* Cellular and Molecular Factors Influencing Tendon Repair

Abstract

Tendons are complex connective tissues that transmit tensile forces between muscles and tendons. Tendon injuries are among the most common orthopedic problems with long-term disability as a frequent consequence due to prolonged healing time. Furthermore, the repair tissue is of inferior quality, predisposing patients to high rates of recurrence following initial injury. Coordinated cellular processes and biological factors under the influence of mechanical loading are involved in tendon healing and our understanding of these events lags behind other musculoskeletal tissues. Tendons are relatively hypocellular and hypovascular, with little or no intrinsic regenerative capacity. Studies have documented fatty degeneration, chondrogenic dysplasia, and ectopic ossification within tendon repair tissue. The underlying pathogenesis for these metaplastic changes that compromise the quality of tendon repair tissue is poorly understood. The purpose of this review is to compile literature reporting molecular processes that regulate/control the phenotype of cells responsible for abnormal matrix deposition at repair site. In addition, recent studies reporting the interplay of mechanotransduction and cellular responses during tendon repair are summarized. Identifying the links between cellular, biological, and mechanical parameters involved in tendon repair is paramount to develop successful therapies for tendon healing.

Introduction

Tendons are dense collagenous tissues that connect muscles to bones and transmit tensile forces from contracting muscles to the skeleton. Tendons consist primarily of collagen fibers (65–80%) oriented in the direction of tensile load. Proteoglycans and glycoproteins combine to form a ground substance, accounting for 1–2% of the dry mass of the tendon.¹ The ground substance surrounds the collagen fibers and plays an important role in cellular interactions and collagen fibrillogenesis.² The cellular fraction of tendons is very small (<1%), but is responsible for the synthesis and turnover of extracellular matrix (ECM), which forms the bulk of the tissue. In addition to mature tenocytes, multipotent tendon progenitor cells (TPCs) reside within tendon tissues, and their activities are regulated by the noncollagenous ECM components.³

Tendinitis/tendinopathy is a common and debilitating injury with a prevalence rate of 15–55% in human and equine athletes.^4,5 Primary healing is prolonged and recurrence rates as high as 30% have been reported.^6,7 Furthermore, tendons have limited reparative/regenerative capacity and the healing process is predominantly scar mediated. Disorganized collagen fiber arrangement, increased noncollagenous ground substance and increased number and rounded morphology of the tenocytes, fatty deposits, and ectopic ossification are recognized features of injured tendon.⁸ Also, during the healing process, the content of large proteoglycans and water in the tendon matrix increases. Collectively, the tissue replaced at the site of injury has decreased elasticity and tensile strength.^6–8 In the past decade, tendon healing research has characterized cytokines and growth factors active during tendon repair, yet our current understanding of the mechanisms controlling tendinopathy pathogenesis and repair is limited. This knowledge will be necessary to design/develop successful therapies for tendon healing.

First, the cellular and biochemical processes and biological factors that are active during endogenous tendon repair. Secondly, this review compiles available information on the influence of mechanobiology on tendon degeneration and healing, and the biological processes that compromise the quality of repair tissue. The objective of this review is to highlight literature reporting aberrant cellular and molecular mechanisms that operate during tendon healing and could be potentially modulated to promote effective healing.

Cellular Responses During Endogenous Tendon Healing

Tendon healing progresses through three sequential and partially overlapping phases: an initial inflammatory stage, followed by a proliferative phase, and then a prolonged remodeling phase. Soon after injury, circulating inflammatory cells, especially monocyte/macrophages, are recruited to the site of injury. Macrophages are classified as M1, classically activated, or M2, alternatively activated. M1 macrophages promote inflammation and ECM deposition and drive fibrosis, whereas M2 macrophages promote cell proliferation and decrease inflammation.⁹ Macrophages are absent in normal equine tendons; however, during the initial period of injury, M1 macrophages are predominant with subsequent transition to M2 phenotype in the remodeling phase.¹⁰ Recent evidence suggests a strong connection between chronic inflammation, macrophage infiltration during tendon healing, and fibrosis/fatty infiltration, mediated by transforming growth factor β (TGFβ) signaling.¹¹ Macrophage polarization during tendon healing is poorly understood and further research is warranted. Given that inflammation and fibrosis are closely associated, modulating macrophage activities at tendon repair site are promising therapeutic targets.

During the proliferative phase, cellular infiltrates at the repair site are predominated by fibroblast-like cells. These cells are thought to originate from endogenous tenocytes and/or TPCs, or extrinsically from paratenon and epitenon.⁹ In mice, experimental flexor tendon defects bridged with live autografts healed with better gliding function than defects bridged with cell-free autografts.¹² This finding highlights the role of endogenous cells in improving the quality of repair tissue. Recent work by Cadby et al. demonstrated that equine paratenon-derived cells isolated from injured tendon were more proliferative in vitro than endogenous tenocytes within the tendon core.¹³ Understanding the lineage of the proliferating fibroblasts, their nature, and biosynthetic activity at the site of repair are important as they are responsible for the ECM deposited in the repair tissue.

