Remodeling of murine intrasynovial tendon adhesions following injury: MMP and neotendon gene expression

Tendon injury frequently results in the formation of adhesions that reduce joint range of motion. To study the cellular, molecular, and biomechanical events involved in intrasynovial tendon healing and adhesion formation, we developed a murine flexor tendon healing model in which the flexor digitorum longus (FDL) tendon of C57BL/6 mice was transected and repaired using suture. This model was used to test the hypothesis that murine flexor tendons heal with differential expression of matrix metalloproteases (MMPs), resulting in the formation of scar tissue as well as the subsequent remodeling of scar and adhesions. Healing tendons were evaluated by histology, gene expression via real-time RT-PCR, and in situ hybridization, as well as biomechanical testing to assess the metatarsophalangeal (MTP) joint flexion range of motion (ROM) and the tensile failure properties. Tendons healed with a highly disorganized fibroblastic tissue response that was progressively remodeled through day 35 resulting in a more organized pattern of collagen fibers. Initial repair involved elevated levels of Mmp-9 at day 7, which is associated with catabolism of damaged collagen fibers. High levels of Col3 are consistent with scar tissue, and gradually transition to the expression of Col1. Scleraxis expression peaked at day 7, but the expression was limited to the original tendon adjacent to the injury site, and no expression was present in granulation tissue involved in the repair response. The MTP joint ROM with standardized force on the tendon was decreased on days 14 and 21 compared to day 0, indicating the presence of adhesions. Peak expressions of Mmp-2 and Mmp-14 were observed at day 21, associated with tendon remodeling. At day 28, two genes associated with neotendon formation, Smad8 and Gdf-5, were elevated and an improvement in MTP ROM occurred. Tensile strength of the tendon progressively increased, but by 63 days the repaired tendons had not reached the tensile strength of normal tendon. The murine model of primary tendon repair, described here, provides a novel mechanism to study the tendon healing process, and further enhances the understanding of this process at the molecular, cellular, and biomechanical level. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 833–840, 2009     

Scleraxis expression is coordinately regulated in a murine model of patellar tendon injury

This study investigated the expression of Scleraxis in a murine model of patellar tendon injury in which the central third of the patellar tendon was unilaterally injured. The presence of tendon pathology was assessed using dual photon microscopy, conventional histology and microCT. Tendon pathology was also quantified noninvasively over a 12‐week period using high‐frequency ultrasound and laser Doppler flowmetry. Gene expression (Scx, Tnmd, and Col1a1) was determined at defined end‐points (1, 4, 8, and 12 weeks) using qPCR on RNA from individual patellar tendons on injured and uninjured sides. There was significant development of tendon pathology as gauged by ultrasound and laser Doppler over 12 weeks. Injured tendons demonstrated significant histological and microCT evidence of pathological change, and disorganized collagen with reduced density. The expression of Scx and Col1a1 was unchanged at 1 week, significantly upregulated at 4 and 8 weeks, and had returned to baseline by 12 weeks. Tnmd expression was unchanged at 1 week, and significantly increased at 4, 8, and 12 weeks. Patellar tendon injury was associated with marked increases in the expression of Scx, Tnmd, and Col1a1. Our data suggest new roles for Scleraxis in coordinating the response to injury in the pathogenesis of tendon disorders. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:289–296, 2011     

Structural and mechanical effects of in vivo fatigue damage induction on murine tendon

The purpose of this study was to develop and validate an in vivo mouse model of tendon fatigue and use this model to investigate and quantify the physical manifestations of fatigue damage in mouse tendon. Patellar tendons of C57BL/6J mice were fatigue loaded at 2 Hz to three endpoints (4 N peak force per cycle for 1 h, 6 N for 1 h, and 4 N for 2 h), during which hysteresis, tangent stiffness, and peak strain of each cycle were measured. Damage accumulation was then quantified using in situ histology, and each tendon was loaded monotonically to failure. Histological damage increased significantly in all three groups (≥2-fold), and monotonic stiffness decreased significantly in the 6 N, 1 h and 4 N, 2-h groups (~25%), suggesting that damage initially manifests as changes to the collagen structure of the tendon and subsequently as changes to the function. For the fatigue loading protocols used in this study, none of the evaluated real-time parameters from fatigue loading correlated with damage area fraction measured structural damage or monotonic stiffness, suggesting that they are not suited to serve as proxies for damage accumulation. In future studies, this model will be used to compare the biological response of mouse tendon to fatigue damage across genetic strains.     

Cycle‐dependent matrix remodeling gene expression response in fatigue‐loaded rat patellar tendons

Expression profiling of selected matrix remodeling genes was conducted to evaluate differences in molecular response to low‐cycle (100) and high‐cycle (7,200) sub‐failure‐fatigue loading of patellar tendons. Using our previously developed in vivo patellar tendon model, tendons were loaded for 100 or 7,200 cycles and expression of selected metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and collagens were quantified by real‐time RT‐PCR at 1‐ and 7‐day post‐loading. Expression profiles were also obtained from lacerated tendons as an acute injury model. The high‐cycle group showed upregulation of TIMP‐1, ‐2, Col3a1, and Col5a1, and downregulation TIMP‐4 at both time points, upregulation of MMP‐2 at 7‐day post‐loading and downregulation of MMP‐13 and ‐14 at 1‐day post‐loading, suggesting overall repair/remodeling. In contrast, the low‐cycle loaded group showed upregulation of MMP‐2, ‐3, ‐13, and Col12a1 at both time points, upregulation of TIMP‐1, ‐2, ‐3, