Recent Scientific Advances Towards the Development of Tendon Healing Strategies

Abstract

There exists a range of surgical and non-surgical approaches to the treatment of both acute and chronic tendon injuries. Despite surgical advances in the management of acute tears and increasing treatment options for tendinopathies, strategies frequently are unsuccessful, due to impaired mechanical properties of the treated tendon and/or a deficiency in progenitor cell activities. Hence, there is an urgent need for effective therapeutic strategies to augment intrinsic and/or surgical repair. Such approaches can benefit both tendinopathies and tendon tears which, due to their severity, appear to be irreversible or irreparable. Biologic therapies include the utilization of scaffolds as well as gene, growth factor, and cell delivery. These treatment modalities aim to provide mechanical durability or augment the biologic healing potential of the repaired tissue. Here, we review the emerging concepts and scientific evidence which provide a rationale for tissue engineering and regeneration strategies as well as discuss the clinical translation of recent innovations.

Environmental Stressors and Tendon Disease

Tendons are fibrous connective tissues which transmit force between muscle and bone to provide skeletal mobility. Functional impairment of tendons (e.g., rotator cuff, patellar and Achilles) represents a major health care problem. Tendons are composed of a dense, hierarchical organization of a parallel arrangement of type I collagen fibers (~70% of dry weight),¹ along with varying, lesser amounts of collagen type III and V. The collagen fibers are embedded within a hydrophilic extracellular matrix consisting of matricellular proteins such as tenascin C, large proteoglycans such as versican and aggrecan, small proteoglycans such as biglycan and decorin, and glycosaminoglycans such as hyaluronan.

Clinically, tendon injuries are typically characterized by pain, swelling, stiffness and immobility. Broadly speaking, they are described as either acute or chronic. Acute injuries are the result of “macro-trauma” to the tendon, such as a laceration or sudden tensile overload, leading to either partial tearing or complete rupture. Chronic injuries, which generally do not involve macro-trauma and present histologically with a conspicuous absence of inflammatory cells,² can arise from one or more conditions, each of which can either initiate and/or perpetuate the pathology. These stressors can be broadly defined as 1) metabolic (e.g. diabetes),³ 2) genetic (e.g. Col5a1 mutations),⁴ 3) vascular imbalance,⁵ 4) hypoxia⁶ or 5) biomechanical (e.g., overuse injuries).^7,8 The attendant cellular responses can include apoptosis, proliferation, migration or differentiation (e.g., adipogenic, chondrogenic, fibrogenic).² Since such cellular responses often alter anabolic and/or catabolic pathways, they can disrupt collagen organization with a loss of tissue material properties, and they can also interfere with cell–matrix interactions involved in the transduction of mechanical signals to the resident cells.

Histopathology of Tendinopathy

A well-recognized pathognomonic feature of tendinopathy is the presence of multiple small groups of cells with rounded nuclei which are distinct from the spindle-like fibroblasts normally seen on the surface of, or between, collagen fibers.⁹ These groups of rounded cells (Figure 1) are most often associated with the accumulation of a chondroid (Safranin-O positive) matrix, which is most highly stained near the cell groups and which appears to locally disrupt the normal linear arrangement of distinct collagen fibers.¹⁰ Electron microscopic analysis of tendinopathic regions has revealed an abnormal buckling of the collagen fascicles, the tendon cells and their nuclei, and it has been proposed that this is due to “loosening” of a sufficient number of fibers to allow a “cell-induced buckling of the tissue.”¹¹ This raises the possibility that one mechanism by which stressors, such as mechanical overloading or hypoxia, can promote tendinopathic change is via the accumulation of cell clusters in a mechanically stiff chondroid matrix. While tendon loading readily deforms elongated cells in tissue regions of aligned collagen, it has a markedly reduced effect on rounded cells in chondroid regions,¹² indicating that cells embedded within tendon chondroid deposits are shielded from tensile strain, which might increase their capacity to disorganize adjacent collagen fibers. Furthermore, a reduction in mechanical stimulation of cells within the chondroid matrix may impede appropriate (e.g., fibrogenic) remodeling of these diseased tendon regions.

Figure 1.

(A) Safranin-O staining and aggrecan immunohistochemical (IHC) localization of human extensor carpi radialis brevis and long head of the biceps tendons. Circled areas highlight chrondroid cell clusters, pericellular aggrecan accumulation, and localized collagen fiber disorganization in tendinopathic tissues. ²¹ (B) Schematic illustration of tendon fibroblasts within dense, aligned collagen fiber network of uninjured tendon (left panel), in contrast to the accumulation of rounded (chondroid) cells which disrupt the normal collagenous architecture in tendinopathic tissue (right panel)

Aggrecan is a Major Component of Tendinopathic Chondroid Deposits

While the precise structure and cell/matrix composition of chondroid deposits in tendinopathic tissue are largely unknown, they appear to be closely related to the tendon fibrocartilage which normally accumulates as an adaptive response to regions of tendon compression.^13,14 Due to its unique intra-tissue osmotic effects, aggrecan is the matrix component which is primarily responsible for the compressive resistance of such normal tendon fibrocartilages.¹⁵ The possibility that it also promotes buckling and mechanical weakening in the body of the tendon is suggested by the finding that aggrecan expression in humans,^16,17 and its chemical abundance in humans,^18,19 horses,²⁰ and mice²¹ are markedly increased in tendinopathic regions (Figure 1). Moreover, cross-sectional data from asymptomatic tendons found that collagen disorganization occurred only in association with cellular changes and chondroid accumulation.²² Additionally, glycosaminoglycan accumulation, largely due to aggrecan chondroitin sulfate, is strongly correlated with advanced clinical symptoms (pain, tenderness, overall weakness),²³ suggesting a direct link of chondroid pathology to mechanical dysfunction and pain.