Since Bi et al.³ showed that small leucine-rich proteoglycans fibromodulin and biglycan contribute to the TPC niche, a few studies have attempted to characterize progenitor cells that give rise to tendon cells during healing. Tan et al. used iododeoxyuridine (IdU) label retention to identify in vivo stem cells in healing rat patellar tendon window defects.¹⁴ Label-retaining TPCs were present in greater numbers in the paratenon compared to tendon midsubstance. These cells migrated to and proliferated at the repair site and expressed tendon markers (scleraxis and tenomodulin). Overall, lower numbers of label-retaining TPCs were detected in the midsubstance and paratenon of uninjured contralateral patellar tendons.

More recent lineage-tracing experiments by Dyment et al. in a murine patellar tendon defect model demonstrated that smooth muscle actin 9-positive (SMA9⁺) TPCs originating from the adjacent paratenon migrated toward and proliferated in the tendon midsubstance within the first week following injury.¹⁵ In contrast, there were few SMA9⁺ cells in the contralateral uninjured patellar tendon. In addition, these SMA9⁺ cells differentiated into scleraxis-positive (Scx⁺) cells that formed a collagenous bridge across the defect. This study showed that SMA9⁺ cells constituted an amplifying progenitor population that generated Scx⁺ cells. Both these studies suggest that paratenon is the likely source for proliferating tendon cells at repair sites; however, further studies are required in this regard. Cells at the repair site may also secrete bioactive trophic or chemotactic signals. In support of this statement, migration of endogenous bone marrow-derived mesenchymal stem cells (BM-MSCs) to flexor tendon repair sites has been demonstrated in a murine tendon transection model, while there were no BM-MSCs in uninjured controls.¹⁶

During the final remodeling phase of tendon healing, the collagen structure and tissue strength of the repair tissue improve; however, they do not return to preinjury values. Matrix remodeling is primarily mediated by proteases acting in the extracellular environment. These include matrix metalloproteinases (MMPs) and aggrecanases from the “a disintegrin and metalloproteinase with thrombospondin motifs” (ADAMTS) family. The MMP activity is regulated by tissue inhibitors of metalloproteinase (TIMPs).¹⁷ Gelatinases, MMP-2 and MMP-9; and collagenases, MMP-1 and MMP-13; are involved in tendon metabolism, through their broad proteolytic capacity. Their activity is reversibly inhibited by TIMPs-1 and TIMPs-2. A balance between the activities of MMPs and TIMPs regulates tendon remodeling.¹⁸ Tenocytes exposed to TGFβ1 supplementation in an in vitro model of flexor tendon repair produce MMP-2, which is associated with ECM remodeling.¹⁹ The cellular processes that control collagen remodeling during tendon homeostasis and healing are largely unknown.

Biological Factors and Their Effects in Tendon Healing

The key determinants of tendon healing are collagen production and organization, with subsequent restoration of hierarchical structure. Growth factors are the primary modulators of collagen production. TGFβ, insulin-like growth factors-I and II (IGF-I and II), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) are all upregulated during tendon healing and work synergistically to regulate the repair process.^20,21 These growth factors have also been applied experimentally and clinically to improve and/or accelerate tendon repair.

TGFβ is released from platelets, lymphocytes, macrophages, endothelial cells, and fibroblasts and stimulates chemotaxis, angiogenesis, and transcription of ECM genes.^20,21 Despite its beneficial effects, TGFβ is implicated in fibrous tissue formation and excessive deposition of disorganized collagen. The TGFβ signaling activity, from TGFβ1 in particular, is increased in injured tendon, as is particularly increased in regions of chondrogenic metaplasia and heterotopic ossification.^22,23 Matrix disruption and altered mechanical forces following tendon injury result in supraphysiological levels of TGFβ, causing proinflammatory effects and tenocyte apoptosis.²⁴ Consistent with these activities, attenuation of TGFβ signaling (TGFβ1 and Smad 3) in a tendon transection model reduced scarring and adhesion formation during healing.²⁵ Chondro-degenerative changes in healing tendons are also dependent on TGFβ signaling,^22,26 and is discussed in the following sections. Therefore, modulating TGFβ signaling in injured tendons could result in a more “regenerative” healing process with less scar formation and improved repair tissue compliance.

