Abstract
Tendon injury may occur suddenly or progressively, and can be divided into tendon rupture or tendinopathy based on the severity of injury. It is frequently found in professional or nonprofessional people who are making repetitive movements. In aged people, tendon degeneration becomes obvious; their tendon injuries are then frequently evident. No effective therapies for tendon injury are currently available. In this article, we review the tendon structure, mechanisms of tendon injury, and tendon healing process. More importantly, cell-based therapies for tendon injury are fully addressed, which will play an important role for tendon therapy in the near future.
Introduction
More than 100 million people worldwide have musculoskeletal injuries each year, and about 30–50% of them are tendon or ligament injuries (59). Tendon areas are formed by special connective tissues and these connective tissues connect muscles to bones, transmit force to the joint, and facilitate articulation in the musculoskeletal system. Tendon injuries may develop either suddenly or gradually. Based on the degree of injury, tendon injuries may be divided into tendon rupture and tendinopathy (81). Tendon injuries are common in professional or amateur athletes (52) or the people with jobs of highly repetitive movements (31,56,59). Tendon degenerations may occur more frequently as people get older and may also increase in the incidence of injuries (32). Based on many clinical experiences, we have found that injured tendons cannot recover to the original normal status as expected (20,81,85). Tendinopathy is the most frequent tendon disorder (1,29,52) and is usually described as an overuse of the tendons (56). The histological evidence of tendinopathy has been reported by Longo et al. and Maffuli et al. (48,55,57).
Tendinopathy can be found in any tendon and is frequently found in the following tendons: Achilles, patellar, shoulder rotator cuff, and forearm extensor tendons (72). The term “tendinosis” represents the degenerative pathological condition without inflammatory changes. On the other hand, tendonitis or tendinitis means an inflammatory process. These conditions can be confirmed by histopathological studies (56). Research studies on possible treatments include randomized controlled trials such as the use of nonsteroidal anti-inflammatory medications (2,3,34), eccentric exercise (19,23,69,74,75), topical glyceryl trinitrate (17,21,33,40,66–68), sclerosing injection treatment (14,42,97,98), nonsteroid materials injections (15), low-intensity pulsed ultrasound (LIPUS) (94,95), and shock wave therapy (25,77,79,88,89). What may appear clinically as an acute tendinopathy is actually a well-advanced failure of a chronic healing response in which neither histological nor biochemical evidence of inflammation appears (57,58). Many challenging theories explain the pathogenesis and the manifestation of tendinopathy in different stages (24,49–51). Cell therapy is one type of new therapy for tendinopathy after tendon injury (83,85). In this review, we focus on the recent cell therapy developments for the tendon injuries in animal studies and clinical trials.
Tendon Structure
Normal tendons are brightening white in color and have a fibroelastic texture. Tendons morphology are variable in forms; they can be rounded cords, straplike bands, or flattened ribbons (5). Tenoblasts and tenocytes account for 90–95% of tendon extracellular matrix network (41). The other 5–10% of cellular contents include chondrocytes, synovial cells, capillary endothelial cells, and smooth muscle cells of arterioles (81). The oxygen consumption of tendon is about 7.5 times less than that for skeletal muscles (93). In tendons, the metabolic rate is quite low and the anaerobic energy generation has been well developed, so that tendons can tolerate energy crisis and avoid ischemia and necrosis. However, the low metabolic rate in tendons can cause a slow healing process after injury (99). The dry mass of human tendons is about 30% of total tendon mass; 65–80% of them are collagen type 1 fibers and only 2% of them are elastin fibers (30,38,64,91). There are proteoglycans, glycoproteins, glycosaminoglycans, and several other molecules inside the extracellular matrix network (41). Proteoglycans are strongly hydrophilic, water-soluble molecules. Adhesive glycoproteins, such as fibronectin and thrombospondin, are important contents involving the tendon repair and regeneration processes (37,46,60). Another important content is tenascin-C in the tendon body or at the osteotendinous and myotendinous junctions (22,73). Tenascin-C is regulated by mechanical strain and is unregulated in tendinopathy (22,45,87). Tenascin-C may be a factor in collagen fiber alignment and orientation (8).