Hypoxia and Tendinopathy

The histologic appearance of the diseased tendon has also been described as an angio-fibroblastic tendinosis, invading and disrupting normal tissue,²⁴ and it has been suggested that a goal of nonsurgical treatment should be a revascularization of the tissue. Indeed, it has been proposed that hypoxic cell injury is a critical pathophysiological mechanism (primary stressor) responsible for most tendinopathies²⁵ (Figure 2). In support of this idea, increased hypoxia-inducible factor 1 alpha (HIF-1α) protein has been observed in cells within, but not outside, tendinopathic lesions²⁵ and it is also abundant at the edge of torn tendons harvested at the time of rotator cuff surgery.²⁶ Further, there are increased intra-tendinous lactate levels in Achilles tendinopathy,²⁶ consistent with the pathology being associated with hypoxic stress and a switch to anaerobic glucose metabolism. Moreover, in the rotator cuff, a critical zone of hypovascularity (predictive of hypoxia) has been identified histologically^27,28 and this coincides with the most common site for cuff tears. Lastly, in vitro studies²⁵ have shown that exposure of tendon cells to hypoxia increases their expression of HIF-1α and collagen type III (Col3a1), both of which are increased in tendinopathic tissues.^11,25

Figure 2.

An hypoxic tissue environment (e.g., Hif-1α upregulation)^25,236 induced by factors such as overuse or vascular stress may promote chondroid deposition,^30–32 leading to collagen disorganization and tendinopathy. Therapeutic approaches such as mechanical loading⁷¹ can restore normoxic conditions and eliminate chondroid accumulation²¹ to restore tendon function.

Does Chondrogenesis Represent a Plausible Link Between Hypoxia and Tendinopathy?

Under normoxic conditions, HIF-1α is maintained at low levels by ubiquitination and proteasomal degradation. However, hypoxic conditions block this degradative pathway leading to high levels of cytoplasmic HIF-1α, which translocates to the nucleus to upregulate a large number of HIF-responsive genes,²⁹ including those required for glucose uptake and anaerobic glycolysis. Such a response maintains high ATP levels in cells and thereby facilitates, among other processes, the synthesis of the UDP-sugar precursors required for synthesis of chondroitin sulfate and hyaluronan. In keeping with this, exposure of adipose-derived stem cells and human chondrocytes to hypoxia stimulates the expression of a wide range of chondrogenic genes,^30–32 consistent with the major use of anaerobic glycolysis by chondrocytes in vivo.³³ Taken together, these findings appear to provide a direct mechanistic link between hypoxic stress, the appearance of chondroid deposits and the loss of tensile strength in tendons (Figure 2).

Cellular Healing Deficiencies in Tendinopathy

Tendinopathic tendons are characterized by chondroid deposition, collagen disruption and loss of tensile strength. The chronicity of the pathology appears to be another example of that which occurs in any non-healing fibrous tissue wound. Significantly, it has been found that deposition of a chondroid matrix can interfere with dermal healing in an excisional murine model.³⁴ Gene expression analysis of the non-healing dermis showed that it was accompanied by deficiencies in the TGF-β1 pro-fibrogenic pathway. In this regard, it has been shown that a markedly higher number of cells in human patellar tendinopathy express TGF-β1, consistent with an attempted fibrogenic wound healing response.³⁵ Consistent with this notion, studies of tendinopathic tissue have shown an increased expression of pericellular components (often with a short half-life) such as fibronectin, tenascin C, aggrecan and biglycan,¹⁹ whereas the expression of constituents more associated with the stable intercellular matrix (decorin and type I collagen), was less altered.^17,36,37 When examined from this perspective the healing deficiencies in tendinopathy appear to result from a chronic stimulation of synthesis of pericellular components (within chondroid deposits) without a concomitant increase in the production of stable, long-lived matrix structures (Figure 2).

Experimental Models of Tendinopathy

While changes which follow acute tendon injury (e.g., tendon transection and repair) have been well described in animal models, the cellular and molecular responses to chronic tendinopathies have been less well studied. As described more extensively in other reviews,^38–40 tendon injury responses have been characterized both in vivo and in vitro, using various approaches which include: (i) surgical tendon lacerations^41–44; (ii) pharmacological induction of tendinopathy (e.g., using bacterial collagenase,^45–47 cytokines or prostaglandins^48–50; and (iii) in vitro^51–53 and in vivo^54–59 mechanical loading models (e.g., treadmill overuse, muscle stimulation, and fatigue loading).

In general, animal tendinopathy models are accompanied by an induction of chondrogenic genes and/or chondroid deposits. For example, collagenase injection into horse⁶⁰ or rat^61–63 tendons produced an inflammatory response with tendon collagen bundle disarray, mucoid deposits, and at the molecular level, expression of Col2a1 and Sox9 by fibroblast-like cells and expression of Bmp-4/7 by chondrocytic cells, suggesting that aberrant differentiation of healing tendon progenitor fibroblasts may account for failed healing. Further, when a mixture of cytokines (e.g., IL-1) and growth factors (TGF-β1, FGF2) was injected into rabbit tendons,⁵³ both fibrosis and mucoid accumulation resulted. This consistent finding of a chondroid response has also been described in mechanically-induced (treadmill overuse) models examining rat rotator cuff^64,65 and mouse patellar and Achilles tendons.⁵⁹

Mechanical Loading Can Promote Tendon Healing

Treatment options for tendinopathy patients are limited since corticosteroid injections and surgical debridement show poor long-term outcomes.⁶⁶ Similarly, effectiveness of modalities such as ultrasound and shock wave therapy has not been proven.^67–69 Rehabilitative exercise, specifically eccentric loading (e.g. heel drop exercises) to lengthen the muscle-tendon unit, is currently the most effective long-term therapy for Achilles tendinopathy, as shown by pain reduction and improved tendon function.^70–72 However, the mechanisms by which mechanical stimulation contributes to tendon healing and restoration of function are currently unknown. In a recent systematic review focusing predominantly on Achilles tendinopathy, Drew and colleagues⁷³ concluded that the clinical literature does not support the notion that observable structural changes in the tendon can explain the therapeutic efficacy of exercise. Hence, it is likely that other factors (e.g., neurogenic or biochemical) play an integral role in healing of tendinopathies, and strategies to target associated mechanisms are warranted.

