is ready for use, as clotting
too early may mitigate it beneficial effects. Of note, thrombin—activated PRP clots have
demonstrated significant contraction whereas a recent study Showed that collagen-
activated PRP clots underwent much less contraction and were equally effective in
stimulating growth factor secretion.(60)

The correct formulation of PRP is critical in order to achieve the greatest benefits
from PRP.(61) Platelets are known to be fragile cells. They should be collected with large
gauge needles and handled gently so as not to induce premature platelet activation. Some
investigators have noted that the effectiveness of PRP seemed to vary among batches,(62)
demonstrating the importance of the preparation methods. One investigator has proposed
that studies that fail to show a significant benefit from PRP are often the result of

improper preparation.(6 l)

Rationale for the Use Platelet-Rich Plasma

The main rationale behind the use of PRP arises from the many bioactive
molecules present within the platelet’s a-granules. Seemingly all of the growth factors
present in platelet a-granules including PDGF, TGF-B, bFGF, VEGF, IGF-1, and EGF

play a significant role in connective tissue healing (Table 1.2).(3, 5, 8, 52, 53, 55, 63)
23

Platelet-rich plasma is also appealing because safety concerns such as immune reactions
and infectious disease transmission are theoretically nullified due to its autologous nature.
Furthermore, PRP is relatively cost-effective and easily obtainable, especially compared
to the aforementioned exogenous growth factor delivery methods. Not only does PRP
provide increased concentrations of several growth factors and cytokines, it also provides
these bioactive molecules in the “normal” physiologic proportions.(55, 59) Maintaining
the physiologic proportional relationship between grth factors is thought to be
important for optimal tissue regeneration.(64, 65) The growth factors that are used for
exogenous delivery such as BMPS are produced by recombinant technology and are often
delivered as a single factor in high or unpredictable doses using carrier vehicles. Due to
the complexity of connective tissue healing, it is unlikely that a single growth factor

delivery system will provide optimal benefits for connective tissue repair.

 

Table 1.2
Growth factors identified within PRP and their role in tissue repair.
Adapted from ref.(55)
Growth Factor Target Cell/T issue Function

 

Endothelial cells, fibroblasts, Cell proliferation, recrurtment,

PD-EGF differentiation, wound closure,
and many other cells types . .
cytokme secretion
Fibroblasts, smooth muscle 2:222:21 tplgorrlltjzraéfeg,is
PDGF A+B cells, chondrocytes, osteoblasts, h f ’ g g . . ’ .
mesenchymal stem cells growt . actor secretion. matrix
formation wrth BMPS
Blood vessel tissue, fibroblasts, Potent collagen synthesis,
TGF-Bl . . . .
monocytes chemotaxis, antrprolrferatrve
. Cell proliferation,
IGF—1,11 Bone, blOOd vessel, Skm’ differentiation, recruitment,
fibroblasts .
collagen synthesrs
VEGF, ECGF Endothelial cells EndOIhCI‘i" cc" PrOI'ferft'on’
chemotaxrs, angrogenesrs
bFGF Endothelial cells, smooth Cell proliferation, chemotaxis,

muscle, skin, fibroblasts, others

angiogenesis

 

24

Numerous studies have demonstrated positive effects regarding the use of PRP to
enhance the healing of connective tissues including bone,(66-68) cartilage,(69-72)
tendon,(62, 73-80) and ligament.(80-84) Particularly well-documented is the positive
effect of PRP on tendon and ligament cell proliferation, gene expression, and matrix
synthesis. Several in vitro studies of human tenocytes cultured in the presence of PRP
have documented that PRP increases cell proliferation, VEGF expression, and total
collagen production.(73, 75, 76) Equine superficial digital flexor tendon and suspensory
ligament explants cultured in PRP have Shown enhanced gene expression of type I
collagen, type III collagen, cartilage oligomeric matrix protein and a decrease in the gene
expression of the catabolic matrix metalloproteinase (MMP) -3 and MMP-l3.(79, 80) In
vivo studies have also demonstrated positive results for tendon and ligament healing.
Lyras et al. have Shown evidence that PRP enhances and accelerates tendon healing via
increased neovascularization in the early phases of tendon healing in rabbits.(85, 86)
There is also evidence that PRP is able enhance the biomechanical properties of injured
tendons in animal models, as increased tendon stiffness and strength following the use of
PRP has been documented.(62, 87, 88) Furthermore, a recent study demonstrated that an
injection of PRP into the patellar tendon injury site resulted in increased recruitment of
circulation-derived cells to the injury site, with a concomitant increase in type I and type
III collagen production.(78) Murray and colleagues have demonstrated that PRP in
concert with a collagen scaffold has shown Significant promise in enhancing anterior

cruciate ligament healing in a variety of large animal models.(81-84, 89, 90)

25

Clinical Applications of Platelet-Rich Plasma

To date, there is abundant evidence documenting the safety and efficacy of PRP
in a wide variety of medical fields. Numerous animal studies, cases reports, and small
case series in oral and maxillofacial surgery,(9l-93) plastic surgery,(94-97)
cardiovascular surgery,(98, 99) and general surgery(100, 101) have examined the role of
PRP in various clinical settings. In general these Studies support the use of various PRP
preparations in the clinical setting, suggesting an overall improvement in soft tissue
healing. However, within the oral and maxillofacial surgery literature there are other
small case reports that argue there is little benefit from using PRP to promote healing or
osteogenesis.(102-104) In general, many of the human clinical studies claim excellent
outcomes but are, at best, limited case series. Consequently, it is difficult to draw
conclusions from these case reports that may or may not have controls, have small
sample sizes, and do not define a standardized preparation of PRP; thus, it is hard to
interpret and compare any of the results.(61) The nature of PRP may vary significantly
from study to study and is somewhat “operator dependent”. Therefore, one must pay
careful attention to the specific method of PRP formulation when interpreting results.

Numerous applications for PRP in clinical orthopaedic medicine have been
proposed including chronic (over-use) tendinopathy,(105-lll) bone healing,(112-116)
osteoarthritis,(117, 118) acute tendon/ligament repair,(119-121) muscle injury,(122) joint
arthroplasty,(123, 124) rotator cuff repair,(125-128) and articular cartilage repair.(7l, 72,
129) However, strong clinical evidence in support the use of PRP for these applications is

currently lacking in peer-reviewed journals. To date, the majority of published clinical

26

orthopaedic studies have investigated the use of PRP for tendon and bone healing. A
review of the clinical studies examining the role of PRP in tendon healing is discussed
next

Elbow T endinosis / Lateral Epicondylitis. Lateral epicondylitis (more commonly
referred to as “tennis elbow”) is a common problem in individuals whose activities
require strong gripping or repetitive wrist movements. It is often a chronic recurring
problem that is refractory to current treatment regimens. Mishra and Pavelko were the
first to publish a cohort study on the use of PRP for chronic severe elbow
tendinosis.(108) Fifteen patients were given a PRP injection as an alternative to surgery
and 5 patients served as controls. Individuals that underwent PRP treatment reported a
60% improvement in pain scores compared with 16% reduction in the controls at 8
weeks. However, 3 of the 5 controls dropped out of the study by 8 weeks to seek
alternative treatment, which limited comparisons. At 6 months pain scores were reduced
81% in the PRP-group. At the final follow-up of 2 years PRP-treated patients reported
93% improvement in pain, and 94% return to sport and work. The authors attributed the
significant reduction in pain to PRP and stated that this treatment should be considered
prior to surgery. No adverse effects or complications were reported.

Recently, Peerbooms et al.(109) sought to determine the effectiveness of a single
PRP injection compared to a corticosteroid injection for chronic lateral epicondylitis in a
prospective randomized double-blind clinical trial. One hundred patients that previously
failed nonoperative treatment (at least 6 months prior) met their inclusion criteria. Their
results showed that 73% (37 of 51 patients) of the PRP-treated patients were considered

to be successfully treated (defined as more than 25% reduction in pain and function

27

scores without reintervention after 1 year) compared to 51% (25 of 49 patients) in the
corticosteroid group. The authors mentioned that the PRP—treated patients progressively
improved with time whereas the corticosteroid group improved initially and then
declined.

Achilles T endinopathy. Often refractory to treatment and rehabilitation, chronic
Achilles tendinopathy is a degenerative disorder thought to occur as a result of over-use
of the tendon and is common in athletes and active individuals. Recently, a placebo-
controlled, double-blinded, randomized controlled trial was conducted to determine if a
PRP injection could improve pain and functional outcomes in individuals with chronic
Achilles tendinopathy while also undergoing an eccentric exercise program (usual
care).(105) Fifty-four individuals met the inclusion criteria and were randomly enrolled
into the PRP and control groups (n = 27 per group). Although an improvement in pain
and function scores was noted in the PRP group, this was not significantly different from
the control group of eccentric exercises alone. The authors concluded that they do not
recommend the use of PRP for chronic Achilles tendinopathy due to the lack of
significant improvement in pain and activity witnessed in their study.

Acute Achilles Tendon Repair. In a case—control study, Sanchez et al.(1 19)
evaluated the intraoperative effect of PRP in athletes that underwent Achilles tendon
repair. The authors used both the PRP clot and supernatant (termed “preparation rich in
growth factors”) to augment open suture repair in 6 patients, which was retrospectively
compared to 6 age-matched controls who underwent the conventional surgical procedure.

Follow-up included physical examination and ultrasonic imaging at regular intervals up

28

to a year. The treated group had earlier range of motion, showed no wound
complications, and took less time to return to gentle running.

Rotator Cufl Repair. Platelet-rich plasma appears to be an attractive adjunct to
many shoulder surgeons, as its potential use for rotator cuff repair has been recently
reviewed.(l27) To date, there are no published clinical trials on the use of PRP for rotator
cuff repair. However, the intraoperative use of PRP to augment rotator cuff repair has
been gaining in popularity among shoulder surgeons.(55) A pilot study by Randelli et
al.(128) is the only published clinical data on the use of PRP for rotator cuff repair, albeit
without a control group. After arthroscopic repair of the torn rotator cuff, 14 patients
received intra-operative autologous PRP combined with an autologous thrombin
component. These patients were followed for 24 months and demonstrated a significant
reduction in pain scores and a significant increase in function scores at 6, 12, and 24-
month follow-ups compared to pre-operative scores. The authors mentioned that there
were no adverse events related to the PRP augmentation procedure and concluded that
the procedure was safe and effective.

Patellar T endinosis/T endinopathy. Chronic patellar tendinopathy, more
commonly referred to as “jumper’s knee”, is a common problem of athletes who undergo
repetitive jumping motions and is characterized by a localized tenderness of the patellar
tendon at its origin on the inferior pole of the patella. To date, two small preliminary
reports have evaluated the use of PRP for jumper’s knee. A recent pilot study examined
the use of PRP in 20 athletes with chronic patellar tendinosis that were prospectively
evaluated at a 6 month follow-up.(107) No adverse effects were observed and significant

improvements were noted in functional scores compared to the pre-treatment period.

29

Filardo et al.(106) conducted a second study on the use of PRP for jumper’s knee
incorporated a control group that underwent conventional therapy alone. Fifteen patients
were treated with multiple PRP injections at 3 instances, 2 weeks apart and 16 patients
were enrolled in the control group. The PRP injections caused significant improvements

in pain and functionality scores and in sport activity level by 6 months.

Platelet-Rich Plasma in Veterinary Medicine.

The use of PRP in veterinary medicine is primarily limited to equine medicine.
Experimental studies have examined the use of PRP for horse skin wounds(l30, 131) and
orthopaedic conditions.(79, 80, 88, 132, 133) Currently, there is one published clinical
trial on the use of PRP for augmentation of suspensory ligament desmitis in horses,(110)
despite its wide-spread use in equine sports medicine. Severe suspensory ligament
desmitis is often a career-ending injury. Waselau et al. examined the effect of a Single
application of PRP in concert with a gradual exercise program (conventional therapy) in
9 Standardbred racehorses with moderate to severe suspensory ligament desmitis and
compared them to the performance of healthy racehorses that raced during the same time
period. They found that all of the horses in the PRP group not only returned to racing but
also had comparable race records, in terms of number of starts, total earnings, and
earnings per star, to healthy horses 3 years post-treatment. Thus, it appears PRP
significantly contributed to the excellent prognosis for returning to racing in these equine

athletes.

30

Limitations of Platelet-Rich Plasma

There are potential limitations regarding the use of PRP as a growth factor
delivery method for connective tissue healing. One potential limitation may be the
injection of liquid unclotted PRP, a commonly used clinical method of delivering PRP to
desired tissues. Using this method there may be significant “washout” or rapid diffusion
of PRP into the surrounding tissue. Therefore, this washout effect may make it difficult to
determine how much PRP was actually delivered to the desired region; thus complicating
the extrapolation of doses or concentrations of PRP from one study or clinical situation to
the next. The injection method may also account for a lack of clinical benefit as
mentioned in a large-scale clinical trial that used a PRP injection for Achilles
tendinopathy.(105)

A second potential limitation of PRP may be a lack of a standardization protocol
in the preparation of PRP, which (again) makes it problematic to extrapolate data from 1
study to the next. This lack in standardization protocol to produce and evaluate PRP in
the literature may help to explain inconsistent and clinical and experimental results. In
addition to a uniform PRP protocol, it has been proposed that future studies should be
required to quantify platelet yields in both whole blood and the PRP preparation used,

and use commercial assays to quantify growth factor concentration.(l34)

Autologous Growth Factor Delivery: Platelet-Rich Fibrin Matrix
The platelet-rich fibrin matrix (PRFM) represents a new alternative autologous
grth factor delivery method for connective tissue healing. Due to the relatively short

half-life and rapid degradation of most growth factors,(47) the local administration of

31

PRP alone may not be able to provide a prolonged release of grth factors that may be
particularly desired in biologically compromised connective tissues.(l35) The PRFM
system was therefore designed to prolong the release of grth factors for connective

tissue healing.