Tendon Injury and Healing Process
Tendon injury can be divided into two categories: rupture injury and nonrupture injury. Nonrupture injuries include tendonitis and tendinosis. Tendonitis is an acute inflammatory condition with swelling of tendons and the accumulation of inflammatory cells. Tendinosis is used to describe any nonruptured tendon problem with histopathologic changes due to degeneration with no evidence of inflammation (12,18,72). Some authorities suspected that tendinosis is a pathologic condition due to the imbalance between the synthesis and degradation of extracellular matrix (47,80). This condition leads to structural deterioration and degeneration of the tendon.
Tendinopathy is a clinical diagnosis including any type of tendon problem with pathologic change and pain. The etiology of tendinopathy remains unclear. Many causes have been investigated. The maximal tensile load in a tendon can cause ischemia. During tendon relaxation, reperfusion can occur, but oxygen-free radicals can also be produced (26,51) and can induce tendon damage to cause tendinopathy (6,51,61). Hypoxia may be another cause of tendon degeneration, since tendons must depend on aerobic metabolism to maintain cellular ATP levels (7). During vigorous exercise, localized hypoxia may induce tenocyte death.
Tendinosis is usually a term to describe the pathologic features of the extracellular matrix network in tendinopathy (70). Histopathological examination shows that tendinopathy tendons contain no inflammatory cells or any healing process with haphazard proliferation of tenocytes. There are also disruption of collagen fibers, increased noncollagenous matrix, scattered vascular ingrowths, and increased interfibrillar glycosaminoglycans in the tendons with tendinopathy (12,18,57,58,82). The reason why a tendinopathic tendon can be injured more easily is due to increased speed of matrix remodeling to cause an unstable mechanical condition (9). There has been little or no evidence of inflammation in the histological findings of the specimens obtained from the tendinopathic tendons (48). In fact, hypercellularity, lack of tightly bundled collagen fibers, and neovascularization can be found in the tendinopathic tendons (53,54). Inflammation could be important in the early stage (before degeneration), but is therefore not important during the developmental process of tendinopathy (71). The repair activity after tendon injury occurs in the epitenon and endotenon cells, but moves into the lesion region to synthesize the new matrix (36). These cells are thought to be derived from a resident population of stem cells, which can differentiate into mesen-chymal tissues, such as cartilage and tendon (76).
Stem Cell Therapy
Stem cells have the potential to develop into many different cell types. Adult stem cells can be used for repairing tissue and treating diseases based on their ability of multilineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The special abilities of stem cells can be applied for the development of regenerative medicine and tissue engineering (62). After the duplication of a stem cell, the new cells can be still a stem cell or become another type of cell with special function such as tenocyte or osterocyte. Recently, the scientists focus on the research on two types of stem cells in either human or animal: embryonic stem cells and non-embryonic (somatic or adult) stem cells. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be “reprogrammed” genetically to assume a stem cell-like state. This new type of stem cell has been called the induced pluripotent stem cell (iPSC) (90).
No FDA-approved stem cell technologies are currently available for the treatment of degenerative conditions such as tendinopathy. Theoretically, stem cells can be isolated and then injected into the area such as an arthritic joint or a degenerative tendon. Once the stem cells are in the desired location, either local signaling or the addition of exogenous factors can drive the stem cells to differentiate into the target cell line. Recently, stem cell technology has been applied in the synthesis of tendon or ligament grafts and to enhancing graft incorporation (39,43,86). Bone marrow (BM) stem cells can be used for treating overuse tendinopathy in the horse for several years with good results, but has not been used in clinical trials for human tendinopathy (83,84). In recent years, animal experiments have been done for multifaceted cell types of the rats and rabbits, such as human embryonic stem cells (hESCs), bone marrow-mesenchymal stem cells (BM-MSCs), bone marrow cells (BMCs), adipose-derived mesenchymal stem cells (ADMSCs), and so on. A series of animal studies have suggested that stem cell technology may have a role in tendon grafting and repair, but still have been uncertain if this technology can be applied successfully for the treatment of human tendinopathy. We need further experimental studies in the future.