There exist numerous reports on the stimulatory effects of mechanical loading on the healing capacity of transected animal tendons.^74,75 Killian et al.⁷⁶ noted that, while controlled mechanical loading is generally beneficial to tendon healing, the magnitude and timing of such loading is dependent on anatomic location (e.g., intrasynovial versus extrasynovial). While these data have important clinical implications on designing rehabilitation protocols following surgical tendon repair, very few models have examined the therapeutic potential of mechanical loading to treat tendinopathy. In a rat Achilles model of collagenase injection,⁷⁷ early voluntary exercise was found to accelerate collagen deposition but prolonged the inflammatory response and led to decreased tendon biomechanical properties. Tendon healing in rats which initiated voluntary exercise at a later post-injection time point showed minimal differences relative to non-exercised controls. Bell et al.²¹ developed a TGF-β1 injection model to induce chondroid accumulation, which was associated with a dramatic decrease in the material properties of the murine Achilles tendon. Following injection, 4 weeks of treadmill exercise suppressed chondroid formation, decreased chondrogenic gene expression (relative to mice which did not receive exercise), and stimulated a restoration of mechanical properties to those of uninjured tendons. The latter study thereby lends strong support to the clinical physiotherapy literature (as illustrated in Figure 2).

Scientific Rationale for New Approaches to Tendon Regeneration

The first-line treatment strategy for tendon tears typically is surgical reattachment of the tendon to its bony insertion site or direct tendon-tendon repair, depending on the injury location and the severity of its impairment on tendon and joint function. In spite of numerous technical advances to date, primary repair in general neither reliably restores pre-injury functional properties nor regenerates the native tendon structure, organization, and composition.⁷⁸ Biologic therapies represent an experimental strategy for providing mechanical support, augmenting tissue healing, and/or promoting tissue regeneration. Tissue engineering strategies arise from a fundamental understanding of tendon structure, function, composition, and the biologic processes involved in tendon homeostasis and healing. Successful tendon regeneration relies on presence of three critical factors: (1) a scaffold to provide a structural framework for cell attachment and matrix production; (2) progenitor cells, which differentiate into specialized tissue in response to appropriate mechanobiologic signals; (3) growth factors that drive the progenitor cells into a tenogenic differentiation pathway. Biological strategies to augment healing of intrinsic or surgical tendon repair involve utilizing one or more of the aforementioned three factors. Another branch of tendon tissue engineering aims to apply knowledge derived from studies of the embryologic tendon development and postnatal remodeling (e.g., mesenchymal stem cell differentiation pathways and transcription signals) towards strategies for repair after injury.^{79–82, 83,84}

Scaffolds For Use in Tendon Regeneration

Scaffolds provide both mechanical reinforcement and augmentation of healing potential to the injured tissue. Scaffolds come in the form of native tendon matrices⁸⁵ and collagen-based constructs,⁸⁶ synthetic polymers,^87–89 and engineered forms of endogenous proteins,⁹⁰ which can facilitate tendon incorporation at the site of injury. These devices should have sufficient mechanical properties to offload and stabilize the repaired tendon during the early postoperative period, and, particularly in the case of ECM-derived scaffolds, should harbor bioactive substances such as growth factors needed to improve the rate and/or effectiveness of tendon healing.⁹¹ Importantly, the scaffold should also be amenable to suture retention.⁹¹ Among the most common natural biomaterial scaffold materials for tendon repair are porcine small intestine submucosa (SIS), silk, human allograft dermis, and decellularized tendon.⁹² Once introduced into the body, the scaffold gradually resorbs and is replaced by ECM products synthesized by the scaffold-attached cells.⁹³ Scaffold design should provide mechanical durability, adequate surface area and porosity, and biocompatibility in order to avoid eliciting a host inflammatory response.⁹⁴ Efforts are underway to engineer new synthetic scaffold designs, such as fibroblast or MSC-seeded polylactic-co-glycolic acid (PLGA) nanofiber⁹⁵ and MSC seeded collagen sponge scaffolds.^96,97

In preclinical large animal models, ECM scaffolds (Restore, Zimmer Collagen Repair, GraftJacket) have shown promising histologic outcomes and tendon-like remodeling but have generally had disappointing biomechanical outcomes with respect to the repaired constructs at 9 to 24 weeks.^98–101 In these canine and ovine models there were no unfavorable host inflammatory responses noted, although some degree of transient inflammation lasted longer with the cross-linked porcine dermis scaffold¹⁰⁰ than with the non-cross-linked porcine SIS^99,101 or non-cross-linked human dermis⁹⁸ alternatives. In contrast, synthetic scaffolds including Synthasome’s X-Repair (a poly-L-lactide patch) and the Biomerix Rotator Cuff Repair Patch (composed of polycarbonate polyurethane-urea), have proven to be both biocompatible and to significantly enhance the biomechanical function of repaired infraspinatus tendons in canine and ovine models, respectively.^102,103

A large number of clinical studies have assessed the therapeutic use of scaffold devices in human subjects (Table 1).^104–120 While scaffolds have been among the most studied biologics in tendon repair, the clinical translation of these devices is nuanced. At present there is a paucity of studies with long-term follow-up data on these devices, and their results have been inconsistent with respect to clinical outcomes and adverse effects. Clinically significant aseptic inflammatory reactions have been reported in roughly one-quarter of patients treated with non-cross-linked porcine SIS scaffolds during rotator cuff repair,^110,118 and as a result the American Academy of Orthopedic Surgeons (AAOS) does not endorse their use for this clinical indication.¹²¹ Variability in clinical outcomes may be attributable to heterogeneity in the design and application of these devices. Studies have variably utilized scaffolds to either augment repair or for interposition between the tendon and bone insertion. Numerous types of materials are available and, although natural ECM scaffolds have been favored initially, preliminary data for synthetic scaffolds are promising,^109,120 and it is not clear which type is optimally suited for each clinical application. With regard to regulated clinical use, because synthetic and ECM scaffolds are prepared from non-human sources, they are allowed into the healthcare marketplace by the Food and Drug Administration (FDA) so long as they exhibit substantial equivalence to a reference device in their biocompatibility, sterility, safety, and performance.¹²² However, manufacturers are not required to demonstrate the efficacy of these nonhuman-derived augmentation devices in preclinical or controlled human clinical testing.¹²² Notably, the use of human-derived ECM scaffolds as transplantable human tissue is protected under the Code of Federal Regulations and is not subject to FDA approval.⁹¹

Table 1.