Formulation of Platelet-Rich Fibrin Matrices

The PRFM essentially combines the benefits of PRP and a dense fibrin matrix and
is created as follows (Figure 1.4). Whole citrated blood is collected and the blood cells
are separated from the platelets and plasma proteins, during an initial low speed
centrifugation step. Next, the supernatant (platelets and plasma proteins in plasma) is
added to a second tube containing calcium chloride. This tube is then centrifuged at a
higher speed. During the second centrifugation step fibrin polymerization ensues due to
the addition of calcium chloride. Calcium chloride causes the conversion of autogenous
prothrombin to thrombin thus initiating the fibrin polymerization process. This final
centrifugation step permits the concomitant concentration of platelets and polymerization
of fibrin resulting in a dense, pliable platelet-rich fibrin matrix that is suspended in a
liquid serum component. The shape of the PRFM can be tailored into a flat circular
membrane (termed, platelet-rich fibrin membrane [PRFMembrane]) or into a cylindrical

construct (termed, PRFMatrix) depending on the shape of the tube/vial (Figure 1.4).

32

PRFMatrix PRFMembrane

Centrifuge 18 mL
G of citrated blood at

1100 gfor 6 min.
-

Centrifuge 9 mL of
(D citrated blood at
1100 gfor 6 min.
- . -
I platelet-nah
plasma \

‘

platelet-rich
plasma \

        
  

Transfer plasma +
platelets to a bottle pre—
loaded with CaCI2 to
initiate fibrin clotting.

Transfer plasma +
platelets to a tube pre-,
loaded with Ca012 to
initiate fibrin clotting.

      

I
i + CBC/2
Centrifuge at 1450 g Centrifuge at 4500 g
for 15 min. for 25 min.
serum
—

   

Figure 1.4 Schematic of the creation of a platelet-rich fibrin matrix (PRFMatrix) and
platelet-rich fibrin membrane (PRFMembrane).
33

A Platelet-Rich Fibrin Matrix Is Not Platelet-Rich Plasma

The PRFM is different from PRP in several ways. Most methods used to create
PRP currently use calcium chloride and excess human or bovine thrombin. However, the
use of excess thrombin may lead to premature platelet activation and degranulation,
causing the immediate release of platelet-derived growth factors.(l26) The PRFM system
(Cascade Medical Enterprises, Wayne, NJ) employs a different strategy, where both
platelets and fibrin are incorporated into a dense matrix without the use of exogenous
thrombin. It is thought that the lack of excess thrombin in the PRFM system prevents
premature platelet degranulation and subsequently intact growth factor-laden platelets
become entrapped within the fibrin matrix allowing for a natural, gradual release of

growth factors due to autologous activators present at the wound site.(126, 135)

Rationale for the Use of Platelet Rich Fibrin Matrices

The main rationale for the use of a PRFM is that it may prolong the release of
growth factors and provide a growth factor reservoir in concert with a dense provisional
fibrin scaffold that is potentially able to facilitate and enhance tissue repair. The PRF M
may provide for a more natural mechanism to delay growth factor release. The prolonged
release of grth factors may be particularly desired in biologically compromised(l35)
or chronically injured tissues, such as in rotator cuff tendon injury.(125-127, 136)
Currently, there are few published studies in the peer-reviewed literature on the use of a
PRFM for connective tissue healing. However, thus far, it has shown promise in assisting

in the closure of chronic lower-extremity ulcers in small human pilot study.(135) An

34

experimental study in dogs found that the PRFM resulted in faster healing of tooth
extraction sites.(l37) Sarrafian et al. examined the use of a PRFM with a collagen
scaffold for repair of acute Achilles tendon rupture in a sheep model.(138) The PRFM
group showed complete bridging of the surgically-created gap and their findings
supported the use of a PRFM to augment Achilles tendon repair. Additionally, a case
report describing the use of 3 PRP M to augment rotator cuff repair in a 53 year-old man
was recently published where clinical examination at 6 months post-operation Showed
good pain relief and a marked increased in range of motion.(136) Interestingly, several
recent reviews on the biologic augmentation of rotator cuff tendon repair have noted that
a prospective randomized patient-blinded clinical trial is underway investigating the

efficacy of the PRFM in augmentation of arthroscopic rotator cuff repair.(125-127)

PURPOSE AND HYPOTHESES

Based upon the above review of the literature, the general purpose of this thesis
was to determine if a PRF M is able to enhance growth factor delivery and, consequently,
augment connective tissue healing. The general hypothesis of this thesis was that a PRF M
will enhance growth factor delivery and thus enhance connective tissue healing. This
general hypothesis was evaluated in three separate but related studies. The specific
purposes of each study were as follows.

Study 1 (Chapter 2): To compare the kinetics and mitogenic activity of growth

factors eluted from a PRFMatrix, a PRFMembrane and a whole blood clot (BC)

over time.

35

Study 2 (Chapter 3): To examine the effect of a PRF Membrane in enhancing and
accelerating tendon healing in a canine central-third patellar tendon defect model.
Study 3 (Chapter 4): 1) To create a bioactive scaffold using PRFM-related
technology, termed growth factor-rich plasma (GFRP), for tissue engineering
applications. 2) To evaluate the ability of GFRP-enriched scaffolds to induce

tendon cell proliferation and matrix synthesis.

The specific hypotheses and specific aims of each study were as follows.

Study 1 (Chapter 2): In study 1 our hypothesis was two-fold. H1: A PRFMatrix
and PRFMembrane will elute a significantly increased concentration of a sentinel
growth factor (TGF 431) when compared to a BC of similar volume, and H2: The
growth factors eluted from a PRFMatrix and PRFMembrane will Significantly
increase fibroblast proliferation in vitro over time when compared to the eluent
from a whole blood clot of similar volume. Our specific aims were to quantify the
amount of TGF-Bl eluted from a BC, PRF Matrix, and PRFMembrane over time
in vitro. We also sought to compare the mitogenic activity of each construct over
time in vitro.

Study 2 (Chapter 3): In study 2 we hypothesized that the PRFMembrane would
enhance the rate and quality of tendon healing in the PT defect when compared to
empty contralateral defects (controls). Our Specific aim was histoligcally compare
the rate and quality of tendon healing in PRFMembrane-augmented tendons and

controls using an in viva canine central-third patellar tendon defect model.

36

Study 3 (Chapter 4): In study 3 it was our hypothesis that GFRP-enriched
scaffolds would significantly enhance cell proliferation and matrix synthesis over
time when compared to serum-enriched scaffolds and scaffolds alone. Our
Specific aims were to combine a synthetic scaffold with GFRP, an autologous
concentration of growth factors derived from a PRP preparation, and to compare
cell proliferation and matrix synthesis in vitro in GFRP-enriched scaffolds,

serum-enriched scaffolds, and scaffolds alone (control).

37

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45

106. F ilardo G, Kon E, Della Villa S, Vincentelli F, Fomasari PM, Marcacci M. Use of
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46

118. Sanchez M, Anitua E, Azofra J, Aguirre JJ, Andia I. Intra-articular injection of an
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131. Monteiro SO, Lepage OM, Theoret CL. Effects of platelet-rich plasma on the
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132. Smith JJ, Ross MW, Smith RK. Anabolic effects of acellular bone marrow,
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134. Grageda E. Platelet-rich plasma and bone graft materials: a review and a
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135. O'Connell SM, Impeduglia T, Hessler K, Wang XJ, Carroll RJ, Dardik H.
Autologous platelet-rich fibrin matrix as cell therapy in the healing of chronic lower-
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136. Maniscalco P, Gambera D, Lunati A, Vox G, Fossombroni V, Beretta R, et al.
The "Cascade" membrane: a new PRP device for tendon ruptures. Description and case
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137. Simon BI, Zatcoff AL, Kong JJ, O'Connell SM. Clinical and Histological
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138. Sarrafian TL, Wang H, Hackett ES, Yao JQ, Shih MS, Ramsay HL, et al.
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48

CHAPTER 2.

Platelet-Rich Fibrin Constructs Elute Higher Concentrations of TGF-Bl and
Increase Tendon Cell Proliferation Over Time When Compared to Blood Clots: A
Comparative In Vitro Analysis

Lance C. Visser, 88‘, Steven P. Amoczky, DVMl, Oscar Caballero, MS],
and Monika Egerbacher, DVM, PhD2

1Laboratory for Comparative Orthopaedic Research
College of Veterinary Medicine
Michigan State University
East Lansing, MI

2Department of Pathobiology
Institute of Anatomy and Histology
University of Veterinary Medicine
Vienna, Austria

49

Abstract

The purpose of this study was to compare the concentration of a sentinel growth
factor (TGF-Bl) eluted from a platelet rich-fibrin matrix (PRFMatrix), a platelet-rich
fibrin membrane (PRFMembrane), and a whole blood clot (BC) over time, and to
compare the mitogenic effect of the eluents from each construct. PRFMatriceS,
PRFMembranes, and whole blood clots of similar volumes were created from each of 4
adult dogs. Each construct was placed in individual tissue culture wells containing media
for 7 days. The media was collected and replenished with fresh media on days 1, 3, 5, and
7. The concentration of TGF-Bl eluted into the media from each construct at each time
point was measured with an ELISA. Additional aliquots of the conditioned media were
added to individual cultures of canine tendon cells and the amount of cell proliferation
compared. At days 1 and 3, the conditioned media from both PRF M constructs contained
Significantly more (p 5 0.026) TGF 431 when compared to the media from the BC. The
PRFMembrane conditioned media contained significantly more (p S 0.05) TGF-Bl than
the PRFMatrix media at similar time points. Conditioned media from both the PRFMatrix
and PRFMembrane produced a significant increase (p 5 0.044) in cell proliferation
compared to the BC at all time points examined except the PRFMatrix at day 7. The
conditioned media from the PRF Membrane produced a significant increase (p 5 0.002) in
cell proliferation over both BC and PRFMatrix at all time points. These results
demonstrate that both PRFM constructs are comprised of a dense fibrin scaffold that
contains increased concentrations of TGF-Bl and are capable of increasing tendon cell

proliferation over time when compared to a blood clot of similar volume. The sustained

50

increase in growth factor availability in PRFM constructs may be beneficial in the

healing of biologically compromised tissues.

51

Introduction

The ability to enhance the repair of connective tissues through the clinical
application of bioactive factors represents an attractive adjunct to the orthopaedic
surgeon.(l, 2) This is especially true in cases where the tissues in question may be
biologically compromised due to the potentially chronic nature of the pathology, such as
in rotator cuff injuries.(3) Recently, the use of platelet-rich plasma (PRP) has been
proposed as a potential method of delivering locally increased concentrations of a variety
of bioactive autologous growth factors in an effort to optimize connective tissue
healing.(2, 4-7)

Platelet-rich plasma (PRP) has been defined as plasma that contains a platelet
concentration above the “normal” physiologic level found in whole blood.(8) The
increased concentration of platelets also yields an increase in the concentration of grth
factors that are stored in the or-granules of platelets.(8) These factors, such as platelet-
derived growth factor (PDGF), transforming growth factor-beta (TGF-B), and vascular
endothelial grth factor (VEGF) have all been shown to be essential in the healing
cascade of connective tissues.(9) Because numerous in vitro studies have shown a direct
dose-response influence of many grth factors on cell migration, proliferation and
matrix synthesis,(10-13) it has been proposed that the local administration of increased
concentrations of these grth factors through the use of PRP could optimize the local
healing environment and thus enhance the ability of biologically compromised tissues to
generate a repair response.(l4) However, due to the relatively short half-life and rapid

degradation of most growth factors,(15) the local administration of PRP alone may not be

52

able to provide a prolonged release of chemotactic and mitogenic factors that may be
particularly desired in biologically compromised tissues.(16)

During the initial phase of wound healing, platelets interact with the fibrin clot to
not only provide a hemostatic plug, but also create a provisional fibrin scaffold that
supports and stimulates cell migration and proliferation.(l7) The fibrin scaffold also
serves as a reservoir for cytokines,(18) which are bound within the fibrin scaffold and are
released over time.(17) Therefore, the creation of a dense, platelet-rich, fibrin construct
may provide a reservoir of growth factors that permits the sustained elution of increased
concentrations of growth factors over time.(16)

The purpose of this study was to compare the concentration of a sentinel growth
factor (TGF-Bl) eluted from a platelet-rich fibrin matrix (PRFMatrix), a platelet-rich
fibrin membrane (PRF Membrane), and a whole blood clot (BC) over time. In addition,

the mitogenic effect of the eluents from these constructs was also compared over time.

We hypothesized that, H1: A PRFMatrix and PRFMembrane will elute a significantly

increased concentration of TGF-Bl when compared to a BC of Similar volume, and H2:

The growth factors eluted from a PRFMatrix and PRFMembrane will significantly
increase fibroblast proliferation in vitro over time when compared to the eluent from a

whole blood clot of similar volume.

Materials and Methods
Preparation of Blood-Derived Products
An overview of the processing techniques of each construct is shown in Figure

2.1. A PRFMatrix, PRFMembrane, and BC were created from each of four adult beagle

53

dogs. Each construct was created from whole blood collected via jugular venipuncture
with a 20 gauge butterfly catheter. The PRFMatrix and PRFMembrane were created
according to the manufacturer’s instructions (Cascade Medical Enterprises, Wayne, NJ)
using a double centrifugation process, where, during the second centrifugation step, fibrin
polymerization ensues (due to the addition of calcium chloride) while the platelets are
concentrated. The PRFMatrix was prepared from 9 mL of whole blood in a
collection/separation tube containing a buffered tri-sodium citrate.(19) Following
centrifugation for 6 minutes at 1,100 g, the blood was separated into a red and white
blood cell component and a platelet-rich plasma (PRP) component as a result of the
proprietary separation material. Next, the supernatant (PRP) was transferred to a new
cylindrical tube containing calcium chloride, which, after a second centrifugation step (15
min. at 1450 g), resulted in a cylindrical-shaped PRFMatrix of approximately 2 mL in
volume suspended in serum. The PRFMembrane was prepared from 18 mL of whole
blood that was collected into two 9 mL tubes each containing trisodium citrate and the
proprietary separator gel.(16) Both tubes were centrifuged at 1,100 g for 6 minutes
creating the PRP supernatant, which was transferred to a 35 mm-diameter flat-bottom
glass vial containing calcium chloride. The bottle was then centrifuged at 4,500 g for 25
minutes, which resulted in a flat, circular membrane (due to the radial centrifugation)
approximately 2 mL in volume that was suspended in serum. Blood clots were created by
allowing 4 mL of whole blood to clot in a cylindrical vacuum tube for 2 hours at room
temperature creating a cylindrical-shaped clot the same volume (2 mL) as the PRFMatrix

and PRFMembrane.

54

Blood Clot , PRFMatrix PRFMembrane

l Centrifuge 9 mL of 3 Centrifuge 18 mL
’ CD of citrated blood at
1100 gfor 6 min.

citrated blood at
1100 gfor 6 min.

1
!
l
l

    

Allow 4 mL or whole platelet-rich 1
blood to dot for at , plasma \ 1
least 2 hours at room . .
temp. ‘ ‘ E

 

Transfer plasma + Transfer plasma +

 

platelets to a tube pre- j platelets to a bottle pre-
é loaded with C3012 to I loaded with CaCI2 to
3 initiate fibrin clotting. l initiate fibrin clotting.

I31

I l I (Q Q
gl + CaCl2 , + CaCI2
I

i <9 Centrifuge at 1450 g I Centrifuge at 4500 g

E ' for 15 min. I for 25 min.
BC

  

 

62) Extract the solid clot.

serum

.
I
I
I 6
. “.7 a
. o,»

 

PRFMembrane

   

   

Figure 2.1 Schematic flow chart illustrating the processing steps for the platelet-rich
fibrin matrix (PRFMatrix), platelet-rich fibrin membrane (PRFMembrane) and whole

blood clot (BC).

55

Study Design

Immediately following its creation, each construct was poured out of its
respective container into a Petri dish and transferred to a 12-well tissue culture plate
where it was submerged in 2 mL of serum-free Dulbecco’s modified Eagle medium
(DMEM) containing 1% antibiotic-antimycotic solution and incubated at 37°C in a
humidified atmosphere containing 10% C02. The eluent (media containing the eluted
factors) from each construct was collected in its entirety and replenished with fresh media
after 24 hours of incubation and then every 48 hours thereafter. The eluent from each
construct at days I, 3, 5, and 7 was saved and stored at -80°C until either assayed for

growth factor concentration or used for cell culture.

Eluent Growth Factor Quantification

TGF-Bl was used as a sentinel growth factor to compare the amount of eluted
growth factor from each construct. The concentration of TGF-Bl was determined in
triplicate aliquots of the collected eluent from each construct (n = 4/construct/time point)
with a commercial sandwich enzyme-linked immunosorbent assay (ELISA) kit
(MBIOOB, R&D Systems, Minneapolis, MN). Samples were processed according to the

manufacturer’s instructions and sample dilutions were determined empirically.

Isolation of Tendon Cells
Canine patellar tendon fibroblasts were harvested via primary explant cultures

from adult mongrel dogs euthanized for reasons unrelated to this study. Cells were

expanded to passage two in 75 cm2 tissue culture flasks in 10% fetal bovine serum (FBS)

56

 

media (Dulbecco’s modified Eagle medium (DMEM) containing 1% antibiotic-

antimycotic solution, 0.02 mg/mL gentamicin, and 0.15 mg/mL ascorbate) incubated at

37°C in a humidified atmosphere containing 10% C02 (tissue culture conditions). Cells

in each flask were cultured until sub-confluent and then detached by trypsinization for

seeding. Only cells from the third passage were used for this study.

Cell Culture Experiment

Canine patellar tendon cells (2.5 x 104) were seeded into wells of a 24-well tissue

culture plate and maintained in 10% F BS media. After 24 hours under tissue culture
conditions, media in each well was replaced with 2% FBS media, with (treatment) or
without (control) eluent 50% (vol/vol) from each construct collected at each time point.
After 48 hours under tissue culture conditions, representative live-cell images of each
group were obtained and cell proliferation and viability was determined with the MTT (3-

[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay.

MTT Cell Proliferation Assay

Media from each well (n = 4/construct/time point) was replaced with DMEM
without phenol red containing 0.5 mg/mL MTT (Sigma-Aldrich, St Louis, MO) and 2%
FBS. Cells were then incubated at tissue culture conditions for 4 hours. Next, the MTT
solution was discarded and acidic isopropanol containing 1% Triton-X 100 was added to
each well. After 40 minutes of mixing on an orbital shaker, all the purple forrnazan
crystals were dissolved. The colorless isopropanol solution changed to varying degrees of

a purple color and the degree of color change is directly proportional to the degree of cell

57

proliferation and viability. Aliquots from each well were transferred to a 96-well plate in
triplicate and then read immediately at 570 nm in a scanning multiwell spectrophotometer

(Bio-Tek Instraments, Winooski, VT).

Histological Evaluation

Additional blood clots, PRFMatrices, and PRFMmembranes were created and
fixed in 10% buffered formalin. Histological sections were cut and stained for fibrin
using phosphotungstic acid-hematoxylin (PTAH) or processed for immunohistochemical
staining for TGF-Bl. Detection of TGF-Bl was performed using the anti-human
polyclonal TGF-Bl antibody (Lifespan Biosciences, Seattle, WA, dilution 1:50) after
heating in TRIS EDTA buffer, pH 9.0 three times for 5 min each for antigen retrieval.
The primary antibody was incubated overnight at 4°C. Subsequently, the PowerVision
poly HRP anti rabbit IgG (ImmunoLogic, Duiven, The Netherlands) was applied as
secondary system followed by detection with Diaminobenzidine (DAB, brown staining).

Sections were counterstained with hematoxylin.

Statistical Analysis

TGF-Bl concentration and MTT optical density values were analyzed with a one-
way ANOVA within each time point followed by a Tukey’s post hoc test. All data are
displayed as mean i: standard error (SE). The threshold of statistical significance was set

to p S 0.05 for all comparisons.

58

 

Results

Growth Factor Concentration

The day 1 and 3 eluent from both the PRFMatrix and PRFMembrane contained a
significantly greater (p 5 0.026) concentration of TGF-Bl compared to the day 1 and 3
BC eluent. The day l and 3 PRFMembrane eluent contained a significantly greater (p S
0.05) concentration of TGF-Bl than the PRF Matrix (Figure 2.2). There were no
significant differences in the concentration of TGF-Bl between the eluents from the
PRFMatrix, PRF Membrane, and whole blood clots at either 5 or 7 days.

18 250.0001
“M31 Ll BC Eluent

u PRFMatrix Eluent
I PRFMembrane Eluent

50.0001
p=0.050

p=0.001

TGF-B1 (nglml)

      

D3 D5 D7

Time Point Eluent Collected
Figure 2.2 Mean concentrations of TGF-BI : SE (n = 4) eluted from a blood clot (BC),
platelet-rich fibrin matrix (PRFMatrix), and platelet-rich fibrin membrane
(PRFMembrane). Statistical significance was not reached (p > 0.05) where p—values for
comparisons are not shown.

59

Cell Proliferation

Cells that were subjected to each construct’s day l eluent for 48 hours under
tissue culture conditions demonstrated obvious differences in cell density (Figure 2.3).
Cell density was greatest in the PRFMembrane eluent group followed by the PRFMatrix
eluent. The cell density of the BC eluent appeared similar to controls. All cells exhibited

a similar morphology typical of tendon cells (spindle-shaped fibroblast-like appearance).

 

l. . I . r ’ I A \, -. ’h‘
-L' /-‘ _ Z .'- _ {1' ' _ 1 ., L. I '. , h ‘
: PRFMatrix EIUM '- PRFMembrane Eluent 1
Figure 2.3 Photomicroscopic live-cell images of representative tendon cell proliferation

results after 48 hours of exposure to day 1 eluents from the respective constructs (Phase
contrast, 100x original magnification).

The results of the MTT assay performed on the tendon cells subjected to the
eluent from each construct at each time point are shown in Figure 2.4. Mean optical
density values, indicating relative cell proliferation and viability, from the eluents of both
PRFM constructs were significantly greater (p 5 0.044) than the BC eluent at each time

point except at day 7. The PRFMembrane eluent consistently induced a significant

60

increase (at least 2-fold) in cell proliferation compared to the PRFMatrix (p 5 0.002) and

BC (p 5 0.0001) at each time point the eluent was collected.

    
 

  
 

8
p<0.0001 u BC Eluent

7 p<0.0001 .
:- “"" " u PRFMatrix Eluent
O
E 6 I PRFMembrane Eluent
U
.5 5 0.0001
'6 p<0.0001
C _
a 4
a; p<0.0001 p<0.0001
r: = p<0.0001 p=0.002
8 ”—0332 p=0.023
E 2 7 JM
.2
‘5.
O 1

0

   

D1 D3
Time Point Eluent Collected
Figure 2.4 Mean optical density values displayed as n-fold difference over control i SE
(n = 4) of tendon cells subjected to eluent from the blood clot (BC), platelet—rich fibrin
matrix (PRFMatrix), or platelet-rich fibrin membrane (PRFMembrane) from the various

time periods. Statistical significance was not reached (p > 0.05) where p—values for
comparisons are not shown.

Histological Evaluation

Histological evaluation of the relative fibrin content revealed a marked increase in
the density of fibrin in both the PRFMatrix and PRP Membrane constructs when
compared to the blood clot (Figure 2.5A, B, and C). Clusters of platelets appeared
trapped among the interconnecting strands of fibrin in the PRF Matrix (Figure 2.58). The
fibrin content of the PRFMembrane appeared subjectively increased and more compact
than that of the PRFMatrix (Figure 2.5C). This increase in fibrin density made it difficult

to delineate the presence of platelets within the PRFMembrane using PTAH staining.

61

 

Figure 2. 5 Photomicroscopic images of a representative blood clot (A), platelet- rich-
fibrin matrix (B), and platelet- -rich fibrin membrane (C) stained for fibrin with a
phosphotungstic acid hematoxylin (PTAH) stain. Fibrin strands are not visible among the
red blood cells in the whole blood clot (A). “Nests” of platelets (arrows) can be seen
among the dense fibrin network of the platelet-rich fibrin matrix (B). The dense
compaction of the fibrin in the platelet-rich fibrin membrane is obvious (C). Scale bar =
50 um and applies to each image.

Immunohistochemical staining for TGF-BI revealed positive staining of the fibrin
network in both the PRFMatrix and PRFMembrane (Figure 2.6A and B). Localized areas
of increased staining intensity in both the PRFMatrix and PRFMembrane were associated

with “nests” of platelets trapped within the fibrin network.

 

Figure 2. 6 Photomicroscopic images of a representative platelet- rich fibrin matrix (A),
and platelet-rich fibrin membrane (B) immuno- -stained for TGF- [31. In both constructs
there is positive staining of the fibrin strands and localized areas of increased staining
intensity which likely represents “nests” of platelets trapped within the fibrin network.
Scale bar = 50 um and applies to both images.

62

Discussion

Fibrin has been shown to be an excellent provisional scaffold providing a
conducive surface for cell attachment, adhesion, and migration during the initial phase of
the healing process.(20) In addition, fibrin has been shown to indirectly bind cytokines
creating a growth factor reservoir within this scaffold which, in turn, prolongs the
biological activity of these factors.(21) Therefore, the ability to increase the concentration
of growth factors as well as fibrin density within a polymerizing fibrin scaffold through
the creation of platelet-rich fibrin constructs may prolong growth factor activity and
availability when compared to a naturally occurring whole blood clot.

In the current study, the addition of platelet-rich plasma, created by the initial
centrifugation of whole citrated blood, to a second tube containing calcium chloride
permitted the activation of endogenous prothrombin to thrombin. This, in turn, initiated
the natural fibrin polymerization cascade.(16, 19) The concurrent centrifugation of this
mixture during the polymerization process resulted in a marked increase in fibrin density
in the platelet-rich fibrin constructs when compared to a naturally occurring whole blood
clot. Increasing the speed and duration of the centrifugation process further increased the
fibrin density in the PRFMembrane when compared to the PRP Matrix.

The PRFMatrix and PRFMembrane demonstrated the ability to elute significantly
increased levels of a sentinel grth factor (TGF [3-1) over a three day period when
compared to a whole blood clot of similar volume. In addition, eluents from both the
PRP Matrix and PRF Membrane were able to significantly enhance tendon cell
proliferation over 7 days when compared to the eluent from whole blood clots of similar

volume. The rationale for using a single sentinel grth factor (TGFB-l) to document the

63

increase in growth factor concentration achieved with the PRFM constructs was based on
the significant association demonstrated between platelet levels and TGF-Bl
concentrations.(22, 23) While the concentration of specific mitogenic factors such as
platelet-derived growth factor (PDGF) and basic fibroblast grth factor (bFGF)
normally stored in platelet or-granules were not measured in the current study, the
increase in tendon cell proliferation observed following the addition of the eluents at
various time periods indirectly suggests that these mitogenic factors are also likely
increased.(24)

The current study was not able to determine if the increase in the duration of
grth factor availability from both the PRFMatrix and PRFMembrane was due to a
persistence of platelet activity within the constructs or merely as a result of prolonged
diffusion kinetics resulting from the increased fibrin density of the constructs.
Immunohistochemical evaluation demonstrated positive TGFB-l staining of fibrin as well
as what appeared to be clusters of platelets trapped within the fibrin network of both
PRFM constructs. However, this histological assessment could not delineate between
intact and activated (degranulated) platelets.

Several investigators have examined the use of platelet-rich fibrin constructs to
enhance connective tissue healing.(5, 16, 19, 25-27) However, the precise biological
mechanism(s) by which the addition of these fibrin constructs may enhance connective
tissue repair has yet to be elucidated. While the concept of adding increased levels of
grth factors to enhance cellular proliferation and matrix synthesis has strong basic
science support,(10-13) the clinical benefits of increased grth factor concentrations on

connective tissue healing in association with surgical repair are less clear.(2, 28) Previous

64

 

studies have suggested that the addition of an exogenous fibrin clot provides the needed
mitogenic and chemotactic factors, as well as the provisional scaffold, needed to initiate
and support a regenerative response in avascular regions of the meniscus.(29, 30)
Therefore, the significance of an autologous fibrin matrix in wound repair cannot be
underestimated.

The healing response of connective tissues is an exquisitely designed continuum
of events that is initiated by bleeding and the interaction of platelets and the polymerizing
fibrin clot to create a growth factor-laden provisional fibrin scaffold on which reparative
cells can migrate, adhere, proliferate, and begin to synthesize a matrix.(9) While this
process of wound repair usually occurs without incident in normal healthy tissues, it may
be inhibited in tissues that have compromised vascularity or limited cellularity such as
chronic rotator cuff tears.(3) In these cases, vascular and cellular proliferation may be
delayed and require prolonged availability of cytokines that Signal such events.(l6)
Therefore, the ability of PRFM constructs to provide a bioactive fibrin substrate that
elutes increased levels of active growth factors for longer periods of time, when
compared to a naturally occurring fibrin clot, may represent a bioactive delivery vehicle
for the optimization of healing in biologically compromised tissues. While a recent study
has shown the ability of a PRFMembrane to induce healing in chronic lower extremity
ulcers,(16) additional clinical studies are needed to determine if the use of platelet-rich
fibrin constructs can enhance the repair of other biologically compromised tissues such as
ligaments and tendons.

A potential limitation of this study may center around the use of canine blood.

Purebred canine beagles were used in this study to minimize variability and permit

65

utilization of assays and immunohistochemical techniques that have been perfected in our
laboratory. While the baseline levels of grth factors in canine whole blood (serum) are
slightly different from that of humans,(24, 31-34) the same compliment of grth factors
is present within the platelets of both species.(35, 36) In addition, the clotting cascade of
canine blood is comparable to that of humans.(37) Therefore, we believe the use of
canine blood in our in vitro culture model was a valid system in which to test our
hypotheses regarding the ability of platelet-rich fibrin constructs to prolong growth factor
activity and release when compared to a whole blood clot.

In conclusion, the results of the current study confirmed our hypothesis that
platelet-rich fibrin constructs elute significantly higher concentrations of TGF-Bl
compared to whole blood clots of similar volume. In addition, the results demonstrated
that eluents from the platelet-rich fibrin constructs were able to Significantly increase
tendon cell proliferation over time when compared to whole blood clots of similar
volume. While these results demonstrate the potential for prolonged delivery of increased
concentrations of growth factors from a bioactive, fibrin scaffold in vitro, additional
translational and clinical studies are needed to determine if the use of such platelet-rich

fibrin constructs can enhance the repair of biologically compromised tissues in vivo.

66

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1. Creaney L, Hamilton B. Growth factor delivery methods in the management of
sports injuries: the state of play. Br J Sports Med.42:314-20. 2008.

2. Foster TE, Puskas BL, Mandelbaum BR, Gerhardt MB, Rodeo SA. Platelet-rich
plasma: from basic science to clinical applications. Am J Sports Med.37:2259-72. 2009.

3. Gulotta LV, Rodeo SA. Growth factors for rotator cuff repair. Clin Sports
Med.28:l3-23. 2009.

4. Mishra A, Pavelko T. Treatment of chronic elbow tendinosis with buffered
platelet-rich plasma. Am J Sports Med.34:1774-8. 2006.

5. Sanchez M, Anitua E, Azofra J, Andia I, Padilla S, Mujika I. Comparison of
surgically repaired Achilles tendon tears using platelet-rich fibrin matrices. Am J Sports
Med.35z245-51. 2007.

6. Dallari D, Savarino L, Stagni C, Cenni E, Cenacchi A, Fomasari PM, et al.
Enhanced tibial osteotomy healing with use of bone grafts supplemented with platelet gel
or platelet gel and bone marrow stromal cells. J Bone Joint Surg Am.89z2413-20. 2007.

7. Randelli PS, Arrigoni P, Cabitza P, Volpi P, Maffulli N. Autologous platelet rich
plasma for arthroscopic rotator cuff repair. A pilot study. Disabil Rehabil.30:1584-9.
2008.

8. Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant
Dent.10:225-8. 2001.

9. Werner S, Grose R. Regulation of wound healing by growth factors and
cytokines. Physiol Rev.83:835-70. 2003.

10. Abrahamsson SO. Similar effects of recombinant human insulin-like growth
factor-1 and II on cellular activities in flexor tendons of young rabbits: experimental
studies in vitro. J Orthop Res.15:256-62. 1997.

11. Costa MA, Wu C, Pham BV, Chong AK, Pham HM, Chang J. Tissue engineering
of flexor tendons: optimization of tenocyte proliferation using growth factor
supplementation. Tissue Eng. 12: 1937-43. 2006.

12. Haupt JL, Donnelly BP, Nixon AJ. Effects of platelet-derived growth factor-BB
on the metabolic function and morphologic features of equine tendon in explant culture.
Am J Vet Res.67:1595-600. 2006.

13. Thomopoulos S, Harwood FL, Silva MJ, Amiel D, Gelberman RH. Effect of
several grth factors on canine flexor tendon fibroblast proliferation and collagen
synthesis in vitro. J Hand Surg [Am].30:44l-7. 2005.

67

l4. Marx RE. Platelet-rich plasma: evidence to support its use. J Oral Maxillofac
Surg.62:489-96. 2004.

15. Chen RR, Mooney DJ. Polymeric growth factor delivery strategies for tissue
engineering. Pharm Res.20:1 103-12. 2003.

16. O'Connell SM, Impeduglia T, Hessler K, Wang XJ, Carroll RJ, Dardik H.
Autologous platelet-rich fibrin matrix as cell therapy in the healing of chronic lower-
extremity ulcers. Wound Repair Regen.16:749-56. 2008.

17. Breen A, O'Brien T, Pandit A. Fibrin as a Delivery System for Therapeutic Drugs
and Biomolecules. Tissue Eng Part B Rev.in press. 2009.

18. Nathan C, Sporn M. Cytokines in context. J Cell Biol.113:981-6. 1991.

19. Simon BI, Zatcoff AL, Kong JJ, O'Connell SM. Clinical and Histological
Comparison of Extraction Socket Healing Following the Use of Autologous Platelet-Rich
Fibrin Matrix (PRFM) to Ridge Preservation Procedures Employing Demineralized
Freeze Dried Bone Allograft Material and Membrane. Open Dent J .3:92-9. 2009.

20. Ahmed TA, Dare EV, Hincke M. Fibrin: a versatile scaffold for tissue
engineering applications. Tissue Eng Part B Rev.l4:199-215. 2008.

21. Macri L, Silverstein D, Clark RA. Growth factor binding to the pericellular matrix
and its importance in tissue engineering. Adv Drug Deliv Rev.59zl366-81. 2007.

22. Zimmermann R, Jakubietz R, Jakubietz M, Strasser E, Schlegel A, Wiltfang J, et
al. Different preparation methods to obtain platelet components as a source of grth
factors for local application. Transfusion.4l :1217-24. 2001 .

23. McCarrel T, Fortier L. Temporal growth factor release from platelet-rich plasma,
trehalose lyophilized platelets, and bone marrow aspirate and their effect on tendon and
ligament gene expression. J Orthop Res.27:]033-42. 2009.

24. Visser LC, Amoczky SP, Caballero O, Kern A, Ratcliffe A, Gardner KL. Growth
factor-rich plasma increases tendon cell proliferation and matrix synthesis on a synthetic
scaffold: an in vitro study. Tissue Eng Part A.16:1021-9. 2010.

25. Anitua E, Sanchez M, Nurden AT, Nurden P, Orive G, Andia I. New insights into
and novel applications for platelet-rich fibrin therapies. Trends Biotechnol.24:227-34.
2006.

26. Anitua E, Sanchez M, Nurden AT, Zalduendo M, de la Fuente M, Orive G, et al.
Autologous fibrin matrices: a potential source of biological mediators that modulate
tendon cell activities. J Biomed Mater Res A.77:285-93. 2006.

27. Sunitha Raja V, Munirathnam Naidu E. Platelet-rich fibrin: evolution of a second-
generation platelet concentrate. Indian J Dent Res.19z42-6. 2008.

68

 

28. de Vos RJ, Weir A, van Schie HT, Bierma-Zeinstra SM, Verhaar JA, Weinans H,
et al. Platelet-rich plasma injection for chronic Achilles tendinopathy: a randomized
controlled trial. JAMA.303: 144-9. 2010.

29. Arnoczky SP, Warren RF, Spivak JM. Meniscal repair using an exogenous fibrin
clot. An experimental study in dogs. J Bone Joint Surg Am.70zl209-17. 1988.

30. Henning CE, Lynch MA, Yearout KM, Vequist SW, Stallbaumer RJ, Decker KA.
Arthroscopic meniscal repair using an exogenous fibrin clot. Clin Orthop Relat Res.64-
72. 1990.

31. Bowen-Pope DF, Hart CE, Seifert RA. Sera and conditioned media contain
different isoforms of platelet-derived growth factor (PDGF) which bind to different
classes of PDGF receptor. J Biol Chem.264z2502-8. 1989.

32. Golledge J, Clancy P, Jones GT, Cooper M, Palmer LJ, van Rij AM, et al.
Possible association between genetic polymorphisms in transforming growth factor beta
receptors, serum transforming growth factor betal concentration and abdominal aortic

aneurysm. Br J Surg.96:628-32. 2009.

33. Kato Y, Asano K, Mogi T, Kutara K, Teshima K, Edamura K, et al. Clinical
significance of circulating vascular endothelial growth factor in dogs with mammary
gland tumors. J Vet Med Sci.69:77-80. 2007.

34. Werther K, Christensen IJ, Nielsen HJ. Prognostic impact of matched

preoperative plasma and serum VEGF in patients with primary colorectal carcinoma. Br J
Cancer.86:417-23. 2002.

35. Tablin F. Platelet Structure and Function. In: Feldman BV, Zinkl JG, Jain NC,
eds. Schalm's Veterinary Hematology. 5th ed. Oxford: Blackwell Publishing; 2006. pp.
448-52.

36. Blair P, Flaumenhaft R. Platelet alpha-granules: basic biology and clinical
correlates. Blood Rev.23zl77-89. 2009.

37. Gentry PA. Comparative aspects of blood coagulation. Vet J .168:238-51. 2004.

69

CHAPTER 3.

The Use of an Autologous Platelet-Rich Fibrin Membrane to Enhance Tendon
Healing: An Experimental Study in Dogs

Lance C. Visser, BS, Steven P. Amoczky, DVM, Oscar Caballero, MS,
and Keri L. Gardner, MS

Laboratory for Comparative Orthopaedic Research
College of Veterinary Medicine
Michigan State University
East Lansing, MI

Visser LC, Amoczky SP, Caballero O, Gardner KL. The use of an autologous platelet-
rich fibrin membrane to enhance tendon healing: An experimental study in dogs. Am J
Vet Res, in press.

70

Abstract

The purpose of this study was to examine the effect of an autologous platelet-rich
fibrin membrane (PRF Membrane) in enhancing healing of a central-third patellar tendon
(PT) defect in dogs. Bilateral central-third PT defects were created each dog. One defect
was implanted with an autologous PRF Membrane and the contralateral defect left empty.
Dogs were euthanized at 4 and 8 weeks post-operatively (n = 4/time period), and tendon
healing was assessed grossly and histologically using a semiquantitative scoring system.
Cross-sectional area of the PTs was also compared. Both treated and control defects were
filled in with repair tissue by 4 weeks. There was no significant difference in the
histologic quality of the repair tissue between control and PRFMembrane-treated defects
at either time point. At both time points, the cross-sectional area of PRFMembrane-
treated tendons was significantly (P _<_ 0.01) greater (at least 2.5-fold) than that of control
tendons. At 4 weeks the repair tissue consisted of disorganized proliferative fibrovascular
tissue originating predominantly from the fat pad. By 8 weeks the tissue was less cellular
and slightly more organized in both groups. A PRF Membrane did not enhance the rate or
quality of tendon healing. However, it did increase the amount of reparative tissue within
the defect. This may be due to the increased concentration and duration of grth factor
exposure provided by the membrane. These results suggest that a PRFMembrane may be
more beneficial in biologically compromised tissues where increased concentrations of

bioactive molecules may be needed for longer periods of time.

71

Introduction

Tendon injuries are a common cause of morbidity in both human and veterinary
medicine and their management poses a significant challenge to the clinician. The injured
tendon is often refractory to treatment, slow to heal, and despite nature’s best repair
efforts, remains functionally inferior to an uninjured tendon.(1, 2) Contemporary research
has revealed that numerous growth factors such as platelet-derived grth factor, TGF-B,
insulin-like grth factor, basic fibroblast growth factor, and vascular endothelial growth
factor play critical roles in tendon healing, including mitogenesis, chemotaxis,
angiogenesis, and matrix synthesis.(3, 4) In addition, increasing grth factor levels
above those normally found in serum have been shown to improve both the rate and
quality of tendon healing.(5-8) Therefore, the use of growth factors as a potential
treatment option to enhance connective tissue healing holds great promise.(9) However,
questions regarding the most effective dose, choice/combination of grth factor(s), and
delivery method to enhance tendon repair remain unanswered.

One potential method of delivering growth factors as well as other beneficial
bioactive molecules to an injured tendon is through the use of PRP. Platelets are known
to contain all of the aforementioned grth factors involved in tendon healing within
their a-granules.(10) Recently, several experimental and pro-clinical Studies have
demonstrated promising results following the use of PRP to enhance tendon healing.(l l-
19) Platelet-rich plasma has been defined as autologous plasma that contains a platelet
concentration above the baseline levels found in whole blood.(20) It is typically created
from citrated whole blood using centrifugation to separate the platelets from the other

blood cells in plasma and to concentrate the platelets in plasma.(20) In addition to

72

providing a convenient source of increased concentrations of autologous growth factors,
PRP also maintains the “normal” physiologic ratios of growth factors and bioactive
molecules,(20) demonstrated to be ideal for wound healing.(21, 22)

While platelets and their associated grth factors are important for initiating the
healing cascade, perhaps of equal importance is the presence of a provisional fibrin
scaffold.(23, 24) Fibrin provides a naturally derived matrix, on which repair cells can
adhere, migrate, proliferate, and deposit matrix.(25) Together with other plasma-derived
proteins such as fibronectin and vitronectin, fibrin is able to bind to many grth factors
present within platelet iii-granules thus creating a grth factor reservoir.(23, 24, 26)
Therefore, the ability to combine an increased concentration of platelets and their
associated grth factors in plasma (PRP), within a fibrin scaffold, may provide an
optimal environment for tissue repair and regeneration.

The purpose of this study was to examine the effect of a PRFMembrane (Cascade
Medical Enterprises, Wayne, NJ) in enhancing and accelerating healing of a central-third
patellar tendon defect in a canine model. It was our hypothesis that the PRFMembrane
would enhance the rate (based on percent of defect fill) and histologic quality of the
repair tissue (based on a semiquantitative evaluation of cellularity, vascularity, collagen
organization, and glycosaminoglycan content) in the PT defect when compared to empty
contralateral defects (surgical controls).

Materials and Methods
All procedures in this study were approved by the Institutional Animal Care and

Use Committee at Michigan State University.

73

Study Design

A central-third PT defect was created in both hind limbs of eight adult male
beagle dogs (average weight, 14.5 i 1.9 kg) and an autologous PRFMembrane was
implanted into one defect and the contralateral defect was left empty as a surgical control
(sham). Four dogs at 4 and 8 weeks post-operatively were humanely euthanized using an

intravenous overdose of pentobarbital.

PRF Membrane Preparation

The autologous PRFMembrane was created from each dog as per the
manufacturer’s instructions as follows (Figure 3.1).(27) Prior to the induction of
anesthesia, 18 mL of whole blood was drawn via jugular venipuncture with a 20 gauge
butterfly catheter into two 9 mL tubes containing trisodium citrate and a proprietary
separator gel. The tubes were centrifuged at 1,100 g for 6 minutes to create a PRP
supernatant. Using an 18 gauge needle and a 20 mL syringe, the PRP supernatant from
both 9 mL tubes was carefully transferred to a 35 mm-diameter flat-bottom glass vial
containing 1.0 M calcium chloride. The vial was immediately centrifuged at 4,500 g for
25 minutes while fibrin polymerization ensued. The result was a dense, flat, circular,
fibrin membrane (due to radial centrifugation) suspended in a liquid serum component

(Figure 3.1).

74

#1 #2

S! - Platelet-rich E! ’-
plasma (PRP)
\

‘

    

Collect 18 mL of whole blood into two citrated
tubes and centrifuge at 1.100 g for 6 min.

 

 

 

3 serum

 

 

PRFMembrane

Centrifuge vial at 4.500 g for 25 min. and the PRFMembrane results.
Figure 3.1 Summary of the creation of the platelet-rich fibrin membrane

(PRFMembrane) used in this study.
Surgical Procedure

After premedication with intramuscular morphine (0.6 mg/kg) and acepromazine
(0.02 mg/kg), the dogs were anesthetized using intravenous thiopental (dosed to effect)
and maintained with isoflurane in oxygen (0.5% to 3%). Next, preservative free
Morphine (0.1 mg/kg) was injected into the epidural Space for post-operative analgesia.
With the animal placed in dorsal recumbency and using aseptic surgical technique, the
patellar tendon was isolated and exposed utilizing a medial parapatellar approach (Figure
3.2A). After measuring the width of the patellar tendon, the central-third of the patellar
tendon was sharply incised from the distal aspect of the patella to the tibial tuberosity and

then resected (Figure 3.23). Care was taken to separate the retropatellar fat pad from the

75

resected portion of the PT. In the treated leg, a bed of 4-0 polydioxanone suture was
placed through the remaining patellar tendon and under the defect using a horizontal
mattress pattern to act as caudal support for the PRFMembrane (Figure 3.2C). The
PRFMembrane was rolled onto itself, placed into the defect and sutured to the remaining
patellar tendon using a simple interrupted pattern of nonabsorbable 5-0 nylon suture
(Figure 3.2D). The surgical site was closed in a routine layered manner. In the sham leg
the surgical site was closed following the resection of the central-third of the patellar
tendon. Carprofen (4 mg/kg, SC) was administered once during surgery and q 24 h as

needed for post-operative analgesia.

 

Figure 3.2 Photographs of the surgical procedure. The patellar tendon was isolated and
exposed (A). A central-third patellar tendon defect was created (B). A bed of sutures was
created in the defect to act as caudal support (C). The autologous platelet-rich fibrin
membrane was rolled onto itself and sutured to the defect (D). The contralateral limb
consisted of only steps A and B.

76

Post-operative Regimen

After recovery, all animals were housed individually in 4 x 1.5 meter runs and
allowed unrestricted activity. Tramadol (2-4 mg/kg, PO, q 8-12 h) was administered for a
minimum of 3 post-operative days for analgesia. No post-operative
bandage/immobilization was applied. The dogs were observed at least twice daily and
general condition, temperature, pulse rate, respiratory rate, attitude, appetite, activity
level, and degree of lameness was recorded daily. The incision sites were checked daily

for signs of swelling, erythema, discharge and dehiscence until complete healing.

Gross Evaluation

Following euthanasia, both stifle joints and PTS were exposed and grossly
evaluated. The patella-patellar tendon-proximal tibial complex was harvested en bloc and
placed in 10% neutral-buffered formalin. Following formalin fixation, the tendons were
bisected at their midpoint in the transverse plane and side-by-side digital photographs of

each dog’s sham and PRFMembrane-treated tendons were taken.

Histologic Evaluation

After routine histologic processing, S-um-thick sections of the proximal segments
of 2 dogs PTS from each time period were sectioned in the coronal plane and the distal
segments were sectioned in the transverse plane, and the vice versa was true for
sectioning the PTS from the other 2 dogs from each time period. Digital photographs of
the transverse hematoxylin and eosin (H&E) —Stained histologic sections at the midpoint

of each PT were taken and the cross-sectional area of each PT (excluding the fat pad) was

77

measured, where the pixels in the digital photographs were converted to area
measurements using Scion Image (Scion Corporation, Frederick, MD) computer software
(Figure 3.3). A modified version of a semiquantitative histologic tendon pathology
scoring system(28) (Table 3.1) was used to compare tendon healing in the central-third
defect, designated “repair tissue (RT)” and in the native patellar tendon, designated
“tendon tissue (TT)”. From 4 different sections of the same tendon, both the RT and TT
were assigned an ordinal score based upon the following scoring variables: cellularity,
vascularity, collagen organization, and glycosaminoglycan content. The histologic
sections were evaluated by one of the authors (LCV) who was blinded to the identity of
the animal and its treatment. The sum of the mean histologic scores for each variable was
used to obtain a total histologic score for both the RT and TT from each patellar tendon.
All sections were stained with H&E, except those evaluated for glycosaminoglycan
content, where sections were stained with Alcian blue (pH 25)/periodic acid-Schiff.

Collagen organization was evaluated using coronal sections under polarized light.

    

Figure 3.3 Photograph of a patellar tendon after histologic processing mounted in cross-
section representing an example of how the cross-sectional area (area within the dotted
line) data were gathered in this study. The number of pixels within the dotted line was
converted to area measurements using imaging software.

78

Table 3.1
Semiquantitative Histologic Scoring System

 

 

 

. Score
Variable O 1 2 3
Cellularity a 100-199 200-299 >300
(100 “HS/”PF cells/I—IPF cells/HPF cells/HPF
Vascularity Normal, vessels Slight increase, Moderate Severe
parallel to transverse increase within increase,
collagen fibers vessels in the tendon including
tendon tissue tissue ‘ clusters 7
Collagen Organized, Moderately Slightly Disorganized,
- - uniform, linear, organized, organized, 20- non-linear,
Organization parallel fibers, >50% linear & 50% linear & complete
coarse even uniform, fine uniform, <50% disarray, no
crimp even crimp 77 7 crimp 7 crimp
GAG No alcianophilia Slight Moderate Severe
contentc alcianophilia alcianophilia alcianophilia

between
collagen fibers

forming blue
lakes

 

aHigh-power field (HPF) = 200x original magnification, bcoronal sections assessed

with polarized light, calcian blue (pH 25)/periodic acid-Schiff staining, GAG =

glycosaminoglycan. Adapted from ref. (28)

Statistical Analysis

All data are displayed as mean :t standard deviation (SD). Mean cross-sectional
area data of sham and treated PTS within each time point were compared using a paired
Student’s t-test. Mean cross-sectional area data of sham and treated PTs were compared
over time with an unpaired Student’s t-test. Means from the histologic scoring system
were compared with a Wilcoxon Signed-rank test using a statistical software program
(GraphPad Software, La Jolla, CA). A power analysis determined that 4 dogs per time
period would suffice in order to detect a 33% difference in the quality of tissue repair at a

confidence level of 95% and a statistical power of 0.8. Values of P S 0.05 were

considered Significant for all comparisons.

79

Results
The post-operative period was uneventful and all of the animals were ambulatory
and weight-bearing following the recovery period. There were no major clinical
complications related to the surgical procedures, and lameness was not observed beyond

the first post-operative week.

Gross Healing Assessment & Cross-sectional Area

Gross examination of the PTs immediately following euthanasia revealed that
after 4 weeks, the central-third PT defect in both groups was filled in with repair tissue,
which appeared contiguous with the retropatellar fat pad. Hyperemia was evident within
the surrounding PT tissue by 4 weeks but was less obvious by 8 weeks. At both time
points the PRFMembrane-treated tendons exhibited a more abundant healing response
than the controls (Figure 3.4). The cross-sectional area of the PRFMembrane-treated
tendons was at least 2.5-fold greater than the sham tendons at both time points (Figure

3.5). At the 4-week time point, the cross-sectional area of the PRFMembrane-treated

tendons (1.82 d: 48 mmz) was significantly greater (P = 0.001) than the sham tendons (73
i 47 mm2). Similarly, at the 8-week time point, the cross-sectional area of the
PRFMembrane-treated tendons (216 i 51 mmz) was Significantly greater (P = 0.012)

than the sham tendons (62 i 28 mmz). The cross-sectional area of the treated and sham

tendons did not significantly differ (P = 0.383 and P = 0.71 1, respectively) with time.

80

Sham PRFMembrane

 

Figure 3.4 Representative photographs of cross-sectional tissue sections of a sham and
PRFMembrane-treated patellar tendons at 4 (A) and 8 (B) weeks after surgery. Note the
difference in size of the PRFMembrane-treated tendons compared to its surgical control
(sham).

81

M Sham l PRFMembrane

300 P= 0.012

P= 0.001

250

200

150

100

PT Cross-sectional Area (mmz)
8

   

4 weeks post-op. 8 weeks post-op.

Figure 3.5 Mean cross-sectional area of the sham and PRFMembrane-treated patellar
tendons 4 and 8 weeks post-operation i SD (n = 4).
Histologic Healing Assessment

When comparing the sham and PRFMembrane-treated tendons at each time point,
they appeared histologically similar (Figure 3.6). After comparing the sham and
PRFMembrane-treated tendons using the semiquantitative tendon pathology scoring
system, there was no significant difference (P > 0.05) in any of the individual scoring
variables (cellularity, vascularity, collagen organization, or glycosaminoglycan content)
or the total histologic score (sum of the individual variables) in the RT or TT at either
time point (Table 3.2). Histologic analysis confirmed the gross observations that by 4

weeks the defects in both groups had filled in with a hypercellular fibrovascular repair

82

tissue. The origin of the RT in both groups appeared to be predominantly from a
proliferative response arising from the retropatellar fat pad and, to a lesser extent, the
paratenon (Figure 3.7). In addition to repair cells, it appeared that the fat pad contributed
a significant amount of the blood supply to the RT within the central-third defect of both
groups at 4 weeks and to a lesser extent at 8 weeks. At 4 weeks the RT was hypercellular
with a whorled fibroblastic appearance, and blood vessels were abundant in several
planes in each group. Although there was some evidence of collagen organization
(moderate linearity and crimp) at 4 weeks, overall, the collagen in the RT of both groups
was disorganized and immature. By 8 weeks both groups exhibited less cellularity and
vascularity, and there was evidence of a more organized collagen pattern (increased
linearity and crimp). An increase in glycosaminoglycan staining was also noted with time
in the RT of both groups. There was evidence of fibrous metaplasia within the
retropatellar fat pad of both groups by 4 weeks. This finding persisted in the 8 week

specimens as well.

83

mm ‘ W21

Sham PRFMembrane Sham PRFMembrane
Fax-4:3.--oxmmmfi thd’fle"..;1’ . . . _.. .21! 51-41.}. 2*
i _ v; i :
‘7 r. . :5 r
Cellularity E; - . ;-
E 7'- ‘ :1
ii 5" ;
mefi—woaa has-Laura” mama‘s—"9.; ‘ as k- n-- a. ‘1
tl.‘ z 2* - 1 :~ 1.1: 1:. v- Jim ‘35} ~ g . . ' 3.;-
i 3 .1’ t 1 :
._ 'l
i I I. .1
’ - r '; :1
Vascularity i t. . . _.
1" i 1 f" l.:
i. ; L .-
B—s u; ‘. 1:531; Lemur--inkp H... j ”0‘1.- -- e ‘u i a. '5. a :- -\ Juli.
nadir-:51- 9‘ -, w»: 1.1 “,3 r...”.i-....4L-4_1-e..m..- Jig pr .- .a 11.33 9:11: r-H - can
11" f z. .1 E: .1" 1' l
“- ‘I i ii 9 ., 3 :i
, a. g. 2* ‘. :1 C ,
Collagen E -.' i 13 g ,1 i. .j
' i. -f '~i f .2
Organizatlonlt . 3 .g :. 3,3
'-= ~ . * “ -'~ 1» .l
E , .3 l. , 3 If: 3 l-
l I ' P: Elsi-“.‘fiammitfi I. . I;

    

 

Collagen
Organization
(polarized llght)

Figure 3.6 Photomicrographs of representative H&E-stained sections of the repair tissue
(RT) within the central-third patellar tendon defect of the sham and PRFMembrane-
treated patellar tendons at each time point. The cellularity, vascularity, collagen
organization, and glycosaminoglycan content (images not shown) of the PRFMembrane-
treated tendons were similar when compared to the sham tendons at each time point. The
white arrows denote blood vessels. Scale bar = 100 pm.

84

Table 3.2

 

 

A 4 weeks post-op.
churr 'l'r'ss'irc (R'l') 'l'cm/nri 'I'r‘sxirc (77')
Variable (0-3) Sham PRFMem P-value Sham PRFMem P-value
Cellularity 2.7 : 0.1 2.6 : 0.4 1.099 I.6 : 0.9 1.9 : 0.5 0.50
Vascularity 2.1 : 0.5 2.6 : 0.6 0.25 1.9 : 0.7 2.0 : 0.7 0.50
Collagen 2.8: 0.3 23:04 0.10 11:04 12:04 0.85
GAGS 08:04 1.1 :03 0.50 09:05 1.1 :05 0.85
Total (0-12) 83:05 86:08 0.63 52:23 6.2: 1.5 0.63

 

 

 

B 8 weeks post-op.
lr'cpirrr' Tissue (R 7:) Tum/rm 'I'i'sslrc ('17)
Variable (0-3) Sham PRFMem I ’-value Sham PRFMem I’-value
Cellularity 1.5 : 0.5 1.8 : 0.4 0.50 1.4 : 0.3 1.8 : 0.4 0.50
Vascularity 1.9 : 0.8 2.0 : 0.5 >099 1.8 : 04 1.4 : 0.5 0.50
Collagen 2.4 : 0.1 2.3 : 0.4 0.77 0.9 : 0.3 1.3 : 0.8 050
GAGS 14:08 23:07 0.13 07:04 13:05 0.25
Total (012) 7.2: 1.1 8.4: 1.1 0.50 47:03 5.7: 2.1 >099

 

Table 3.2 Results of the semiquantitative histologic scoring system used to assess tendon
healing at 4 (A) and 8 (B) weeks post-operation. The ordinal data is displayed as mean
score :1: SD (n = 4). An unoperated (“normal”) tendon would have a perfect score of 0.
Repair tissue (RT) refers to the regenerated healing tissue within the central-third patellar
tendon defect and tendon tissue (TT) refers to the native patellar tendon tissue. GAGS =
glycosaminoglycans, PRFMem = platelet-rich fibrin membrane.

85

. . __ PRFMembrane,

 

Figure 3.7 Representative photographs of the sham (A) and PRFMembrane-treated (B)

patellar tendons 4 weeks after surgery mounted in cross-section. Note that the origin of

the repair tissue was primarily from a proliferative response arising from the fat pad and

paratenon (asterisks) in both groups, albeit more abundant in the PRFMembrane group.
Discussion

The results of the current study did not support the hypothesis that a
PRFMembrane would enhance the quality and rate of healing of a central-third patellar
tendon defect when compared to control defects. While both the control and
PRFMembrane-treated defects healed with a fibrovascular repair tissue of similar
histologic quality, the cross-sectional area of the PRFMembrane-treated tendons was
significantly greater (at least 2.5-fold) than that of the controls at both 4 and 8 weeks.

The increase in cross-sectional area in the PRFMembrane-treated defects may be
associated with the prolonged presence of increased levels of growth factors, including
TGF-Bl. Transforming growth factor-Bl plays a major role in the early phases of wound
healing where it is involved in the recruitment of inflammatory cells and fibroblasts and,
at later stages, promotes collagen production.(29) Transforming growth factor 451 is
known to be secreted from platelets as a latent precursor where continuous latent

activation is thought to occur up to 14 days post-injury.(29) Platelets may provide a long-

terrn source of TGF-Bl activity, as they are thought to activate only a small fraction of

86

the latent TGF-Blthey release.(30) Moreover, numerous studies have associated
. increased fibroplasia with increased levels of TGF-BI .(31-33) Although TGF-Bl was not
quantified in the PRFMembrane in the current study, a recent study has shown that a
PRFMembrane is able to elute significantly increased concentrations of TGF-Bl and
enhance tendon cell proliferation over time when compared to a blood clot of similar
volume.(34) Therefore, it is possible that the significant increase in fibroplasia (and
resultant increase in cross-sectional area) associated with the application of a
PRFMembrane in the current study could be the result of increased levels (and a
prolonged duration) of TGF-Blavailable to the repair cells.

In addition to the chemotactic and mitogenic stimuli provided to tendon cells by
increased levels of various grth factors in PRP,(11, 14, 18, 35) it has been suggested
that the addition of PRP in the initial period following acute tendon injury could actually
exacerbate inflammation and pain.(10) A recent study using PRP to treat acute skin
wounds of horses found that the PRP led to the development of excess (granulation)
tissue and actually slowed wound healing.(36) The authors suggested that the
development of excess granulation tissue could be due to a prolonged expression of TGF-
[31 from PRP via two mechanisms. First, although platelets secrete approximately 95% of
their growth factors within 1 hour after activation, they have demonstrated to
continuously synthesize small amounts of growth factors for the remainder of their
lifespan (5-10 days).(20, 37) Secondly, it was noted that TGF-Bl, unlike other growth
factors, can regulate its own production by monocytes and activated macrophages in an
autocrine manner, resulting in a persistent expression at the wound site following a single

exogenous application.(38) These results suggest that because of the increased

87

concentrations of growth factors present in PRP, such preparations may be better suited
for more chronic wounds where a fresh exogenous source of growth factors would be
more beneficial.(36)

A recent experimental study(39) on the spatiotemporal expression of TGF-Bl
after an acute PT injury (transverse incision of the medial-half of the mid-body patellar
tendon) may also help explain the abundant repair tissue witnessed. The study found that
the expression of TGF-Bl propagates out and away from the wound Site to the nearby
uninjured tendon tissue with time and as healing progresses. Interestingly, the authors
also noted that the expression of TGF-Bl is transiently enhanced over the entire length of
the patellar tendon as early as 7 days post-injury.(39) Therefore, the reported
spatiotemporal mechanism of TGF-Blexpression, coupled with the prolonged elution of
increased concentrations of TGF- BI from the PRFMembrane may, help explain the
abundant healing tissue witnessed in the current study.

In this study the healing response in both the control and PRFMembrane-treated
defects appeared to arise predominantly from the retropatellar fat pad. An increase in
cross-sectional area in conjunction with a significant amount of repair tissue originating
from the fat pad has been documented by numerous investigators using similar central-
third patellar tendon defect models.(40-45) The retropatellar fat pad is thought to
significantly contribute to PT healing due to their close proximity and shared blood
supply.(46, 47) In addition to contributing a blood supply, it is possible that the fat pad
also contributes reparative progenitor cells to the patellar tendon healing process, as the

fat pad has been recently demonstrated to be a significant source of highly proliferative

88

adipose-derived mesenchymal stem cells potentially able to respond to tendon injury.(48,
49)

A previously published study using a similar defect model reported healing of the
defects at three months post-operation.(42) However, contrary to our hypothesis, the
PRFMembrane did not accelerate the healing or enhance the quality of the repair tissue in
a canine central-third PT defect at the earlier time periods examined in the current study.
This may have occurred because the central-third PT defect in this model was not of
critical size as the control defect was filled in with a similar quality repair tissue at the
earliest time point examined in this study. The results of the current study suggest that
since the retropatellar fat pad appears to provide an adequate supply of repair cells,
vasculature, and growth factor stimulus in acute PT defects, the PRFMembrane is not a
significant adjunct to the healing process in these situations. It is possible that the
PRFMembrane may be of more benefit in biologically compromised or chronically
injured tissues where a fresh source of bioactive molecules may be needed for longer
periods of time in order to enhance the repair process. A Similar conclusion was reached
in a study comparing the use of PRP in the healing of acute cutaneous wounds.(36) In
that study, the histologic quality of the control and PRP-treated defects was also not
Significantly different.(3 6)

The outcome metrics in the current study were limited to the semiquantitative
histologic assessment of the repair tissue using a previously published grading scale.(28)
While only 4 animals in each group were evaluated at each time period, an a priori power
analysis revealed that this number would be sufficient to detect a 33% difference in repair

tissue quality, which was felt to be clinically relevant. The absence of any functional

89

assessment, such as biomechanical analysis of the healed tendons, could also be
considered a limitation of this study. Since the animals did not Show any obvious
untoward clinical signs (i.e., abnormal activity or gait) related to the surgical procedure, a
clinically relevant impact on joint function was not apparent. Finally, the 4 and 8 week
time points used in the current study did not permit any assessment of the long-tenn
impact of PRP Membrane augmentation on the remodeling of the repair tissue.

In summary, the PRP Membrane did not enhance the rate and quality of healing in
central-third PT defects at either 4 or 8 weeks post-operation. While the PRFMembrane
produced an increased amount of fibroblastic repair tissue, as determined by a significant
increase in cross-sectional area, this tissue was not significantly different, with respect to
cellularity, vascularity, collagen organization, and glycosaminoglycan content from the
naturally occurring repair tissue. It is possible that a PRFMembrane may be of more
benefit in larger defects where a naturally occurring provisional fibrin scaffold may not
adequately fill the defect or in biologically compromised tissues where the prolonged
release of growth factors may be required to induce and sustain a repair response from
adjacent tissues. Further basic science and experimental studies are warranted to help

determine the precise role of PRP preparations in tendon healing.

90

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42. Burks RT, Haut RC, Lancaster RL. Biomechanical and histological observations
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43. Kamps BS, Linder LH, DeCamp CE, Haut RC. The influence of immobilization
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Healing of the patellar tendon donor defect created after central-third patellar tendon

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94

CHAPTER 4.

Growth Factor-Rich Plasma Increases Tendon Cell Proliferation and Matrix
Synthesis on a Synthetic Scaffold: An In Vitro Study

Lance C. Visser, BS,1 Steven P. Amoczky, DVM,1 Oscar Caballero, MS,l Andreas Kern,
PhD,2 Anthony Ratcliffe, PhD,2 and Keri L. Gardner, MS1

1Laboratory for Comparative Orthopaedic Research
College of Veterinary Medicine
Michigan State University
East Lansing, Michigan

ZSynthasome, Inc.
San Diego, CA

Visser LC, Amoczky SP, Caballero O, Kern A, Ratcliffe A, Gardner KL. Growth factor-
rich plasma increases tendon cell proliferation and matrix synthesis on a synthetic
scaffold: An in vitro study. Tissue Eng Part A. 2010;16(3):1021-9.

95

Abstract

Numerous scaffolds have been proposed for use in connective tissue engineering.
Although these scaffolds direct cell migration and attachment, many are biologically inert
and thus lack the physiological stimulus to attract cells and induce mitogenesis and
matrix synthesis. In the current study, a bioactive scaffold was created by combining a
synthetic scaffold with growth factor-rich plasma (GFRP), an autologous concentration
of growth factors derived from a platelet-rich plasma preparation. In vitro tendon cell
proliferation and matrix synthesis on autologous GFRP-enriched scaffolds, autologous
serum-enriched scaffolds, and scaffolds alone were compared. The GFRP preparation
was found to have a 4.7-fold greater concentration of a sentinel growth factor (TGF -[31)
compared to serum. When combined with media containing calcium, the GFRP produced
a thin fibrin matrix over and within the GFRP-enriched scaffolds. Cell proliferation
assays demonstrated that GFRP-enriched scaffolds Significantly enhanced cell
proliferation over autologous serum and control groups at both 48 and 72 hours. Analysis
of the scaffolds at 14, 21, and 28 days revealed that GFRP-enriched scaffolds
significantly increased the deposition of a collagen-rich extracellular matrix when
compared to the other groups. These results indicate that GF RP can be used to enhance in

vitro cellular population and matrix deposition of tissue-engineered scaffolds.

96

Introduction

Scaffolds play an essential role in engineering biological substitutes for the body
in vitro and/or orchestrating tissue regeneration and remodeling in vivo.(l) To date, a
variety of scaffolds derived from both natural and synthetic materials have been proposed
for use in connective tissue engineering.(2) While these scaffolds provide a conductive
surface for cell attachment and migration, many are biologically inert and thus lack the
ability to induce chemotaxis, stimulate cell proliferation, and coordinate matrix synthesis
on their own. The addition of bioactive factors to such materials could result in more
rapid cellular repopulation and matrix synthesis in vitro and, in turn, optimize scaffold
incorporation in vivo. Previous studies have incorporated recombinant growth factors,
transfected cells, or plasmid DNA with polymer or hydrogel scaffolds in an attempt to
increase the bioactivity of these scaffolds.(3) Although these techniques appear
promising for the controlled release of specific growth factors, questions regarding the
choice and concentration of growth factors to be utilized have yet to be answered.(4, 5)

One potential method for providing the entire complex of grth factors involved
in tissue repair and regeneration might be through the use of platelet-rich plasma (PRP).
Platelet-rich plasma is defined as an autologous concentration (above baseline) of
platelets and their associated growth factors.(6) Several recent studies have demonstrated
the effectiveness of PRP in the repair and regeneration of a variety of tissues including
bone,(7-9) cartilage,(10-12) tendon,(l3-18) and ligament.(19-22) The use of PRP for
tissue engineering applications is appealing because it is safe (autologous), easily
obtainable, and a cost-effective source of bioactive molecules.(23, 24) Most importantly,

PRP not only provides an increased concentration of several growth factors, it also

97

maintains the physiologic proportions of individual growth factors to each other.(6)
Maintaining the physiologic proportional relationship between growth factors is thought
to be important in tissue regeneration.(25, 26) Combining the increased concentration of
growth factors found in PRP with a synthetic scaffold could create a bioactive construct
that is able to optimize cellular repopulation and matrix synthesis onto the scaffold in
vitro.

Therefore, the purpose of this study was to evaluate the ability of growth factor-
rich plasma (GFRP) -enriched scaffolds to induce tendon cell proliferation and matrix
synthesis compared to serum-enriched scaffolds and scaffolds alone. Growth factor rich
plasma was created by degranulating the platelets in a standard PRP preparation by a
freeze-thaw process thereby liberating all the growth factors contained in the platelets. It
was our hypothesis that GFRP-enriched scaffolds would significantly enhance cell
proliferation and matrix synthesis when compared to serum-enriched scaffolds and

scaffolds alone.

Materials and Methods
Scaffold Composition and Preparation
The scaffold used in this study was a synthetic biodegradable poly-L-lactic acid
(PLLA) scaffold constructed in a woven pattern that is used for tendon and connective
tissue repair (Synthasome Inc., San Diego, CA). Eight-mm-diameter disks were isolated
from a larger scaffold sample with a heated biopsy punch (Figure 4.1). Scaffold-disks
were soaked in a 70% ethanol solution, rinsed with phosphate buffered saline (PBS), and

then air-dried overnight under UV light to sterilize.

98

 

        

Dir. Fil '1

Figure 4.1 Photographs of the synthetic biodegradable polymeric scaf old used in this
study showing an 8-mm-diameter scaffold-disk (A) isolated from a larger sample of the
scaffold (B). Scanning electron photomicrographs of the scaffold at 20x (C) and 100x (D)
magnification.

Preparation of Growth Factor-Rich Plasma (GF RP) and Serum

GFRP was prepared from 18 mL of canine whole blood as follows. Blood was
collected via jugular venipuncture with a 19G butterfly catheter in 9 mL increments in
two separate vacuum tubes containing trisodium citrate. Red blood cells and plasma
containing platelets (supernatant) were separated by centrifugation for 6 minutes at 1,100
g. The supernatant from both tubes was then transferred to one 15 mL test tube. Platelets
were concentrated in the plasma with a second centrifugation step at 3,000 g for 15
minutes. A platelet pellet was evident and the platelets were resuspended in 2 mL of

plasma. The resultant PRP was snap-frozen in liquid nitrogen and then allowed to thaw to

99

room temperature. The freeze-thaw process was repeated to ensure total platelet
activation and thus release the maximum amount of growth factors available from the
platelets.(27) The resulting preparation was termed growth factor-rich plasma and was
always used immediately following its creation. Serum was obtained from 9 mL of whole
blood at the same time and from the same animal used to create GFRP. The collection
method was similar, except blood was drawn into a plain vacuum tube, allowed to
coagulate for at least 2 hours at room temperature, and then centrifuged for 15 minutes at
3,000 g. Samples of both blood products were separated into aliquots and stored at -80°C

until assayed for grth factor concentration.

Isolation of Tendon Cells
Canine patellar tendon fibroblasts were harvested via primary explant cultures

from adult mongrel dogs euthanized for reasons unrelated to this study. Cells were

expanded to passage two in 75 cm2 tissue culture flasks in Dulbecco’s modified Eagle

medium (DMEM) containing 1% antibiotic-antimycotic solution, 0.02 mg/mL
gentamicin, and 0.15 mg/mL ascorbate (DMEM media) containing 10% fetal bovine
serum (F BS) incubated at 37°C in a humidified atmosphere containing 10% C02. Cells

in each flask were cultured until sub-confluent and then detached by trypsinization for

scaffold seeding. Only cells from the third passage were used for this study.

Scaffold Enrichment and Cell Culture
Scaffolds were divided into three treatment groups and 50 uL of either: 1) DMEM

media containing 2% FBS (control), 2) serum, or 3) GFRP was pipetted onto the surface

100

of the scaffolds in a 24-well tissue culture plate. Scaffolds were allowed to soak in their
respective treatments for approximately 10 minutes. Next, 105 canine patellar tendon
fibroblasts suspended in DMEM media containing 2% FBS were drip-seeded onto the
scaffolds and maintained in DMEM media containing 2% FBS (total volume per well
was 2 mL) at 37°C in a humidified atmosphere containing 10% C02. Upon the addition
of the culture media, the calcium in the media triggered the conversion of pro-thrombin
to thrombin and the clotting cascade.(28) The subsequent polymerization of fibrin

produced a thin coating of fibrin over the surface and within the interstices of the

scaffolds (Figure 4.2).

 

      

.91” I' i ,ggp
Figure 4.2 Scanning electron photomicrographs of a GFRP-enriched scaffold after
combining with cell culture media and prior to cell-seeding at 20x (A), 50x (B), 80x (C),
and 3500x (D) magnification. Notice the thin fibrin-matrix coating throughout the
scaffold surface that results from fibrin polymerizing around the scaffold when combined
with culture media containing calcium.

   

101

After 72 hours, the scaffolds were maintained in DMEM media containing 10%
FBS and the media was replenished every other day throughout the remainder of the
study. Scaffolds were transferred to new plates once per week to avoid cell confluence
around the scaffolds in the bottom of the wells. Care was taken to maintain the
orientation of the scaffolds; i.e., the cell-seeded side of the scaffold was designated

“surface.”

T GF -/3 1 Enzyme-Linked Immunosorbent Assay

TGF-Bl was used as a sentinel growth factor to document the extent of growth
factor enrichment in GF RP compared to serum. The concentration of TGF-fil in aliquots
of GFRP and serum from the same dog was compared using a commercial sandwich
enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s
instructions (MBIOOB, R&D Systems, Minneapolis, MN). Sample dilutions were

determined empirically.

DAPI and Rhodamine Phalloiden Staining

To compare cell density on the scaffolds 24 hours post-cell seeding, cells on the
scaffolds (n=3/group) were fixed in 10% neutral-buffered formalin for 20 minutes. The
cells were rinsed with PBS and then incubated in 10% sucrose for 15 minutes. After a
second rinsing with PBS, cellular actin filaments were stained with rhodamine phalloidin
(50 U/mL) (Invitrogen, Carlsbad, CA) for 20 minutes. Scaffolds were mounted cells-up
on a glass slide following an additional PBS rinse. Vecta-mount with DAPI (4’,6-

diamidino-2-phenylindole) (Vector, Burligame, CA) was added to counter-stain the

102

nuclei of cells on each scaffold. Scaffolds were photographed through a #1 coverslip on

an inverted microscope (Zeiss, Oberkochen, Germany).

MTT Assay

Tendon cell proliferation and viability throughout the scaffolds was compared
using the MTT (3-[4,S-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
colorimetric assay.(29) After 48 and 72 hours, scaffolds (n=5/group/time point) were
transferred to a 24-well tissue culture plate prior to the assay. DMEM without phenol red
and containing 0.5 mg/mL MTT (Sigma-Aldrich, St Louis, MO), and 2% FBS was added

to each well, and the plates were incubated at 37°C in a humidified atmosphere

containing 10% C02 for 4 hours. Next, the MTT solution was discarded and acidic

isopropanol containing 1% Triton-X 100 was added to each well. After 40 minutes of
mixing on an orbital shaker, aliquots from each well were transferred to a 96-well plate in
triplicate and then read immediately at 570 nm in a scanning multiwell spectrophotometer

(Bio-Tek Instraments, Winooski, Vermont).

Histological Evaluation

After 14, 21, and 28 days in culture, scaffolds (n=4/group/time point) were
processed for histology to compare tissue neogenesis on the scaffolds and cellular
invasion into the scaffolds. All samples were fixed in 10% neutral-buffered formalin for
at least 3 days. Samples were transected in half, processed for routine histology, and cut
at 6-um increments in cross-section. The sections were stained with hematoxylin and

eosin (H&E) or picrosirius red (PSR).

103

Cross-sectional tissue thickness on the surface of the scaffolds was measured
using Scion image software (Scion corp., Frederick, MD) at three separate locations from
representative photomicrographs of H&E-stained sections from all of the scaffolds. All

images were obtained with the 20x objective using a Zeiss Axioscope.

Scanning Electron Microscopy

To examine the morphology of GFRP-enriched scaffolds prior to cell-seeding, a
representative sample was obtained and processed for scanning electron microscopy
(SEM). Additionally, after 14, 21, and 28 days in culture, scaffolds (n=2/group/time
point) were processed for SEM. All SEM samples were fixed in 4% glutaraldehyde
buffered with 0.1M phosphate buffer (pH 7.4) for 36 hours at 4°C. After rinsing in the
buffer, samples were dehydrated in a graduated ethanol series. Samples were then dried
in a critical point drier (Balzers CPD, Lichtenstein), mounted, and osmium coated (Pure
Osmium Coater, Neoc-An, Meiwa Shoji Co., Japan). Prepared samples were examined

using a Jeol 6400V scanning electron microscope.

Hydroxyproline and Dimethyl-Methylene Blue Assay

Hydroxyproline and dimethyl-methylene blue assays were performed to compare
the total collagen and glycosaminoglycan (GAG) content respectively amongst the
scaffolds. The samples were digested with Proteinase K at 60°C and aliquots were taken
for the hydroxyproline assay or dimethyl-methylene blue assay. To determine total
collagen, the Proteinase K digest was hydrolyzed by heating with an equal volume of

12M HCI at 107°C for 18 hours, dried, and the hydrolysates were assayed for

104

hydroxyproline using a colorimetric procedure.(30) Total sulfated-GAG content was

determined using the dye 1,9-dimethyl methylene blue.(3 1)

Statistical Analysis

MTT optical density values, surface tissue thickness, total collagen, and total
GAG content were each analyzed with a one-way ANOVA within each time point
followed by a Tukey’s post hoc test. All data are displayed as _mean :1: standard error (SE).

The threshold for statistical significance was set to p < 0.05 for all comparisons.

Results
Growth F actor-Rich Plasma
The concentration of the sentinel growth factor (TGF-Bl) in the GFRP
preparation (315 ng/mL) used in the study was approximately 4.7-fold greater than the

concentration of TGF-Bl in serum (67 ng/mL).

Cell Proliferation

Histologic examination of the scaffolds at 24 hours after cell-seeding using DAPI
and rhodamine phalloidin staining demonstrated an obvious increase in cell density on
the GFRP-enriched scaffolds compared to the other two groups (Figure 4.3). In the
control and serum groups, cells appeared to be orientated along the axes of the polymer
fibers (Figure 4.3A and B) while cells in the GFRP group appeared less restricted and

were spread out over the thin fibrin matrix coating (Figure 4.3C).

105

 

Figure 4.3 Representative photomicrographs of the cell-seeded surface of a control (A)
serum—enriched (B) and GFRP-enriched (C) scaffold stained rhodamine phalloidin and
DAPI after 24 hours in culture. Note the dramatic increase in cell density of the GFRP-
enriched scaffold compared to the other groups.

The results of the MTT assays performed on the scaffolds after 48 and 72 hours in
culture are shown in Figure 4.4. Optical density values indicating relative cell
proliferation and viability were significantly higher for the GFRP-enriched scaffolds
compared to serum-enriched scaffolds (p 5 0.029) and controls (p < 0.0001) at both time
points examined. At 72 hours the GFRP—enriched scaffolds had a 7.8-fold higher optical
density value compared to the controls. Serum-enriched scaffolds also induced a

significant (p 5 0.013) increase in cell proliferation compared to controls at both time

points.

106

p<0.0001

    

0.14 -
El Control p<0.0001

E 0.12 - EISerum
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48 hr 72 hr

Figure 4.4 Results of the MTT assays performed on the scaffolds after 48 and 72 hours
in culture. Optical density values indicate relative cell proliferation. Bars represent mean
optical density value :1: SE (n = 5).
Tissue Morphology and Morphometry

Tissue neogenesis appeared most abundant on the surface of the GFRP-enriched
scaffolds compared to the other groups at each time point examined (Figure 4.5). Figure
4.6 shows a comparison of the scaffolds in each group after 21 days in culture with H&E
and picrosirius red (PSR) staining and SEM. Histological evaluation of the H&E-stained
scaffolds showed that cells were able to invade into the scaffolds equally well in all three
groups as cells were noted amongst the polymer fibers throughout the depths of the
scaffolds in each group. Examination of sections stained with picrosirius red revealed that
a large proportion of the extracellular matrix on the surface of the scaffolds was

composed of collagen. Analysis of the scaffolds with SEM confirmed the observations

found with histology.

107

Control Serum GFRP

 

028

    

. i _ _
54w 3;!»

Figure 4.5 Photomicrogr phs of representative transected scaffolds stained with
hematoxylin and eosin (H&E) from each group after 14, 21, and 28 days in culture. Note
the relative differences in the surface tissue thickness at each time point. Scale bar = 100

pm.

108

Control Serum GFRP

 

Figure 4.6 Photomicrographs of representative transected scaffolds from each group
after 21 days in culture. Tissue neogenesis on the surface of the scaffolds appeared most
abundant on the GFRP-enriched scaffolds. Hematoxylin & Eosin (H&E) -staining (top
row) showed that cells were able to invade into the scaffold equally well in all three
groups. Picrosirius red (PSR) -staining (middle row) suggested that the majority of tissue
generated was collagen. The bottom row shows scanning electron micrographs of the
scaffolds in each group. Scale bar = 100 um.

Surface tissue thickness of the GFRP-enriched scaffolds was significantly greater
than controls (p 5 0.038) at all three time points and significantly greater than serum-
enriched scaffolds (p < 0.0001) at days 14 and 21 (Figure 4.7). Surface tissue thickness of

serum-enriched scaffolds was significantly greater (p 5 0.017) than controls at day 14.

109

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D14 D28

Figure 4.7 Cross-sectional tissue thickness measurements on the surface of the scaffolds
after 14, 21, and 28 days in culture. Bars represent the mean surface tissue thickness 3: SE
(n = 4). Statistical significance was not reached (p Z 0.05) where p-values for
comparisons are not shown.
Total Collagen and GA Gs

The total amount of collagen in each group increased with time (Figure 4.8A).
Both the serum-enriched and GFRP-enriched scaffolds had significantly (p S 0.026)
greater amount of total collagen than the control scaffolds at each time point. Collagen
content was significantly greater (p < 0.0001) on the serum-enriched scaffolds compared
to the GFRP-enriched scaffolds at days 14 and 21. However, the GFRP-enriched
scaffolds had a significantly greater (p < 0.0001) amount of collagen than the serum-
enriched scaffolds at day 28.

The total sulfated-glycosaminoglycan (S-GAG) content remained unchanged with

time (Figure 4.88). At the day 14 time point, both the serum-enriched and GFRP-

enriched scaffolds contained significantly more (p < 0.0001) S-GAGS than the controls,

110

and the serum-enriched scaffolds contained significantly more (p < 0.0001) S—GAGs than
the GFRP-enriched scaffolds. There were no statistically significant differences at the
other two times points examined.

CI Control El Serum I GFRP e<0.0001

 

 

 

 

 

 

 

 

3 12° p<0.0001
FE» 3 E<°-°°°1
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D14 D21 028
Figure 4.8 Total collagen (A) and GAG (B) deposition by tendon cells seeded onto plain
scaffolds (control), serum-enriched scaffolds, or GFRP-enriched scaffolds after 14, 21,
and 28 days in culture. Bars represent the mean total collagen (A) or GAG (B) content
among the scaffolds : SE (n = 4). Statistical significance was not reached (1) Z 0.05)
where p-values for comparisons are not shown.

111

Discussion

Combining growth factors known to stimulate tissue repair and regeneration with
scaffolds has the potential to significantly enhance host incorporation of scaffolds and
more efficiently regenerate tissues.(3) The results of the current study suggests that
GFRP-enriched scaffolds significantly enhanced early cell proliferation and collagen
matrix synthesis on a synthetic scaffold when compared to serum-enriched scaffolds and
scaffolds alone. However, GFRP failed to consistently enhance GAGS in the matrix over
time when compared to serum-enriched scaffolds.

The creation of GF RP from platelet-rich plasma by the addition of a freeze-thaw
step was done to ensure total platelet activation and thus release the maximum amount of
growth factors available from the platelets.(27) This produced a GFRP solution that
contained a 4.7-fold increase in the concentration of a sentinel growth factor (TGF-Bl)
compared to autologous serum. The rationale for using only a single growth factor to
document the increase in concentration achieved by the creation of the GFRP is based on
the significant association that has been demonstrated between platelets and TGF-Bl
concentrations.(32, 33) Similar results have been reported regarding the ability to
concentrate TGF-[31(10, 17, 33-35) in addition to other growth factors such as platelet-
derived growth factor (PDGF)(17, 33, 34, 36) and vascular endothelial grth factor
(VEGF)(34, 36) in various PRP preparations compared to other blood-derived
components. While the concentration of specific mitogenic growth factors (e.g., PDGF,
insulin-like growth factor (IGF) -1, and basic fibroblast grth factor (bFGF)) normally
stored in platelet (it-granules were not measured in the current study, the increase in cell

proliferation observed as a result of the addition of GFRP indirectly suggests that these

112

mitogenic factors might also be increased. Thus, it is likely that, similar to platelet-rich
plasma (PRP), GFRP provides the entire compliment of growth factors present in serum
but in significantly higher concentrations.(6)

Several investigators have examined the ability of individual growth factors or
various combinations of growth factors to increase tendon cell proliferation and matrix
synthesis in a dose-dependent manner in vitro.(3 7-40) The results of the current study are
consistent with other investigators who examined the ability of PRP preparations to
increase tendon cell proliferation and matrix synthesis over time.(34, 36, 41) Most
importantly, PRP not only provides an increased concentration of several grth factors,
it also maintains the normal relative proportions of individual grth factors to each
other.(6) Maintaining the normal proportional relationship between grth factors is
thought to be important in encouraging tissue regeneration over scar formation.(25, 26)

The ability of GFRP-enriched scaffolds to significantly increase cell density
within 24 hours of cell-seeding could be related to an increase in chemotactic growth
factor concentration as well as the creation of an optimal surface for cell attachment by
way of a fibrin coating on the polymer surface. As a result of combining GFRP with
media containing calcium, the clotting cascade was initiated creating a thin fibrin matrix
coating throughout the scaffolds. Fibrin has been shown to be an excellent provisional
scaffold in a variety of applications providing a naturally-derived matrix for cell
adhesion/attachment and migration.(42) Several elegant studies have demonstrated that
fibrin coating of synthetic substrates resulted in an increase in initial cell adherence
which, in turn, led to an increase in cellular proliferation.(43-46) Therefore, it is possible

that the fibrin coating of the polymeric scaffold created by the plasma component of the

113

GFRP enhanced initial cell adherence and thus contributed to the increased cellular
proliferation observed in this study.

While fibrin provides an excellent conductive surface for cells, fibrin itself (in the
absence of growth factors) does not induce cell proliferation and promote cell
viability.(47, 48) Additionally, the concentration of adhesion proteins such as fibronectin
is not increased in fresh frozen plasma when compared to serum.(49) Therefore, the
significant increase in cell and tissue proliferation seen in the GFRP group in the current
study may not be a result of the fibrin coating alone but rather the combination of
increase initial cell adhesion as well as the increased concentration of mitogenic grth
factors present in the fibrin scaffold.(33, 34, 38, 40) Fibrin has been Shown to indirectly
bind growth factors, potentially creating a growth factor reservoir and prolonging their
biological activity.(50) While the potential elution of growth factors from the precipitated
fibrin matrix was not evaluated in the currently study, it is possible that a sustained
release of mitogenic agents could occur. Additional studies are needed to evaluate this
possibility.

In conclusion, the results of this study suggest that growth factor-rich plasma can
enhance the bioactivity of a synthetic scaffold in vitro. The increase in grth factor
concentration of GFRP over normal serum levels as well as the fibrin coating of the
polymeric surfaces created by the interaction of the GF RP and the calcium within the
culture media contributed to enhanced cell adhesion and proliferation as well as enhanced
matrix synthesis on the surface of a bio-inert scaffold when compared to serum-enriched
scaffolds and controls. While GFRP was able to Significantly increase cell proliferation

and collagen matrix deposition over serum-enriched controls, the in vivo benefit of this

114

effect has yet to be proven. Additional studies are needed to determine the ability of
GFRP to accelerate in viva host incorporation and remodeling of tissue-engineered

scaffolds.

115

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119

CHAPTER 5.

Concluding Discussion

120

Concluding Discussion

The goal of this thesis was to investigate the role of an autologous platelet-rich
fibrin matrix (PRFM) in enhancing growth factor delivery for connective tissue healing.
Combining platelets and their associated growth factors with a fibrin scaffold represents a
promising method to enhance growth factor delivery for tissue repair and tissue
engineering applications. The work presented in this thesis supports the hypothesis that a
PRF M will enhance growth factor delivery for connective tissue repair and regeneration.
However, as evident in chapter 3, the benefit of a PRFM may be situation-dependent and
be of greater benefit in chronically injured or biologically compromised tissues. I

The results of the study 1 presented in chapter 2 of this thesis demonstrated that
two different formulations of the PRFM (PRFMatrix and PRFMembrane) were able to
elute a significantly greater concentration of a sentinel grth factor (TGF-Bl) over time
when compared to whole blood clots of similar volume. The eluents from the platelet-rich
fibrin constructs were also able to significantly increase tendon cell proliferation over
time when compared to whole blood clots of similar volume. This sustained increase in
growth factor elution from the PRFM in vitro supports the idea that a PRFM may be
beneficial for connective tissue repair in viva, particularly in tissues where a prolonged
increase in increased concentrations of growth factors is desired.

Study 2 presented in chapter 3 examined the use of a PRFM, specifically the
PRFMembrane, in viva. The PRFMembrane did not enhance the rate and quality of
healing in central-third patellar tendon defects at either 4 or 8 weeks post-operation.
While the PRFMembrane produced an increased amount of fibroblastic repair tissue, the

quality of this tissue was not significantly different from the naturally occurring repair

121

tissue when assessed histologically using a semiquantitative scoring system. The results
of study 2 suggest that a PRFMembrane may be of more benefit in larger defects where a
naturally occurring provisional fibrin scaffold may not adequately fill the defect or in
biologically compromised tissues where the prolonged release of growth factors may be
required to induce and sustain a repair response from adjacent tissues.

In chapter 4 a bioactive scaffold using PRFM-related technology, termed growth
factor-rich plasma (GFRP), for tissue engineering applications was created. The GFRP-
enriched scaffolds significantly enhance early cell proliferation and the deposition of a
collagen-rich extracellular matrix over time compared to serum-enriched scaffolds and
controls. The results of this study indicate that a PRFM-related preparation (i.e., GFRP)
can be combined with a synthetic scaffold to create a bioactive scaffold. This scaffold can
potentially be used for tissue engineering applications to enhance cell proliferation and

matrix synthesis in vitro and optimize scaffold incorporation in viva.

Future Directions
A number of important questions remain about the basic biologic mechanisms of
the PRP M and represent potential areas of future study:
0 What are the diffusion kinetics of other growth factors (in addition to TGF-Bl)
eluted from PRFMS?
o Are viable (unactivated platelets) trapped within the PRFM following the current
preparation methods? Is the prolonged growth factor availability a result of

sequential degranulation of viable platelets over their 5-10 day half-life or merely

122

the result of prolonged diffusion kinetics from the increased fibrin density of a
PRFM?

o What are the optimal platelet and growth factor concentrations in a PRF M? Are
more growth factors necessarily better?

0 Given that the fibrin scaffold may act as a growth factor reservoir, how important
is the contribution of the fibrin scaffold to the proposed benefits of the PRFM?

0 Would the PRFM be of greater benefit in biologically compromised tissues, such
as in chronic tendinopathies or desmopathies or patients with endocrinopathies
e.g., Cushingoid or diabetic patients with delayed wound healing

0 When is the best time to intervene with a PRFM following an injury?

Although autologous PRFM constructs appear promising, further in vitro
characterization and experimental studies are necessary as the study of these constructs
has merely just begun. Moreover, just as with platelet-rich plasma, well-control
prospective randomized clinical trials under rigorous scientific scrutiny are necessary

prior to the wide-spread clinical use of these products.

123

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