Human Embryonic Stem Cells
Human embryonic stem cells (hESCs) are ideal cells for tissue regeneration. Chen et al. investigated the efficacy of using hESCs for tendon regeneration (10). The hESC-derived MSCs (hESC-MSCs) regenerated tendon tissues in both in vitro tissue engineering model and in vivo ectopic tendon regeneration model, which were confirmed by the tendon-specific genes and structure. In rat patellar tendon repair model, injured tendon treated with hESC-MSCs had much better structural and mechanical properties than controls. Furthermore, hESC-MSCs remained visible at the tendon wound site for at least 4 weeks. The authors also mentioned that the remaining cells secreted human fetal tendon-specific matrix components and differentiation factors, which activated the endogenous regeneration process in tendons. No teratomas were found in any sample, and these findings demonstrate a safe and practical method of applying ESCs for tendon regeneration and may be applicable for treating tendon diseases in the future (10,11). Watts et al. used fetal derived embryonic-like stem cells (fdESCs) to study horse superficial digital flexor tendon (SDFT) tensile region lesion, which was prepared by collagenase induction (96). Serial ultrasound and final analysis at 8 weeks including magnetic resonance imaging (MRI), biochemical assays, gene expression, and histology were performed. No differences were found in tendon matrix specific gene expression, total proteoglycan, collagen, or DNA of tendon lesions. Tissue architecture, tendon size, tendon lesion size, and tendon linear fiber pattern were significantly improved on histologic sections and ultrasound in the fdESC-treated tendons (96).
Bone Marrow-Mesenchymal Stem Cells
MSCs are the first reported and mostly used stem cell type to help tendon repair. MSCs are one of the most commonly occurring stem cells. They can be easily derived from most tissues and have the potential to differentiate into tenocytes in vitro and in vivo. The use of MSCs in animal studies includes all of the previously mentioned animals such as horses, rabbits, rats, etc. These tendon studies include Achilles tendon, SDFT, patellar tendon, and rotator cuff tendon. Practical standardized treatments were used for the SDFT central cord area overuse tendinopathy in horses (16,85). In the horse clinical treatment studies, the authors evaluated the effect of implanting BM-MSCs on the progression of tendon injury in overused disease. However, this study did not provide strong evidence since they did not have a control group in their study. In addition, they did not apply detail and acceptable evaluations outcome measures. Therefore, we still have no strong evidence to apply this technique for treating tendon diseases in humans (83). The authors would like to further understand the important roles of MSCs and insulin-like growth factor 1 (IGF-I) gene-enhanced MSCs (AdIGF-MSCs) in the in vivo process of tendon healings. They used collagenase to induce bilateral tendinitis in equine SDFTs. Then they applied MSCs or AdIGF-MSCs to treat it. In their control group, the tendon was injected with 1 ml of phosphate-buffered saline (PBS). Horses were euthanized at 8 weeks and SDFTs were evaluated for biochemical composition and histologic characteristics. Both MSC and AdIGF-MSC injections resulted in significant improvement of tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF-MSCs for the treatment of tendinitis (78). In rabbit studies, Awad et al. implanted MSCs into a surgically induced defect in the donor rabbit's right patellar tendon (4). A cell-free collagen gel was implanted into a control defect in the left patellar tendon. Repair tissues were evaluated biomechanically and histomorphometrically at 4 weeks after surgery. The MSC-mediated repair tissue demonstrated significant increases of 26% (p < 0.001) in maximum stress, 18% (p < 0.01) modulus, and 33% (p < 0.02) strain energy density compared to their matched controls. A minor increase in the number of tenocytes and larger and more mature-looking collagen fiber bundles were identified from the histological appearance of some of the MSC-mediated repaired tendons. There were no significant left-right differences in nuclear aspect ratio or nuclear alignment. Therefore, delivering a