Clinical studies using scaffolds in tendon repair.

Study design	Type of scaffold	Study groups	F/U	Outcomes
Metcalf et al114 Case series	Restore	Open RCR + scaffold (n = 12)	24 mos	Repair failure in 1/12; UCLA scores doubled
Sclamberg et al116 Case series	Restore	Open RCR + scaffold (n = 11)	6 to 10 mos	Repair failure in 10/11; no improvement in ASES scores
Iannotti et al110 RCT	Restore	Open RCR Scaffold (n = 15) No scaffold (n = 15)	12 to 26 mos	Scaffold group had lower healing rates; no difference in Penn scores
Malcarney et al113 Case series	Restore	Open RCR + scaffold (n = 25)	6 mos	RCT aborted due to postoperative inflammatory reactions in 4/25 causing repair failure and requiring open debridement
Walton et al118 Retrospective case-control	Restore	Open RCR Scaffold (n = 10) No scaffold (n = 12)	24 mos	No difference in tendon healing rates, satisfaction scores, or range of motion
Burkhead et al107 Case series	GraftJacket	Open RCR + scaffold (n = 17)	14 mos	Repair failure in 3/12; improvement in UCLA scores, strength, and range of motion and satisfaction in 14/17 patients
Dopirak et al108 Bond et al106 Case series	GraftJacket	Arthroscopic RCR + scaffold (n = 16)	12 to 38 mos	Incorporation into tendon in 13/16; improvement in UCLA and Constant scores, strength, and range of motion; 15/16 satisfied
Wong et al119 Case series	GraftJacket	Arthroscopic RCR + scaffold (n = 45)	24 to 68 mos	Update to above study; continued favorable outcomes
Barber et al105 RCT	GraftJacket	Open RCR Scaffold (n = 22) No scaffold (n = 20)	12 to 38 mos	Tendon healing rates and Constant and ASES scores higher in scaffold group; no difference in UCLA scores
Rotini et al115 Case series	ADM	Open or arthroscopic RCR + scaffold (n = 5)	12 to 18 mos	Incorporation into tendon in 5/5; improvement in Constant scores
Lee et al111 Lee et al112 Case series	GraftJacket	Achilles tendon repair + scaffold (n = 9, n = 11)	≥ 20 mos	All patients able to recover full ROM at 3 months and perform single heel raises at 6 months; improvement in AOFAS scores at 12 months; no re-ruptures, recurrent pain, or reoperations at ≥ 20 months
Soler et al117 Case series	Zimmer Collagen Repair	Open RCR + scaffold (n = 4)	3 to 6 mos	All patients had failed incorporation into tendon, inflammatory reaction, graft disintegration, and tissue necrosis at time of revision surgery
Badhe et al104 Case series	Zimmer Collagen Repair	Open RCR + scaffold (n = 10)	36 to 60 mos	Incorporation into tendon in 8/10; improvement of Constant scores, pain, ROM, and abduction strength; 9/10 satisfied; no adverse events
Encalada-Diaz et al109 Case series	Biomerix RCR Patch	Open RCR + scaffold (n = 10)	12 mos	Intact tendon healing in 9/10; improvement in ASES and SST scores, pain, and ROM; no adverse events
Proctor120	X-Repair	Arthroscopic RCR + scaffold (n = 18)	42 mos	Intact tendon healing in 14/18; improved ASES scores

RCT = randomized controlled trial; ADM = acellular dermal matrix (commercial name not specified); RCR = rotator cuff repair; UCLA = University of California, Los Angeles; ASES = American Shoulder and Elbow Surgeons; AOFAS = American Orthopaedic Foot and Ankle Society; SST = Simple Shoulder Test; ROM = range of motion

Gene Therapy Approaches to Tendon Regeneration

Gene therapy is a promising method for facilitating trafficking of growth factors to the injured site,¹²³ potentially allowing sustained temporal delivery.¹²⁴ This is accomplished using either direct gene transfer or viral or non-viral vectors coding for growth factors, whose delivery can be achieved by local injection. While these in vivo forms of gene therapy allow accurate introduction of genetic products, they are inconsistent in their efficiency of transduction and expression.¹²⁵ Ex vivo gene therapy relies upon the extraction and viral transduction of autologous cells, cell expansion and antibiotic selection in culture, and reinoculation into the injured tissue.¹²⁶ While the latter method is more labor intensive and time consuming, it provides a means for quality control and greater efficiency.¹²⁷

Preclinical models have given credence to this emerging therapeutic modality. Transfection of tenocytes with BMP12 in a chicken flexor profundus tendon transection model significantly improved the tissue tensile strength and failure load at 4 weeks.¹²⁸ Bone marrow-derived mesenchymal stem cells (BMSCs) transduced with adenovirus carrying human TGF-β1 cDNA accelerated healing of rabbit Achilles tendon defects.¹²⁹ Implantation of BMP12- and TGF-β1 transfected muscle flaps similarly enhanced healing in transected rat Achilles tendons.^130,131 In addition to growth factor-expressing genes, genes involved in tendon cell development and maturation, such as scleraxis,⁷⁹ tenomodulin,⁸⁰ tenascin-C,⁸¹ and Smad8⁸² have also been highlighted for their tissue engineering potential. The clinical importance of such genes in tendon repair was demonstrated in a murine model of flexor digitorum longus (FDL) transection that showed increased Smad8 expression at 4 weeks after tendon repair, in association with neotendon formation, remodeling of intrasynovial adhesions, and improvement of range of motion.¹³² Gulotta et al.¹³³ reported that MSCs transduced with adenoviral-mediated scleraxis conferred improved biomechanical properties of repaired rat supraspinatus tendons up to four weeks post-surgery.