IGFs are prominent in the early stages of tendon healing.^27,28 The primary effect of IGF on tendon healing is mitogenesis, stimulating fibroblast and tenocyte proliferation at the site of injury.²⁹ Exogenously applied IGF-I stimulated replication, with associated increases in collagen and proteoglycan synthesis in healthy rabbit flexor tendons.³⁰ In an equine collagenase-induced superficial digital flexor (SDF) tendinitis model, intralesional injections of IGF-I increased cell proliferation and collagen synthesis, reduced overall lesion size, and trended toward increased mechanical strength in treated tendons compared to control tendons.³¹ Accepting these outcomes, a direct correlation of increased cellularity and increased or improved matrix synthesis in healing tendons has not been established and the value of IGF-mediated cell proliferation during tendon healing remains unclear.

VEGF regulates angiogenesis by mediating the breakdown of vascular basement membranes, stimulating vasodilatation and increasing vascular permeability, endothelial cell proliferation, and monocyte migration. In tendons, VEGF is expressed in tendon sheath fibroblasts and its expression increases in early healing process.^32,33 Intralesional VEGF injection in a murine model of Achilles tendinopathy significantly increased the tensile strength of healing tendons compared to control tendons during the early stages of healing.³⁴ In addition, VEGF also increased TGFβ1 expression during early stages of tendon repair, indicating both direct and secondary beneficial consequences of VEGF signaling activity.

PDGF acts as a chemoattractant and mitogen for fibroblasts and endothelial cells.²⁰ PDGF may exert some effects through IGF-I, as it upregulates IGF-I and its receptors in target cells.²¹ PDGF administration to in vitro tenocyte cultures stimulates collagen, proteoglycan, and DNA synthesis.³⁵ bFGF stimulates angiogenesis and proliferation of fibroblasts.²⁰ FGF induces fibroblasts to produce collagenase and stimulates proliferation of capillary endothelial cells, which are important for initiation of angiogenesis. Although VEGF, PDGF, and bFGF signaling activities are important during initial stages of tendon repair, there is little information regarding their therapeutic value in modulating tendon healing.

Mechanobiologic Influences on the Quality of Repair During Tendon Healing

Mechanical properties of healing tendons are primarily determined by the collagen architecture and the intrinsic viscoelastic property of collagen.³⁶ Tendon homeostasis and healing are regulated by tensile loading of tendons, thereby affecting the structure and composition of the tissue.³⁷ The number and size of collagen fibrils, and cross-sectional area of murine digital flexor tendons exercised on treadmills increased compared to sedentary mice.³⁸ Change in phenotype and ECM gene expression of endogenous cells and subsequent protein synthesis are few factors associated with anabolic structural and compositional changes in tendon repair sites.³⁷ In support of this, unloading the repair site completely through immobilization decreases the biomechanical strength of healing tendons, largely as a consequence of decreased ECM synthesis.³⁹ Furthermore, rotator cuff tendon unloading following tenotomy and denervation induces fatty degeneration and increased fibrosis in the repair tissues.⁴⁰ Conversely, overloading and/or excessive exercise results in tenocyte apoptosis and collagen proteolysis.⁴¹ These studies collectively illustrate the sensitivity of repair response to mechanical loading and emphasize the fact that a fine balance between underloading and overloading must be maintained to optimize tendon repair. To support this concept, Andersson et al. demonstrated that low-level mechanical stimulation improved the mechanical properties of the repair tissue in a rat Achilles tendon transection model, whereas, overloading stimulated callus formation and rupture of repair tissue during mechanical testing.⁴²

Tenocytes respond to mechanical loading in a load intensity-dependent manner. Physiologic mechanical loading is essential for their normal function, whereas excessive or lack of loading is detrimental to their bioactivity.⁴³ Thorpe et al. demonstrated that altering mechanical stimuli results in a phenotypic shift of cells involved in tendon repair.⁴³ In tendon fascicles exposed to repetitive in vitro loads to induce ECM disruption, tenocytes adopted a rounded morphology and ECM damage.⁴³ A possible mechanistic explanation for these cellular and biochemical changes was suggested by a recent in vitro study by Lavagnino et al., wherein actin cytoskeleton depolymerization and concomitant collagenase upregulation were seen in tenocytes exposed to high-magnitude cyclic strains.⁴⁴

TPCs, like terminally differentiated tenocytes, are also sensitive to experimental repetitive tensile loading.^45,46 Short-term treadmill exercise increased the number of TPCs in murine Achilles and patellar tendons.⁴⁶ In addition, TPCs isolated from exercised mice had higher in vitro biosynthetic activities than TPCs isolated from control mice. In vitro exposure of TPCs to 4% tensile strain promoted tenogenic differentiation, whereas 8% tensile strain induced osteogenic differentiation, emphasizing the importance of tensile modulation during tendon repair.⁴⁶ Applying in vitro biaxial mechanical stress induces the expression of the proteoglycans, fibromodulin, lumican, and versican in tenocytes and TPCs.⁴⁷ These studies indicate that nonphysiologic loading affects the quality of reparative matrix by altering the biosynthetic functions of tenocytes and TPCs. Contrary to this body of evidence, recent evidence from Blomgran et al. demonstrated that loading delayed the shift of M1 to M2 macrophages in a rat Achilles tendon healing model.⁴⁸ As described previously, M1 macrophages promote inflammation and M2 macrophages support healing. The clinical implication of mechanical loading prolonging inflammation reported in this study is unclear and does not provide a mechanistic explanation for beneficial effects of loading on tendon healing. From a clinical perspective, understanding the role of mechanobiology on tenocyte activities can be applied to optimize postinjury rehabilitation and physical therapy protocols for patients.

There is little information regarding the molecular events behind mechanical stimulation of healing, a process termed “mechanotransduction.” Inflammatory mediators PGE₂, COX-2, and IL-6 are upregulated in tendon fascicles exposed to cyclic loading. Interestingly, these mediators were concentrated in the vicinity of highly cellular interfascicular matrix.^43,49 Exercise stimulates IGF-I expression in rat Achilles tendon and, as mentioned earlier, IGF-I stimulates cell proliferation and collagen synthesis.⁵⁰ In vitro experiments have demonstrated sustained release of MMPs and inhibition of TIMPs when tenocytes are deprived of mechanical stress, stimulating a matrix remodeling response to unloading.^49,51 Further research is clearly necessary to understand the complex interplay of mechanotransduction and cellular bioactivity during tendon repair.

TGFβ has been identified as one of the key molecular players linked to mechanotransduction in tendon homeostasis and healing. Landmark work in mice by Maeda et al. demonstrated that tensile load induces expression of Scleraxis, a tenogenic transcription factor, by TGFβ/Smad2/3 transactivation.²⁴ Conversely, scleraxis expression was decreased in Achilles tendons transiently unloaded with botulinum toxin, and secretion of collagen type I and COMP was also decreased in these tendons. Consequently, the mechanical properties of the Achilles tendons were compromised. Although TGFβ is important for tendon homeostasis, supraphysiologic levels of TGFβ in tendons are associated with adhesions, fibrous and chondroid tissue deposition.⁵² Intratendinous TGFβ1 injections in mouse Achilles tendons accompanied by strenuous treadmill exercise decreased tendon collagen content and mechanical strength.⁵³ These findings suggest that the threshold-dependent effects of mechanical loading on quality of repair is mediated by TGFβ signaling.

Dysregulation of chondro-osteogenic bone morphogenetic protein (BMP) expression is linked to ectopic calcification of tendon midsubstance in natural and experimental tendinopathies.^53,54 Tendon fibroblast cultures exposed in vitro to a combination of tensile loading and recombinant BMP-12 had higher collagen type I and type III, and decreased decorin gene expression than control fibroblasts cultures.⁵⁵ TPCs exposed to in vitro mechanical loading increased BMP-2 expression and had an increased osteogenic potential compared to unloaded TPCs.⁴⁶ Furthermore, treating rat Achilles tendon defects with growth and differentiation factor-2 (GDF-2), another BMP family ligand, induced tendon-like tissue when the tendons were loaded, whereas a bony callus was formed in the absence of loading.⁵⁶ These findings provide a mechanistic explanation for ectopic calcification that occurs as a result of altered mechanical loading. That is, a combination of aberrant loading, BMP upregulation, and increased noncollagenous protein expression in endogenous cells favor chondro-osteogenic degeneration in tendon repair tissue.^22,23,53,54 These findings suggest threshold-dependent effect of BMPs and mechanical loading on quality of tendon repair, similar to TGFβ.

Tenogenesis as a Model for Optimal Adult Tendon Healing

The critical events and regulation of tendon development are not well understood, in contrast to other musculoskeletal tissues like articular cartilage and bone. This is largely due to an absence of tendon-specific markers that can be used to follow tenogenic progression. Delineating mechanisms that govern fetal tendon fibrillogenesis are integral to developing successful therapeutic strategies in adult tendon, as the molecular factors and signaling regulators (described below) involved in tendon development could be applied to implement a regenerative form of adult tendon healing. Scleraxis (Scx), a basic helix-loop-helix transcription factor, first described in 2001, is expressed by all tendon and ligament progenitors.⁵⁷ Scx expression is seen in early stages of tendon development, although deletion of Scx did not result in tendon loss. Although Scx^-/- mutants were viable and generated tendinous structures, they had minimal mobility due to absence of important energy-strong and weight-bearing long tendons.

Scx also drives the expression of later tenogenic markers, Col1α1 and Tenomodulin;^58–60 however, tendon induction is not exclusively dependent on Scx.⁵⁷ In the context of tendon healing, Scx transfection enhances tenogenic differentiation of mesenchymal stem cells in vitro.⁶¹ Intratendinous injection of TPCs transduced with Scx (TPC-Scx) into rat patellar tendon window defects significantly improved the histological and biomechanical properties of the repair tissue compared to tendons treated with naive TPCs.⁶² Collectively, these findings demonstrate that Scx regulates critical aspects of tendon development, homeostasis, and disease; however, Scx is not the master regulator of tendon cell fate and studies exploring the interplay of Scx with other molecular and biological factors of tenogenesis are required to fully elaborate the factors that regulate tenogenesis.

Mohawk (Mkx), an atypical homeodomain transcription factor, was later found to have a critical role in tendon differentiation.⁶³ Tendons of Mkx^-/- mutants are predominantly normal during embryogenesis, but, postnatally, the collagen fibrils remain smaller compared to wild-type mice, suggesting suppression of collagen matrix organization and maturation. Similarly, to Scx, ectopic expression of Mkx in mesenchymal cells enhances in vitro tenogenic capacity.⁶⁴ As the biomechanical properties of tendon correlate with collagen structure, greater understanding of Mkx's functions during tendon differentiation may be critical to improving repair tissue organization and consequent functional capacity. Early growth response (Egr) 1 and 2, both zinc finger transcription factors, regulate certain aspects of matrix formation, however, they do not play a major role in vertebrate tendon specification and will not be discussed further. Sine oculis-related homeobox (Six) and sex determining region Y-box 9 (Sox-9) are pivotal to myotendinous and bone–tendon development, respectively, but these activities are beyond the scope of this review.⁶⁵

In contrast to adult tendon healing, injured fetal tendons restore the native structure of injured tendon, stimulating the investigation of factors responsible for regenerative-type or “scarless healing.” The ovine model of scarless tendon healing has been utilized to identify the fundamental differences between fetal and adult tendon healing. Intrauterine partial tenotomy of fetal extensor tendons in pregnant ewes healed through a regenerative response with complete reconstitution of collagen structure.⁶⁶ In contrast, analogous tenotomies in maternal limbs healed through reparative response characterized by granulation and scar tissue formation. TGFβ1 expression and inflammatory cell infiltration were markedly higher in adult healing tendons than in fetal tendons. As the fetal tendon defect size increased, the healing response shifted from a regenerative to a more reparative outcome.⁶⁷ To further investigate this disparity, injured ovine fetal and adult tendon grafts were transplanted into a subcutaneous pouch of female adult SCID mice. Despite the adult environment, regenerative healing occurred in fetal tendons, but, healing through scar formation occurred in adult tendons.⁶⁸ Similar to the previous study, TGFβ1 and bFGF levels and inflammatory cell infiltrates were increased in adult tendon tissue only. These findings suggest that the absence of TGFβ1 upregulation and inflammatory response during fetal tendon healing are critical for a regenerative healing response.

Degenerative Changes in Tendon Repair Tissue: Structural and Compositional Changes and Mechanisms Underlying These Changes

Fatty degeneration, chondrogenic dysplasia, and ectopic calcification within the repair tissue of chronically injured tendons are well documented.^69–74 A recent study by Liu et al. demonstrated that fibroplasia and fatty infiltration after rotator cuff tendon injury is a consequence of proliferation of two distinct cells types; Tie2+ muscle progenitor cells and PDGFRα+ fibroadipogenic progenitor cells, respectively.⁷⁵ However, the contributions of these cell types to repair of tendon injuries distant from musculotendinous junctions remain to be determined. Ectopic chondro-ossification in the midsubstance of Achilles and patellar tendons occurs through endochondral ossification,^22,23 indicating a major phenotypic shift in cells engaged in tendon repair. The underlying pathogenesis for these metaplastic changes in chronic tendinopathy is poorly understood. Abnormal matrix deposition likely occurs from extrinsic cells that migrate to the site of injury⁷⁶ or from native tenocytes and/or TDPCs that undergo transdifferentiation to nontenogenic phenotypes. This section compiles the information regarding biochemical changes, cellular and biological processes responsible for chondrodegeneration, and ectopic ossification that occurs during tendon healing.

Chondrodegeneration in healing tendons

Chondrogenic metaplasia, characterized by increased proteoglycan (Fig. 1) and collagen type II content, and cellular “rounding” (Fig. 2) are seen in both naturally occurring and experimental models of tendinitis.^8,23,77 Histochemical studies have demonstrated upregulation of inflammatory cytokines like IL-1β, IL-6, and TNFα in areas of chondrogenic metaplasia.^78,79 These cytokines, in turn, induce inflammatory mediators COX-2, PgE2, and collagenases MMP-1 and −13; all involved in tendon matrix degradation.^16–18 Although these changes in inflammatory mediators are ubiquitous to tendon injury/overuse, their cause–effect relationship in tendon chondrodegeneration has not been clarified.

FIG. 1.

Toluidine blue staining of equine superficial digital flexor tendon sections shows the characteristic metachromatic (purple) hue within injured tendons indicative of increased proteoglycan at 10 weeks postcollagenase injection. Scale = 5 mm. Color images available online at www.liebertpub.com/teb

FIG. 2.

Tenocytes with elongated spindle-shaped nuclei (block arrow) in normal equine superficial digital flexor tendon is replaced by tenocytes with rounded nuclei (line arrows) within injured tendons at 5 and 10 weeks postcollagenase injection. Scale = 100 μm.

In normal tendons, decorin is the most abundant proteoglycan and regulates collagen fibrillogenesis and assembly.⁷⁷ Samiric et al. demonstrated that expression of large aggregating proteoglycans, versican and aggrecan, and small proteoglycans, biglycan and fibromodulin, increased in pathologic tendons compared to normal tendons, whereas decorin messenger RNA (mRNA) was similar in both.⁸⁰ These compositional changes led to water retention and decreased elastic and tensile strength of the repair tissue. Proteoglycans such as biglycan, decorin, fibromodulin, and lumican, although constituting a very small portion of tendon ECM, are active participants in collagen fibrillogenesis^81,82 and can also bind and sequester growth factors such as TGFβ⁸³ and IGF-I^28,29 to modulate tendon cell bioactivities. Therefore, biological processes that influence changes in constituent proteoglycans in tendon pathology and healing must be recognized with an overall goal of improving reparative response in healing tendons.

At the cellular level, endogenous cells in injured and/or repair tissue sites assume a “rounded” phenotype similar to chondrocytes. These cells replace the elongate spindle-shaped tenocytes (seen in normal tendons) amidst disorganized collagen fibers. In athletes, rounded tenocytes were commonly seen during histological screening of asymptomatic patellar tendon tissue collected for anterior cruciate ligament (ACL) reconstruction.⁸ Furthermore, this study also demonstrated that tenocyte rounding occurred in areas of increased proteoglycan accumulation. This finding suggests cartilage-like matrix synthesis by these abnormal cells, likely due to their chondrogenic shift. Also, in these asymptomatic tissue samples, tenocyte rounding was more prevalent than overt collagen disruption.⁸ It is possible that this early chondrogenic metaplasia is indicative of early tendon injury. Work by Attia et al. provides a possible explanation for chondrocytic phenotype of tendon cells, where in, they demonstrated that an increased glycosaminoglycan deposition was associated with heparin affin regulatory peptide (HARP), a cytokine necessary for chondrocyte formation, in a rat overuse supraspinatus tendinopathy model.⁸⁴ HARP protein levels were increased in injured tissue, along with increased mRNA expression of the chondrogenic transcription factor, Sox-2. This study is among very few that attempt to delineate the molecular process responsible for the drift toward chondrocytic phenotype. Further research is clearly necessary, given that this chondrogenic shift might reflect a very early pathophysiologic transition in tendon.

Experimental evidence suggests that tendon injury/healing can also alter the phenotype of resident TPCs. Bi et al. demonstrated that TPCs isolated from biglycan–fibromodulin double-knockout mice had increased collagen type II and aggrecan expression compared to wild-type TPCs.³ When TPCs isolated from these mice were subcutaneously implanted in nude mice, calcified tendon-like tissue was formed, whereas wild-type TPCs formed only tendon-like tissue in the same in vivo model.³ These findings graphically demonstrate that noncollagenous proteins exert significant regulatory influences on the phenotype of TPCs, similar to mature tenocytes. More recently, Asai et al. showed that intratendinous administration of TPCs into experimental tendon lesions resulted in their transdifferentiation to chondrogenic cells, giving rise to chondrodegenerative repair tissue.²⁶ Furthermore, the chondrodegenerative changes at the repair site were more severe when TPCs isolated from injured tendons were administered intralesionally compared to TPCs isolated from healthy tendons. In this study, TPCs isolated from injured tendons were classified into two subtypes, based on the expression of TGFβ coreceptor, CD105, or endoglin. CD105^- TPCs were more chondrogenic in vitro and induced larger chondrodegenerative lesions in vivo than CD105⁺ TPCs. Last, TPCs isolated from injured tendons had a higher in vitro chondrogenic capacity than control TPCs. Collectively, these findings suggest that the phenotype of endogenous tendon cells is closely associated with the characteristics of the ECM at the site. However, the cause–effect relationship of aberrant cellular phenotype and chondrogenic ECM synthesis remains unclear. Further research on this issue is required before advocating the clinical use of TPCs from pathological tissue for cell-based therapies.