large number of MSCs to a wound site can significantly improve its biomechanical properties by only 4 weeks (4). In an Achilles tendon study, Young et al. cultured autologous and marrow-derived MSCs, which were suspended in a collagen gel delivery vehicle; the cell–gel composite was subsequently contracted onto a pretensioned suture (100). The resulting tissue prosthesis was then implanted into a 1-cm-long gap defect in the rabbit Achilles tendon, the contralateral tendon receiving only the suture material as a control. Significantly greater load-related structural and material properties were seen at all time intervals in the MSC-treated tendons than in the contralateral, treated control repairs (p < 0.05), which contained suture alone with natural cell recruitment. The values were typically twice of those for the control tissues at each time interval. Load-related material properties for the treated tissues also increased significantly over time (p < 0.05). The treated tissues had a significantly larger cross-sectional area (p < 0.05), and their collagen fibers appeared to be better aligned than those in the matched controls. The results indicate that the biomechanics, structure, and function of the inured tendon can be significantly improved after the implantation of MSC-contracted, organized collagen into the site of large tendon defects (100). Chong et al. used this injury model to make transaction at the Achilles tendon midsubstance (13). The transected tendon was immediately repaired with the use of a modified Kessler suture and a running epitendinous suture. Both limbs were used, and each side was randomized to receive either bone marrow-derived mesenchymal stem cells in a fibrin carrier or fibrin carrier alone (control). There were no differences between the two groups with regard to the gross morphology of the tendons. The fibrin had degraded by 3 weeks. It was found in cell tracing that the labeled bone marrow-derived MSCs remained viable and presented in the intratendinous region for at least 6 weeks, becoming more diffuse at later time periods. At 3 weeks, collagen fibers appeared more organized and there were better morphometric nuclear parameters in the treatment group (p < 0.05). Biomechanical testing showed improved modulus in the treatment group as compared with the control group at 3 weeks (p < 0.05) but not at subsequent time periods. At 6 and 12 weeks, there were no differences between the groups with regard to morphometric nuclear parameters. He concluded intratendinous cell therapy with bone marrow-derived MSCs following primary tendon repair can improve histological and biomechanical parameters in the early stages of tendon healing (13). In the rat studies, the Gulotta group used 98 Lewis rats, which underwent the unilateral detachment and the attempted repair of the supraspinatus tendon; 10 rats were used for MSC harvest (28). Eight animals were used for cell tracking with adenovirus expressing β-galactosidase (Ad-LacZ). The remaining animals received either 106 MSCs in a fibrin carrier, the carrier alone, or nothing at the repair site. Animals were sacrificed at 2 and 4 weeks for histologic analysis to determine the amount of fibrocartilage formation and the collagen organization at the insertion. There were no differences in the amount of new cartilage formation or collagen fiber organization between groups at either time point. There were also no differences in the biomechanical strength of the repairs, the cross-sectional area, peak stress to failure, or stiffness. The conclusion of these studies is the addition of MSCs to the healing rotator cuff insertion site did not improve healing process in all characteristics of rotator cuff tendon (27). In another study, same authors used Ad-Scx (adenoviral-mediated scleraxis)-transduced MSCs to Lewis rats that underwent the unilateral detachment and the repair of the supraspinatus tendon. There were no differences between the Scx and MSC groups in terms of histologic appearance at 2 weeks. However, the Scx group had higher ultimate stress-to-failure and stiffness compared with the MSC group. At 4 weeks, the Scx group had more fibrocartilage, higher ultimate load to failure, higher ultimate stress to failure, and higher stiffness values as compared with the MSC group (28).
Bone Marrow Cells
The Okamoto group used the bone marrow cells to treat the Achilles tendon rupture in rats (65). They used nine Fisher rats, which were the source of bone marrow cells and MSCs, and two syngeneic transgenic male rats expressing green fluorescence protein (GFP) as the source of bone marrow cells. Eighty-seven Fisher rats were used for the experiments. The rats were divided into three groups and injected the materials around the tendon in two groups. The three groups were the BMC group, the MSC group, and the nontreated control group (incision only). They found that transplanted GFP-positive cells could be detected around the incised region not only at 7 days but also at 28 days after incision. These findings suggest that the donor-derived cells were able to migrate and differentiate into fibroblasts, thereby accelerating the tendon healing process. In the MSC group, the ultimate failure load at 7 and 14 days after incision was significantly higher than that in the nontreated group, although the ultimate failure load at 28 days after incision was not significantly greater than that in the nontreated group. In contrast, in the BMC group, the ultimate failure load at 28 days after incision was significantly greater than that in the nontreated group. These findings indicate that bone marrow cell transplantation therapy is more effective than transplantation of mesenchymal stem cells alone in achieving Achilles tendon healing in this rat model (65).
BMMN Cells
The Crovace and Lacitignola groups compared cultured bone marrow mesenchymal stem cells (cBMSC), bone marrow mononucleated cells (BMMNCs), and placebo for repairing collagenase-induced tissue damage in an equine model of experimental tendonitis (16,44). Histological and immunohistochemical staining with H&E, Herovici, and collagen types 1 and 3 revealed mature type 1 collagen with normal architecture in tendons treated with cBMSCs and BMMNCs, while random collagen type 3 organization was observed in the tendons treated with placebo (16,44).
Adipose-Derived Stem Cells (ASCs)
Uysal et al. established a suitable experimental model for the primary tendon repair using ASCs (92). Topical administration of ASCs to the site of injury accelerates tendon repair, as exhibited by a significant increase in tensile strength, direct differentiation of ASCs toward tenocytes and endothelial cells, and increases in angiogenic growth factors. These findings suggest that ASCs may have a positive effect on primary tendon repair and may be useful for future cell-based therapy (92). James et al. investigated the effect of growth differentiation factor-5 (GDF-5) on cell proliferation and gene expression by primary rat adipose-derived stem cells (ADSCs) that were cultured on a poly(dl-lactide-co-glycolide) PLAGA fiber scaffold and compared to a PLAGA 2D film scaffold (35). The electrospun scaffold mimics the collagen fiber bundles presenting in tendon tissue and supports the adhesion and proliferation of multipotent ADSCs. Gene expression of scleraxis was upregulated seven- to eightfold at 1 week with GDF-5 treatment when cultured on a 3D electrospun scaffold and was significantly higher at 2 weeks compared to 2D films with or without GDF-5 treatment. They concluded stimulation with GDF-5 can modulate primary ADSCs on a PLAGA fiber scaffold to produce a soft, collagenous musculoskeletal tissue that fulfills the need for tendon regeneration (35). Nixon et al. used collagenase to induce tendinitis in the superficial digital flexor tendon of one forelimb in study horses (63). Four horses were treated by the injection of autologous ADNC (adipose-derived nucleated cell) fractions, and four control horses were injected with the PBS solution. The histological evaluation revealed a significant improvement in tendon fiber architecture and reductions in vascularity, inflammatory cell infiltrate, collagen type III formation, and tendon fiber density and alignment in ADNC-treated tendons (63).
Conclusion
Cell-based tissue engineering for musculoskeletal tissues repair and regeneration holds great promise for the future. MSCs are still the major development stream for treatment, and the source of MSCs varies from BM to self-derived adipose tissue or peripheral blood. In addition, the studies showed that fetal derived embryonic stem cell applications have just begun. In the future research studies, it is important to find how to identify cell unique markers and mapping lineage development. Based on the development of stem cell biology, MSCs will play an important role in clinical applications and tissue engineering. In the near future, the research direction would be expected to the application of adult stem cells for the human studies in phase I and phase II.
Footnotes
Acknowledgment
The authors declare no conflict of interest.