Growth factors and tendon healing

Francesco Oliva,1Stefano Gatti2, Giuseppe Porcellini 3, Nicholas R. Forsyth4, Nicola Maffulli 5

1 Department of Orthopaedics and Traumatology, University of Rome "Tor Vergata" School of Medicine, Viale Oxford 81, Rome, Italy.
2 Department of Orthopaedics and Traumatology, University of Siena School of Medicine, Strada Delle Scotte, 53100 Siena, Italy.
3 Operative Unit of Shoulder and Elbow Surgery, “D. Cervesi Hospital”, Via L. Van Beethoven 46 Cattolica, Rimini, Italy.
4 The Guy Hilton Research Laboratories, Keele University Medical School, Stoke on Trent, ST4 7QB, UK
5 Centre for Sports and Exercise Medicine Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Mile End Hospital, 275 Bancroft Road, London E1 4DG, UK.

Correspondence:
Nicola Maffulli, MD, MS, PhD, FRCS(Orth) Centre for Sports and Exercise Medicine Queen Mary University of London Barts and The London School of Medicine and Dentistry Mile End Hospital 275 Bancroft Road London E1 4DG, UK.
Tel: + 44 20 8223 8839
Fax: + 44 20 8223 8930
n.maffulli@qmul.ac.uk


Abstract

Recent attention has focused on the biological pathways by which tendons heal leading to the identification of some growth factors (GFs) with involvement in this process. No studies have been published on the time course of the various GFs during tendon healing process in vivo, in humans. We review what is known about these GFs and their role in tendon healing.

Keywords:
Growth factors, tendon healing, tendinopathy, review


Introduction

The basic cell biology of tendons is still not fully understood, and the management of tendon injury is still challenging. Tendon injuries can be acute or chronic, and are caused by intrinsic or extrinsic factors, either alone or in combination. In acute trauma, extrinsic factors predominate. Overuse injuries and chronic tendon disorders generally have a multifactorial origin.1 The pathologic label 'tendinosis' has been in use for more than two decades to describe collagen degeneration in tendinopathy, which is only one of the features of tendinopathy.2

Growth factors are synthesized and secreted by a wide variety of inflammatory cells, platelets, fibroblasts, epithelial cells, and vascular endothelial cells. In a very specific fashion, they bind to external receptors on the cell membrane, which leads to intracellular changes in DNA synthesis and expression. In this manner, growth factors directly affect cellular mitogenesis and chemotaxis and are able to influence the healing cascade. Large-scale clinical trials have not been successful, possibly through inadequate delivery strategies failing to meet the physiological requirements of the tissue repair processes. These requirements are likely to comprise multiple growth factors over a specific temporal pattern at an optimized ratio to meet the specific needs of the physiological process involved in tissue healing3,4 through processes which remain to be clarified.5

The growth factor requirements of tendon cells have been investigated for over three decades. Initial experiments focussed on the actions of epidermal growth factor (EGF), insulin, and PDGF in in vitro condition.6,7 Platelet-rich plasma (PRP), developed in the late 1990’s,8 a means to concentrate endogenous growth factors, is now used in North America9 and Continental Europe.10,11

Tendon repair
Tendon repair can occur either intrinsically via the resident tenocytes12 or via extrinsic mechanisms, whereby cells from the surrounding sheath or synovium invade the tissue.13 Three biologically and temporally overlapping phases are described during tendon repair.14 The healing process starts with:
1) The inflammatory phase: haematoma, platelet activation and invasion of cells that form a granuloma. Usually, this phase occurs three to seven days after the injury, cells migrate from the extrinsic peritendinous tissue such as the tendon sheath, periosteum, subcutaneous tissue, and fascicles, as well as from the epitenon and endotenon.15
2) The formative phase, when cells proliferate and differentiate, continues for eight weeks after the initial injury.16 The migrated fibroblasts in the granuloma produce collagen (mostly collagen type III), which gradually increases its mechanical strength, so that loading can lead to elastic deformation, which allows mechanical signalling to start to influence the process. Production of collagen type I gradually takes over, and the repair callus reaches its largest size. The large transverse area compensates for tissue weakness, so that considerable traction forces can be sustained. Tenocytes become the main cell type, and collagen is continuously synthesised over the next five weeks.
3) Finally, in the remodelling phase, collagen type III is reabsorbed and replaced to produce better organisation, and cross-linking increases. Finally, the callus transverse area gradually decreases as the mechanical tissue properties improve. Despite intensive remodelling over the following months, complete regeneration of the tendon is never achieved. The tissue replacing the defect remains hypercellular. The diameter of the collagen fibrils is altered, favouring thinner fibrils with reduction in the biomechanical strength of the tendon.17

No studies have been published on the time of appearance of GF in the three different phases of the tendon healing process in humans. In a rabbit model, during healing of acute midsubstance rotator cuff tears, Kobayashi et al. assessed semiquantitatively the time expression of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) 1, platelet-derived growth factor (PDGF), and transforming growth factor (TGF-β) for 28 days. IGF-1 and TGF-β appear first in the blood cells in the inflammation phase; bFGF and PDGF appear later during the formative phase. TGF-β was present for all the phases of the healing process, but the distal stump of the tear was lacking of these GF.18

Recently, Würgler-Hauri et al.19 studied bFGF, BMP-12, BMP-13, BMP-14, cartilage oligomeric matrix protein (COMP), connective tissue growth factor (CTGF), PDGF-B, and TGF-β1 in tendon-to-bone healing in a rat supraspinatus model for 16 weeks. Immunoassays showed an increase in the expression of all growth factors at one week, followed by a return to control or undetectable levels by 16 weeks in both the insertion and midsubstance. Of additional note in the insertion, COMP peaked at one week, followed by a decrease in expression at two weeks. BMP-12 and CTGF were moderately expressed across all time points. Furthermore, BMP-12 and PDGF-B were moderately expressed over time in the midsubstance.19

Growth factors in tendon healing
Numerous growth factors are involved in tendon repair. These include BMPs, EGF, FGF1, FGF2, IGF-I, IGF-II, PDGF-AA, PDGF-BB, PDGF-AB, TGF-β. These may be produced locally by cells in areas of injury, growth and repair, or may be delivered by blood.
Basic fibroblastic growth factor

bFGF is a single chain polypeptide of 146 amino acids, and is a member of the part of heparin-binding growth factor family. In humans, 22 members have been identified all of which are structurally related signaling molecules. It is angiogenic20 and has mitogenic effects on many mesenchymal cells such as ligament fibroblasts.21

This growth factor has been shown to be involved in wound healing. Specifically, Chan et al.22 showed that, in vitro, supplementation of bFGF increases the proliferation of rat patellar tendon fibroblasts. Chang et al. showed that bFGF mRNA is upregulated in the tendon wound environment, and upregulated in tenocytes as well as in tendon sheath fibroblasts and inflammatory cells in vivo.23

bFGF has also a stimulatory effect on human rotator cuff tendon cells in vitro, but suppresses collagen synthesis.24 Kobayashi et al. show an immunohistochemical peak expression of bFGF in the first week in a midsubstance injury of supraspinatus tendon in the rabbit, and it was suggested that bFGF could be used to promote the healing process of a torn rotator cuff tendon.25 In a rabbit supraspinatus tendon bone injury, Würgler-Hauri et al. supported this finding and, in addition, they detected another increase of bFGF at eight weeks.19

Bone morphogenetic proteins
BMPs are a group of factors of the TGF-β superfamily that stimulate bone formation but also stimulate tendon cell mitogenesis and tendon healing. Although it is clear that BMPs stimulate tendon healing the mechanism remains unclear.26,27 A combination of BMP signalling and influences of mechanical loading are likely crucial for tendon healing. This is reinforced by the BMP-14 knockout mice studies where a delayed tendon healing response and irregular Type I collagen fibrils were observed.27 However, in rat Achilles tendons injury models, BMP-13, not BMP-14, appeared to be involved in early tendon healing, BMP-14 was primarily required for the maintenance of homeostasis of mature tendons, and BMP-12 was required for both.28 BMP-12, BMP-13 and BMP14 have all been detected in intact human tendons by immunohistochemistry.29,30

Ovoid tendon cells (tenoblasts) in patellar tendons display elevated levels of both BMP-12 and BMP-13 when compared to the elongated tendon cells (tenocytes).30 This suggests that tenoblasts are more active in matrix remodelling and healing than tenocytes. In addition, BMP-2, BMP-7, and BMP-12 all participate in tendon-bone healing and improve formation of new bone and fibrocartilage at the healing tendon attachment site, resulting in an improved load to failure.31
Recently, gene therapy with the BMP-12 cDNA muscle graft showed histologically better organized and homogeneous pattern of collagen fibers at all time points than the control groups.32 Recombinant BMP-12 on a sponge carrier also stimulated rotator cuff repair in the sheep model.33 Although BMP-14 did not appear to be involved directly in healing in the Achilles tendon model, BMP-14 gene therapy did increase tendon tensile strength in a rat model of Achilles tendon injury.34

Insulin-like growth factor
Insulin-like growth factor, or IGF, is named after its hypoglycemic effect after intravenous administration. The stimulatory effects of IGF-I have been demonstrated in many cell types, including cartilage, bone, muscle and tendon cells.35 During tendon healing, its role seems to stimulate the proliferation and migration of the tenoblasts during the inflammatory phase, while its increase in the remodelling phase seems to be clear.36 In addition to the mitogenic effect, IGF-I can also stimulate selected components of matrix synthesis, and its expression was noted in avian tenocytes.37 Complementary to this are the observations that IGF-I induced tenocyte migration, division, matrix expression and accelerated functional recovery from Achilles tendon injury in a rat model (38, 39, 40). However, IGF-1 was not observed in tenocytes on day 10 following injury in a canine flexor tendon repair model, but was instead located in the surrounding inflammatory cells.41 Similar observations were noted in studies concerning lesions of the flexor digitorum superficialis tendons of both forelimbs.42 Similar to the canine model, IGF-1 levels had decreased by approximately 40% when compared to normal tendon at two weeks. Continued analysis then revealed a substantial upregulation of IGF-1, exceeding normal tendons. By four weeks, IGF-1 levels had increased substantially and were maintained through to eight weeks. IGF-I also improved aspects of healing in an equine model of collagenase-induced flexor tendinopathy.43

The expression of the insulin-like growth factor 1 binding proteins (IGFBPs) has been studied in equine flexor tendons after acute injury and during healing over time: mRNA and protein expression for IGFBP-2, -3, and -4 was detected in normal tendon, and showed a marked increase following injury.44

Platelet-derived growth factor
PDGF was first isolated from platelets, but can be produced by many different cells, including smooth muscle cells.45 PDGF is a basic protein of approximately 30 kD formed by two subunits (α and β chain) that exist in three isoforms. Most studies are focussed on the homodimer PDGF-BB isoform, which has stimulatory effects on both cell division and matrix synthesis. An investigation into the consequences of administration of PDGF-BB directly into the wound gap of rat patellar tendons showed that early supplementation (day three post-injury) was not beneficial to restoration of mechanical properties,46 probably from an increase in cell proliferation without matrix production. Later supplementation with PDGF-BB (on day seven post-injury) did stimulate matrix production, with an accompanying higher peak load and pyridinoline content. PDGF-BB stimulated matrix and DNA synthesis in a dose-dependent manner in intrasynovial intermediate and proximal segments of deep flexor tendons, and extrasynovial peroneal tendons of rabbits during short-term cultures. PDGF-BB stimulated collagen synthesis and noncollagen protein synthesis in proximal intrasynovial tendon segments more than in extrasynovial peroneal tendon segments, and DNA synthesis less in proximal than in intermediate intrasynovial tendons.47

Exogenous PDGF genes can be transferred effectively into intrasynovial tenocytes. The transfer increases significantly the expression of genes for PDGF and type I collagen.48
PDGF holds particular promise in combination with other growth factors. Tendon cells express the receptor for PDGF, but do not normally express PDGF itself.50 When applied with IGF-1 TGF-β and bFGF, robust stimulation of tendon fibroblast migration and cell division are produced.38, 39, 49, 50-53 In a tissue engineering study, PDGF-BB transduced cells stimulated adjacent rat tendon fibroblasts to increase collagen synthesis by 300% at 24 hours compared to a 28% increase in IGF-I transduced cells.54

In a study of intrasynovial canine tendon, PDGF-BB and bFGF significantly increased flexor tendon fibroblast proliferation, collagen production and matrix synthesis when each was applied on its own.55 In a study on the expression of growth factors in normal canine flexor tendon healing, PDGF-AA, PDGF-BB and VEGF appeared in the whole tendon section at 10 days following tendon injury.41 Mechanical stretching of tendon fibroblasts also promoted increased concentrations of TGF-β, PDGF and bFGF, suggesting that cyclical mechanical stretching may have a positive influence on tendon and ligament healing through stimulation of cell proliferation, differentiation and matrix formation.50 PDGF in association with hypoxia exerts a synergistic effect which increases the expression of VEGF in Achilles tendon fibroblasts.56 In another study, PDGF gene therapy was more beneficial to tendon healing than VEGF gene therapy in an in vitro study of rat intrasynovial tendons.57

Transforming growth factor β
Originally, TGF-β was thought to be related to cellular transformation events prior to neoplastic growth. It is now clear that TGF-β has numerous physiological effects.58,59 The expression of TGF-β appears closely tied to the expression of a differentiated phenotype in many cell lines including the mesenchymal precursor. Tendon and ligament formation has been tied directly to factors belonging to the TGF-β superfamily.60 Proliferation, matrix synthesis and differentiation have also been affected in tenoblasts, chondroblasts and osteoblasts.61 Whether this is an inhibitory or stimulatory effect depends on the stage of differentiation, presence of other growth factors, and assay system used.62, 63 TGF-β is a weak stimulator of tendon cell migration and mitogenesis, but can stimulate robust expression of extracellular matrix.64 TGF-β affects gene expression primarily through the activation of the Smad signaling pathway. The first step in the Smad pathway is the expression of TGF-β inducible early gene (TIEG). Healing of tendons in the TIEG knockout mouse suggests the possibility of tendon healing in the absence of the Smad pathway, and the existence of TIEG independent routes.65 TGF-β1 significantly increased the amount of SMA (alpha-smooth muscle actin) in nonvascular cells in seven human rotator cuffs, suggesting that SMA-containing cells could contribute to the retraction of the torn ends of a ruptured rotator cuff and play an important role in healing.66

In a canine flexor tendon injury repair model, TGF-β was detected at and proximal to the repair site.41 Complementary to these findings was the observation that in Achilles rat tendon healing models the failure load and stiffness of the healing tendon were increased by administration of TGF-β1 at two and four weeks.67 Furthermore, the application of TGF-β1 significantly increased the tangent modulus and the tensile strength of the fibrous tissue produced in the rabbit patellar tendon after resecting the central portion, suggesting a role of TGF-β1 in in vivo tendon regeneration.68

TGF-β may control the switching point in the healing process from normal to pathological.69 All three TGF-β isoforms significantly increase collagen I and III production in cultured tendon fibroblasts.70 TGF-β1 induced a greater degree of contraction in tendon fibroblasts cultured in collagen gels as compared with TGF-β3.71 This might explain the finding that TGF-β1 induces scar tissue formation, whereas TGF-β3 reduces it.72 Intraoperative infiltration of neutralizing antibody to TGF-β1 improves flexor tendon excursion, but simultaneous infiltration of neutralizing antibody to TGF-β2 nullifies this effect. Therefore, TGF-β isoforms may interact with one another to modulate collagen synthesis in healing tendons.73

The temporal and spatial distribution of three TGF-beta receptor isoforms (RI, RII, and RIII) was analysed in a rabbit zone II flexor tendon wound healing model. This demonstrated that TGF-beta receptors were up-regulated after injury and during repair.74 Receptor production was concentrated in the epitenon, tendon sheath and along the repair site. Peak levels of TGF-beta receptor expression where noted on day 14 and persisted until day 56.

Formation of nitric oxide is an important event in the course of tendon healing, and its inhibition results in chronic inflammation and fibrosis due to an imbalance in TGF-β expression in vivo.75 Examining the expression of inducible nitric oxide synthase (iNOS) and TGF-β in macrophage infiltrates within crush-injured digital flexor tendon and synovium of rats during normal tendon healing, the levels of TGF-β are high at first, and gradually decrease after three weeks of injury.75

Vascular endothelial growth factors
These are important signaling proteins involved in both vasculogenesis and angiogenesis. The broad term 'VEGF' covers a number of proteins from two families, that result from alternate splicing of mRNA from a single, 8 exon, VEGF gene. The most important member is VEGF-A, a glycosylated protein of 46–48 kDa composed of two disulphide-linked subunits. Other members are Placenta growth factor (PIGF), VEGF-B, VEGF-C and VEGF-D. The latter ones were discovered later than VEGF-A, and before their discovery VEGF-A was called just VEGF. All members of the VEGF family stimulate cellular responses by binding to tyrosine kinase receptors (the VEGFRs) on the cell surface, causing them to dimerize and become activated through transphosphorylation, although to different sites, times and extents.76 Bidder et al. using in situ hybridisation in a canine model of tendon injury, identified cell populations within the repair site expressing message for VEGF, suggesting their potential for organising the angiogenic response during the early postoperative phase of wound tendon healing.77 Recently, Pufe et al. demonstrated that VEGF concentrations are negligible in healthy human adult Achilles tendons, but high in ruptured and embryonic ones.

Discussion
In vitro cell-based studies and animal models have clarified the role of single and group of GFs administered to enhance cell proliferation and chemotaxis,78 aid angiogenesis,79 and influence cell differentiation during wound healing.80 Few studies have been conducted to delineate the timing of GF expression during the various phases of the healing process in tendons.19,81 Some authors highlight the necessity to understand the timing of administration of growth factors and their dosage for designing effective growth factor therapy. 5,82 It is necessary to understand the overall reactions which growth factors promote on the various cells involved in tendon healing, considering not only the timing of expression of GFs, but also their up- and down-regulation from the time of injury.83 Some studies considered a single GF during biological tendon healing in relation with cell proliferation and cellularity, without considering others GFs.84 Other studies have characterized the effect of GFs only in vitro,71 while others have characterised the timing of GF expression.85 Further investigations should be conducted to advance our knowledge on expression of GFs during healing before administration of single or multiple GFs in tendon healing trials.

The time course of GF expression is important in wound healing, and a better understanding of how, where, when and for how long such factors are expressed may help in the development of methods to manipulate their expression, accelerate healing, and reduce adhesions. The use of growth factors in soft tissue problems remains, for the time being, largely experimental.

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