A handful of recent studies address the molecular mechanisms that control the phenotypic shift of tendon cells and aberrant matrix synthesis in injured/repair sites. The role of TGFβ signaling in fibrosis, scar formation in connective tissues, and pathogenesis of tendon injury has already been detailed in this review. Administration of the inflammatory mediator, PgE2, to in vitro cultures of mature tenocytes stimulated active TGFβ1 secretion into the culture media.^73,85 In vivo, chondrodegenerative lesions induced by injured TPCs in healing tendons were dependent on TGFβ signaling.²⁶ The spontaneous in vitro chondrogenic capacity of TPCs isolated from injured tendons in micromass cultures was inhibited when TGFβ inhibitor was added to the culture media. This indicated that TGFβ signaling is responsible for the enhanced chondrogenic capacity of TPCs isolated from injured tendon tissue. In support of this in vitro finding, attenuation of TGFβ signaling in vivo by targeting TGFβ1, CTGF, and Smad 3 with antisense oligonucleotides reduced scarring and adhesion formation in a murine flexor tendon repair model.²⁵ These studies provide compelling support for modulating TGFβ signaling during tendon healing to improve the quality of repair.

Ectopic ossification in healing tendons

Pathological bone formation, also known as ectopic ossification, within tendons has been reported in cases of natural and experimental tendinopathy. A few studies have investigated the cause of ectopic ossification in healing tendons. Radiographic evidence of intratendinous ossification 5–10 weeks following horizontal transection was noted in a rat Achilles tendinopathy model.²³ In this study, a chondrogenic phase at 3–5 weeks after tendon transection with Sox-9 mRNA upregulation, along with synthesis of collagen type II and aggrecan, was evident. This phenotypic transition was followed by an osteogenic phase at 5–8 weeks, with upregulation of the osteogenic transcription factor, Runx2, histological evidence of chondrocyte hypertrophy, and lamellar calcification of surrounding matrix. In normal Achilles tendons, Sox9 and Runx2 mRNAs were not detected.

Most recently, Suzuki et al. reported spontaneous ossification of Achilles tendons in 5 week-old Mohawk (Mkx), knockout rats.⁸⁶ This finding suggests that Mkx is required for maintaining tenogenic lineage commitment in progenitors. Mohawk is an atypical homeodomain tenogenic transcription factor required for collagen maturation during embryonic development and also shown to enhance the tenogenic capacity of mesenchymal stem cells.⁷¹ Ossification within the Achilles tendons of Mkx knockout rats in this study was preceded by a chondrogenic phase (present at birth), reflecting an endochondral process for intratendinous ossification, similar to the results reported by Lin et al.²³ TPCs isolated from these Mkx knockout rats had higher in vitro chondrogenic and osteogenic capacities than wild-type cells. These aberrant nontenogenic differentiation capacities were ameliorated with Mkx overexpression. These results suggest that chondrogenic and osteogenic genes are putative targets of tenogenic transcription factor, Mkx.

Evidence for alternate mechanisms for heterotopic ossification in addition to endochondral ossification has also been proposed. Zhang et al. reported endothelial–mesenchymal transition as the cause for heterotopic ossification in experimental Achilles tendon lesions.⁸⁷ This was supported by downregulation of endothelial markers, vascular endothelial cadherin, and CD31 mRNAs and upregulation of mesenchymal markers, neural cadherin, and vimentin. In addition, dense ossified trabecular structures were present in histological sections obtained from 6- and 10-week healing tissue.

Chondro-osteogenic BMPs (BMP-2, -4, -6, and -7), promote bone, cartilage, and enthesis repair.^88–90 Several lines of evidence implicate dysregulation of BMP and TGFβ activity as a cause of ectopic calcification in tendinopathy. BMPs are upregulated in naturally occurring and experimental models of calcifying tendinopathy.^69,70 In addition, intratendinous administration of rhBMP-2 results in ectopic calcification.⁹¹ Murine TPCs exposed to BMP-2 during in vitro culture before subcutaneous implantation into immune-deficient, mice-generated, enthesis-like elements with both tendon-like and osseous tissues.⁴⁶ Furthermore, BMP pathway-related genes (BMP receptor-1a, BMP receptor 2, Smad1, and Smad5) were upregulated in ossified tendon repair sites in Mkx knockout rats.⁸⁶ This finding also suggests that Mkx-mediated chondro-osteogenic changes during tendon repair are dependent on BMP activities.

On the other hand, osteopontin upregulation was reported without concomitant BMP-2 upregulation in tissue samples obtained from clinical cases of calcifying rotator cuff tendinopathies.⁹² Furthermore, the gene expression of BMP-4 and BMP-6 in these calcific areas was decreased. Zhang et al. demonstrated that ectopic ossification by endothelial–mesenchymal stem cell transition in a rat Achilles transection model was driven by TGFβ1/Smad7 signaling.⁸⁷ As described previously regarding chondrogenic effects mediated by TGFβ, the cause–effect relationship between BMPs and ectopic ossification is unclear. A complex interplay of excessive loading, aberrant BMP signaling, and alterations in noncollagenous protein expression could collectively generate conditions favoring ossification at healing sites at the expense of tenogenesis.

Future Directions and Therapeutic Approaches to Promote a Regenerative Response During Tendon Healing

Over the last decade or so, various stem cell-based and regenerative therapies have been evaluated in experimental tendon repair models and naturally occurring tendinitis/tendinopathy lesions. The outcomes of these studies have been recently reviewed by several groups.^93–96 Histological improvement in longitudinal collagen fiber orientation is a consistent finding in these experimental studies. Interestingly, data from recent cell-tracking studies demonstrate that exogenous stem cells are cleared from the injection site within a few weeks and do not directly contribute to the pool of tenocytes and/or progenitor cell engaged in tendon repair/regeneration.^97–99 These studies further highlight the importance of identifying cytokines and/or trophic factors secreted by exogenous cells that mediate their sustained therapeutic effects. This is an active area of research and further studies are necessary to simplify and “decellularize” current biologic therapeutic approaches.

To date, markers specific for tenogenic cells and the identity of cells responsible for tendon matrix synthesis are poorly defined. The cell population(s) in tendon are heterogeneous; therefore, developing a single marker that can definitively identify these cells is likely not feasible; a panel of coexpressed markers to identify tenoprogenitors is a more realistic objective. Understanding the mechanisms involved in maintaining a tenogenic phenotype is an important prerequisite for developing protocols to direct authentic tenogenic regeneration and prevent aberrant transdifferentiation of cells involved in tendon healing that impact the matrix characteristics at the repair site.

TGFβ signaling is responsible for maintaining lineage-specific transcription factor expression in several mesenchymally derived lineages such as tenocytes, chondrocytes, osteocytes, and myocytes.^{3,22,24,26,57,64,88,91} TGFβ acts through Smads 2 and 3 in regulating the tenogenic transcription factor, Scleraxis.^24,25 However, as described in detail above, TGFβ signaling is also implicated in chondrodegeneration and pathological ossification during tendon healing. These observations suggest that gradations of TGFβ activity are involved in cellular differentiation and subsequent tissue synthesis during tendon healing. Modulating TGFβ/BMP signaling to establish a “Goldilocks” balance during tendon healing is likely to be more beneficial than an “all-or none” approach and holds considerable promise for designing therapies for tenogenic healing responses. In support of this hypothesis, Loiselle et al. demonstrated that completely abolishing Smad 3 signaling decreased adhesion formation, but also suppressed collagen matrix deposition and reduced strength of the healing tendon, relative to wild-type mice.²⁵

Over time, as molecular changes associated with tendon healing have been deciphered, several gene deletion and gene therapy approaches in experimental models of tendon healing have been evaluated. Although this approach does not have direct clinical translation, they facilitate identification of the molecular processes suitable for therapeutic intervention. As an example, adenovirus-mediated transfer of siRNA targeting Runx2/Cbfa1 successfully prevented ectopic ossification in a rat Achilles tendinopathy model.¹⁰⁰ Similarly, local delivery of the BMP antagonist, noggin, inhibited ectopic ossification stimulated by BMP-4.¹⁰¹ Given that the bioactivity of tenocytes is heavily influenced by ECM, gene therapy coupled with tenoinductive scaffolds can be developed to control cell differentiation and matrix synthesis, and enhance the quality of repair.

The importance of loading on tendon development, homeostasis, and healing has been recognized for many years; however, our current understanding of the effects of mechanical loading on bioactivity of tendon cells and mechanisms of mechanotransduction is far behind that of other musculoskeletal tissues. Historically, preliminary studies evaluating cell-based therapies for tendon healing did not address posttreatment exercise regimes. However, recent literature stresses the importance of controlled exercise in enhancing therapeutic benefits of biologic therapies.^93,94 The provocative finding that controlled exercise can induce chondroid matrix removal^51,52 should stimulate further research into the specific biomechanical mechanisms to promote healthy tendon repair, particularly given that the rehabilitation aspects of tendon repair can be closely controlled, at least in human patients.

In conclusion, this review highlights the importance of understanding and potentially exploiting the heterogeneity of the cellular environment during tendon healing to develop successful therapeutic strategies. Most of the study outcomes described here are based on in vitro cell culture models and in vivo murine models of tendon repair. While extrapolating results from these studies, it must be kept in mind that all established models of experimental tendinitis/tendinopathy do not fully replicate the course of events occurring in natural disease.

Footnotes

Disclosure Statement

No competing financial interests exist.

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