Gene therapy remains a highly experimental treatment approach that has yet to be extended to the clinic. To our knowledge, no human studies have employed gene therapy in the treatment of tendon repair. The challenges to implementing gene therapy in human patients include establishing the most efficient method for delivering genetic material (viral or non-viral vectors versus direct gene transfer), selection of candidate genes for enhancement or repression, the timing of such treatments and minimizing off-target effects.

Growth Factor Treatment Strategies for Tendon Healing

Once administered to an injured tendon, selected growth factors have the capacity to promote differentiation of resident stem cells into tendon fibroblasts for augmentation of tendon healing. Successful therapeutic use of growth factors requires optimal targeting to, and mechanical stimulation of, the site of pathology.¹³⁴ Growth factors shown to contribute to tendon healing include transforming growth factor-β (TGF-β1 and TGF-β3), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF-BB), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and growth and differentiation factor (GDF)-5, -6, and -7.^135,136

While TGF-β has been shown to promote type I and III collagen synthesis, scar formation, and further growth factor elaboration in vitro,¹³⁷ the endotenon-derived cells appear to down-regulate fibrogenic gene expression in comparison with epitenon and sheath cells.¹³⁸ Data from in vivo models indicate a positive effect of TGF-β1 on healing of central third patellar tendon defects in rabbits,¹³⁹ but given that TGF-β1 has been implicated in adhesion formation (fibrosis) of intrasynovial tendons,¹⁴⁰ strategies to modulate its expression have recently received attention in the literature.¹⁴¹ IGF-I expression increases during the inflammatory phase of wound healing, and this growth factor also promotes cell proliferation and migration, and subsequently promotes collagen and ECM synthesis during tissue remodeling.^142–144 Schnabel et al.,⁴⁵ in an equine model of collagenase-induced injury of the superficial digital flexor tendon (SDFT), observed that both MSCs and IGF-I gene-enhanced MSCs improved histologic indices of healing, although few differences were noted when comparing these two groups.

PDGF has been shown to contribute to tendon healing through cell proliferation and upregulation of collagen and ECM synthesis,^145–147 however it secondarily induces inflammatory cytokine expression which can promote scarring. The extent of scar formation depends upon the timing and duration of PDGF administration.¹⁴⁷ Interest in PDGF therapy has been heightened by several animal studies showing a positive effect in aiding the repair of a tendon defect¹⁴⁸ as well as ruptured^149,150 and collagenase-injected^151,152 tendons. Thomopoulos and coworkers^146,153 developed a novel fibrin/heparin-based delivery system for controlled release of PDGF-BB. This approach led to increased cell density, proliferation, collagen I gene expression, and reducible collagen crosslinks favorable for improving intrasynovial flexor tendon repair.

In a rat model of Achilles transection and repair, exogenous application of VEGF, a regulator of angiogenesis, improved tensile strength of the healing tendon at one and two weeks post-injury, potentially via an increased expression of TGF-β.¹⁵⁴ However, VEGF may contribute to tendon pathology,⁵ as it has been shown to be increased in ruptured Achilles tendons.¹⁵⁵ Acting at the site of injury, bFGF facilitates angiogenesis and mesenchymal cell proliferation.^156,157 Also, following transection and repair of rabbit flexor tendons, subcutaneous administration of human recombinant bFGF led to improved collagen indices (fibril diameter and density) and biomechanical properties.¹⁵⁸ Central region patellar tendon defects in rats treated with bFGF exhibited a dose-dependent increase in cell proliferation and collagen III expression but did not improve mechanical function.¹⁵⁶ bFGF showed promise in the early stages of rat supraspinatus tendon to bone healing via acceleration of bone in growth into the scar tissue interface.¹⁵⁹

BMP family members promote cell proliferation, collagen production, and ECM organization,¹⁶⁰ and play a role in the transcriptional regulation of tendon repair,¹³⁵ while also blunting the expression of inflammatory genes.¹⁶¹ Three members of the BMP family (GDF-5,6, and 7) have shown promise in in vivo pre-clinical studies for accelerating tendon repair.^{128,162–165} In a rat model of Achilles tendon repair, adenovirus-mediated gene therapy with GDF-5 increased the number of tendon progenitor cells at the site of healing and improved the tensile strength of the healed tendon.¹⁶⁶ In a murine model using FDL allografts, GDF-5 transduction led to more rapid healing and reduced adhesion formation than controls.¹⁶⁷ In two separate rat Achilles tendon repair studies,^163,164 GDF5-coated sutures augmented the healing response; however, the authors noted the appearance of chondroid regions at the repair site and speculated that this could be corrected by modifying the GDF5 dosage or the local mechanical loading environment.

An immense degree of clinical interest has been directed in recent years towards the use of autologous platelet-rich plasma (PRP) for healing of skeletal tissues including tendon and ligaments, particularly in the context of sports injuries.¹⁶⁸ PRP can be defined as blood plasma containing supra-physiologic levels of platelets, which is rich in numerous growth factors and cytokines considered integral to wound repair,¹⁶⁹ and many pre-clinical reports of efficacy are now available. 24 weeks after creating surgical lesions in the equine SDFT, Bosch and coworkers¹⁷⁰ observed, relative to saline controls, improved histologic, biochemical, and biomechanical properties following a single intratendinous PRP treatment at 7 days post surgery. Beneficial effects of PRP on tendon healing, including increased collagen deposition and improved collagen fiber orientation and mechanical properties were also reported in rat Achilles tendon defect,⁷⁵ rabbit patellar tendon defect,¹⁷¹ rat Achilles tendon transection,¹⁷² and rabbit chronic rotator cuff tear¹⁷³ models. In apparent support of these findings, evidence from several in vitro studies^174–178 suggests that PRP administration stimulates cell proliferation, increases collagen I and III synthesis, and upregulates the expression of PDGF and TGF-β. While animal models generally support the premise of PRP use for tendon healing in clinical applications, not all studies have shown positive effects. For example, in a sheep model of deep digital flexor tendon injury, addition of PRP to autologous mesenchymal stromal cells did not provide a synergistic regenerative response following injection treatment.¹⁷⁹ Furthermore, potential variability in methods of PRP preparation across studies as well as the use of acute tendon injury models should be noted as limitations of such pre-clinical studies.

Clinical outcomes for PRP and its related formulations known as plasma rich in growth factors (PRGF), platelet-rich fibrin matrix (PRFM), and plasma-leukocyte membrane (PLM) have been published, primarily for rotator cuff repair (Table 2).^180–198 There remains uncertainty regarding the mechanism of action of PRP and related therapies, as PRP contains a plethora of bioactive growth factors and other compounds. In addition, conflicting results must be reconciled, as these treatments have variably been superior, non-superior, and inferior to controls in different studies, suggesting the need for better patient selection, standardization of treatment protocols, and/or standardization of radiological and clinical outcome measures. Indeed, there is great heterogeneity in commercially available growth-factor preparations.¹⁸³ Further basic science and clinical investigations will be needed to better elucidate which particular growth factors or combinations of factors provide the greatest therapeutic benefit. Moreover, future clinical trials must clarify not only the optimal dosing and timing of administration but also the most efficient vehicle for delivering growth factors to injured tissue, be it direct injection of soluble factors, introduction of genetic material coding for growth factors, or surgical placement of scaffolds embedded with these factors. A practical challenge is to ensure efficient localization of growth factors to the site of injury as well as adequate retention of the necessary concentration over a managed timeframe.

Table 2.

Clinical studies using growth factor therapies in tendon repair.

Study design	Type of therapy	Study groups	F/U	Outcomes
Sanchez et al190 Retrospective case control	PRGF	Open Achilles repair PRGF (n = 6) No PRGF (n = 6)	50 mos (control) 32 mos (PRGF)	PRGF group had earlier recovery of ROM and earlier return to activity
Randelli et al187 RCT	PRP	Arthroscopic RCR PRP (n = 26) No PRP (n = 27)	3 to 24 mos	PRP group had lower short-term pain scores and significantly higher SST, UCLA, and Constant scores and external rotation strength at 3 months
Jo et al185 RCT	PRP	Arthroscopic RCR PRP (n = 24) No PRP (n = 24)	12 mos	PRP group had lower retear rate, larger CSA, and better overall function; no differences in ASES, Constant, UCLA, DASH, SST, or SPADI scores
Malavolta et al186 RCT	PRP	Arthroscopic RCR PRP (n = 27) No PRP (n = 27)	24 mos	No difference in retear rates or VAS or Constant scores; PRP group had better UCLA scores at 12 months
Filardo et al197 Case series	PRP	Chronic Achilles tendinopathy PRP (n = 27; 7 bilateral)	2, 6, 54.1 mos	Improved VISA-A, Tegner scores
Murawski et al193 Case series	PRP	Chronic Achilles tendinopathy PRP (n = 32)	6 mos	25 of 32 were asymptomatic and returned to sport/activity at 6 mos; 7 required surgery
Kearney et al196 Randomized pilot	PRP	Chronic Achilles tendinopathy PRP (n = 10) Eccentric loading program (n = 10)	1.5, 3, 6 mos	No difference in VISA-A scores (attributed to pilot design)
Ferrero et al198 Case series	PRP	Chronic Achilles (n = 30) or patellar (n = 28) tendinopathy US-guided PRP (n = 58)	6 mos	Improved VISA scores; reduction of hypoechoic areas in 26 tendons on US
Mishra et al194 RCT	PRP	Chronic lateral epicondylitis PRP (n = 116) Active control (n = 114)	12, 24 wks	Better VAS pain improvement and lower rates of elbow tenderness in PRP group at 24 wks, but not 12 wks
Krogh et al195 RCT	PRP	Chronic lateral epicondylitis PRP (n = 20) CSI (n = 20) Saline (n = 20)	3 mos	Pain improved in all groups at 3 mos, but no differences; CSI improved pain more than PRP at 1 mo; CSI reduced color Doppler activity and tendon thickness more than PRP
Thanasas et al192 RCT	PRP	Chronic lateral epicondylitis US-guided PRP (n = 14) Autologous blood (n = 14)	6 wks, 3 mos, 6 mos	Greater VAS pain improvement with PRP only at 6 wks; no difference in Liverpool elbow scores
Weber et al191 RCT	PRFM	Arthroscopic RCR PRFM (n = 30) No PRFM (n = 30)	12 mos	No differences in structural integrity of repair; VAS pain, SST, or ASES scores; or range of motion
Rodeo et al188 RCT	PRFM	Arthroscopic RCR PRFM (n = 40) No PRFM (n = 39)	≥ 12 mos	No difference in intact tendon healing rate or ASES or L’Insalata scores; PRFM use predicted tendon defect at 12 weeks
Antuna et al180 Randomized pilot	PRFM	Arthroscopic RCR PRFM (n = 14) No PRFM (n = 14)	≥ 24 mos	No difference in retear rates or Constant scores
Barber et al181 Comparative series	PRFM	Arthroscopic RCR Two sutured PRFM constructs (n = 20) No PRFM (n = 20)	24 to 44 mos	Persistent tendon defects in more of control than PRFM patients Rowe scores higher in PRFM group, but no differences in ASES, SANE, SST, or Constant scores
Castricini et al183 RCT	PRFM	Arthroscopic RCR PRFM (n = 43) No PRFM (n = 45)	16 mos	No differences in repair integrity or Constant scores
Bergeson et al182 Prospective cohort	PRFM	Arthroscopic RCR	27 mos (control) 13 mos (PRFM)	Retears more common in PRFM group No differences in ASES, UCLA, SANE, Constant, or WORC scores
Ruiz-Moneo et al189 RCT	PRGF	Arthroscopic RCR	12 mos	No differences in rates of tendon healing, UCLA scores, or satisfaction
Gumina et al184 RCT	PLM	Arthroscopic RCR PLM (n = 39) No PLM (n = 37)	13 mos	PLM group had better repair integrity and Constant scores

RCT = randomized controlled trial; PRP = platelet-rich plasma; PRFM = platelet-rich fibrin matrix; PRGF = plasma rich in growth factors; PLM = plasma-leukocyte membrane; RCR = rotator cuff repair; UCLA = University of California, Los Angeles; ASES = American Shoulder and Elbow Surgeons; SANE = Single Assessment Numeric Evaluation; WORC = Western Ontarior Rotator Cuff Index; SST = Simple Shoulder Test; VAS = visual analog scale; SPADI = Shoulder Pain and Disability Index; DASH = Disabilities of the Arm, Shoulder, and Hand; VISA-A = Victorian Institute of Sport Assessment-Achilles; PRTEE = Patient-Rated Tennis Elbow Evaluation; CSA = cross-sectional area; ROM = range of motion; US = ultrasound; CSI = corticosteroid injection

Use of Mesenchymal Stem Cells for Tendon Healing

Adult stem cells are characterized by their distinct ability for self-renewal. Friedenstein et al. were among the first investigators to demonstrate the presence of fibroblast-like stem cells in the bone marrow that were plastic-adherent and capable of differentiating into multiple phenotypes.¹⁹⁹ The fibroblast-like cells were later termed mesenchymal stem (or stromal) cells (MSCs). Two commonly used subtypes of MSCs include bone marrow- and adipose-derived MSCs. Adult stem cells have been harvested from other tissues including muscle, periosteum, adipose tissue, vascular pericytes, dermis and peripheral blood.²⁰⁰ At present the majority of cell therapies focus on the use of MSCs which have multipotent differentiation potential²⁰¹ and can essentially serve as tissue grafts. Once extracted from the connective tissue or peripheral blood of the patient, undifferentiated MSCs can be injected to give rise to tendon fibroblasts at the site of healing.²⁰² Injected MSCs can replenish and/or augment the reparative cellular pool at the site of tissue repair and also exhibit anti-inflammatory and anti-apoptotic properties.²⁰³ Among the disadvantages of BMSCs are the pain associated with bone marrow harvesting and the potential for low cellular yield.²⁰¹

A number of in vivo studies have explored the potential of BMSCs for tendon regeneration. Transected rat Achilles tendons were treated with MSCs cultured under either hypoxic or normoxic conditions,²⁰⁴ and the failure load of the hypoxia-treated group was significantly higher than that of the normoxic group at both 2 and 4 weeks following injury, although immunostaining of collagen types I and III were similar between the groups. Juncosa-Melvin et al. demonstrated the efficacy of MSC-seeded collagen gel constructs for the treatment of both rabbit Achilles²⁰⁵ and patellar tendon defects.^96,206 These studies also highlighted the importance of construct cell density and pre-implantation mechanical stimulation to the biomechanical properties of the healing tissue. Smith and colleagues²⁰⁷ treated tendinopathic horses with an injection of autologous BMSCs suspended in marrow supernatant. Encouragingly, this treatment conferred improvements in tissue structural organization, composition, and vascularity.

Adipose tissue is an attractive site for isolating MSCs due to its abundance and availability, as well as its comparatively minimal harvest-site morbidity.²⁰¹ In a rabbit model, adipose-derived MSCs (ADSCs) augmented the tensile strength of Achilles tendon repair and increased the expression of type I collagen, FGF, and VEGF.²⁰⁸ Injection of an adipose derived stromal vascular fraction into surgically repaired rabbit flexor tendons enhanced the biomechanical properties of the healing tissue.²⁰⁹ In a rabbit model of chronic subscapularis tendon tears,²¹⁰ repairs augmented by ADSCs exhibited decreased muscle fatty infiltration and a trend towards improved mechanical properties relative to a saline treatment control group. Encouraging results have also been reported with tissue engineering strategies whereby ADSCs were seeded onto synthetic (PLGA) scaffolds^97,211 or acellular tendon ECM.²¹² However, ADSCs did not improve mechanical properties of healing supraspinatus tendon to bone repairs in rats.²¹³

A cell population described as tendon-derived stem cells (TDSCs) have been isolated and characterized from rotator cuff tendon, patellar tendon, paratenon, subacromial bursa and Achilles tendon.^214–217 When TDSCs were isolated and implanted as TDSC-fibrin glue constructs into patellar tendon defects using a rat model,²¹⁸ the collagen content, fiber orientation, and biomechanical properties were significantly improved in the TDSC-group relative to fibrin only controls at four weeks post-injury. Pietschmann et al.²¹⁹ created rat Achilles tendon defects which were bridged by PGA and collagen type I scaffolds seeded with either tail tendon-derived cells or BMSCs. Analysis of DNA from the tendon repair site showed that the implanted cells survived at 16 weeks. Consistent with earlier MSC studies, ectopic ossification was found in tendons of both treatment groups. Despite this, the biomechanical properties and collagen alignment of the regenerated tendon were improved relative to those of the MSC-only or scaffold-only groups. Chen et al.²²⁰ developed a rabbit model to repair massive rotator cuff tendon defects using an interpositional graft consisting of autologous tendon cells seeded onto either a porcine SIS or a collagen types I/III bioscaffold. An inflammatory reaction was noted at 4 weeks post-surgery, though its severity was diminished at 8 weeks. While histologic features of the repair tissue of both tendon cell-seeded groups was inferior to that of autograft controls, the authors noted a superior healing response of cell-seeded scaffolds relative to that of scaffolds alone.

In comparison to scaffolds and growth factors, there is a paucity of clinical studies using stem cells for augmentation of tendon repair or for treating tendinopathy (Table 3).^221–225 Stem cells for clinical use have been harvested from multiple sources including but not limited to bone marrow, adipose tissue, dermis, tendon tissue, periosteum, subacromial bursa.^{214,215,226,227} The stem cells can be used as a stand-alone therapy or in combination with other tissue engineering approaches (growth factors and scaffolds). Stem cells can be used directly after harvest or can be concentrated to increase the cell count before use at the site of repair. Some sources, such as tendon or dermis, give low cell yields such that the cells need to be expanded in culture prior to use. Culture expansion of stem cells has its own limitations and safety issues. There is a theoretical risk of infection and prolonged ex vivo culture expansion can induce senescence and lead to loss of the multipotent potential of stem cells. Moreover, the culture expansion step may not be cost-effective.²²⁸ Although the stem cells can be genetically modified ex vivo using viral gene therapy, there are safety reasons (replication competence of viral vectors, insertional mutagenesis, and immune response) that prohibit the use of this strategy for augmenting tendon repair and healing.^229,230

Table 3.

Clinical studies using stem cell therapies for tendon disorders.

Study design	Type of stem cell	Study groups	F/U	Outcomes
Hernigou et al223 Retrospective cohort	BMSCs	45 pts with SR ARCR treated with BMSCs 45 matched controls treated with ARCR alone	≥ 120 mos	BMSC group had higher healing rates at 6 mo and 10 yr on MRI compared to matched controls
Wang et al224 Case series	Patellar tendon derived tenocytes	17 pts with refractory lateral epidcondylitis were treated with patellar tendon derived tenocytes expanded in vitro	12 mos	Signficant improvement in pain score, DASH scores, MRI grading of tendinopathy
Gomes et al225 Case series	BMMC	14 consecutive pts treated with miniopen transosseous RCR + BMMC	≥ 12 mos	Significant postoperative increase in UCLA score, Intact repairs on MRI imaging (100%)
Clarke et al221 RCT	Skin derived tenocyte like cells	60 pts with refractory patellar tendinopathy were randomized to receive USG guided injections of plasma + autologous dermal fibroblast derived tenocytes (n=33) or plasma alone (n=27) in the patellar tendon	6 mos	Significant improvement in the VISA scores in the tenocyte group
Connell et al222 Prospective clinical pilot	Skin derived tenocyte like cells	12 pts with refractory lateral epicondylitis underwent USG guided injections of	12 mos	Significant improvement in PRTEE scores, reduction in number of tears, and thickness of tendon

RCT = randomized-controlled trial; BMMC = mononuclear cell fraction of bone marrow aspirate; USG = ultrasonography; SR = single-row; RCR = rotator cuff repair; ARCR = arthroscopic rotator cuff repair; DASH = Disabilities of the Arm, Shoulder, and Hand; UCLA = University of California, Los Angeles; VISA = Victorian Institute of Sport Assessment; PRTEE = Patient-Rated Tennis Elbow Evaluation

Hernigou et al. retrospectively evaluated a cohort of 45 patients that were treated with concentrated BMSCs as an adjunct to the arthroscopic single row rotator cuff repair (RCR).²²³ A significant difference between the MRI documented healing rates of RCRs treated with MSCs (100% at 6 months and 87% at minimum 10 years) was noted in comparison to matched controls (67% at 6 months and 44% at minimum 10 years). Two studies demonstrated improved clinical results with the use of dermal fibroblasts (suspended in autologous plasma) for treatment of patellar tendinopathy and lateral epicondylitis (Table 3).^221,222 The use of autologous patellar tendon derived tendon cells for the treatment of refractory lateral epicondylitis was demonstrated by Wang et al.²²⁴ A significant improvement in the pain scores, DASH score and MRI grading of tendinopathy was present at one-year follow-up after ultrasound guided injection into the lateral epicondyle.

The use of stem cells for tendon repair and regeneration is an intriguing strategy. Preclinical and clinical studies have demonstrated safety and biologic potential for their use. However, the precise mechanisms by which stem cells participate in tendon healing are unknown,²¹⁴ so that this approach requires further optimization before robust clinical results can be expected. The ideal stem cell type, cell number and ideal scaffold material are topics of ongoing debate and investigation. Improved methods of cell harvest, which can obviate the need for ex vivo expansion is desirable. Strategies for cell harvest which enable their use on the same day and at same time as the index surgical procedure, will facilitate transplantation of highly active cells at the site of repair, and this approach is expected to improve the likelihood of clinical success.

Summary and Future Directions

Despite a myriad of clinical advances in treating tendinopathies, there remains an unmet need for effective therapeutic strategies to augment intrinsic and/or surgical repair. The majority of clinical studies to date have been performed in patients undergoing RCR, and as such these treatment concepts must be recapitulated for other common tendon injuries. The most robustly tested treatment modalities have been PRP and related formulations harboring growth factors as well as ECM-based scaffolds to augment or interpose between the tendon repair. To date, tissue engineering strategies have been harnessed to augment tendon healing in vitro and in vivo with some success. These strategies include the use of scaffolds, cell therapies, gene therapies, and growth factor therapies. Many of these applications have shown early promise in the clinical setting in patients undergoing tendon repair, and merit continued study in prospective randomized trials. Preclinical large-animal models have provided a substantial amount of data to allow extension of nascent biologic therapies into clinical models, though their limitations must be kept in mind, including discrepancies in tendon anatomy and size, age, injury acuity, ability to simulate postoperative rehabilitation protocols within animals as compared to human subjects.²³¹ Moreover, whereas the vast majority of pre-clinical studies utilize acute injury models, more commonly the human tendon disease “phenotype” is chronic and degenerative.

Therapeutic strategies which have received minimal attention include the use of disease-modifying agents such as chondrolytics for targeted removal of mucoid deposits, which are among the most common histopathologic features of tendinopathy (Figure 1). In addition, the further delineation of disease-linked human mutations^232–234 and analysis of epigenetic changes in animal models^235–237 and human cohorts are expected to facilitate future research into targeted therapeutics and potentially circulating DNA biomarkers of early-stage tendinopathies. Further clinical translation will require improved functional integration of these therapies toward more robust regeneration of native tissue structure, composition, and function. In addition, combination therapies simultaneously employing two or more tissue engineering strategies should be further studied as they may prove to be synergistic in addressing this pathologically complex problem.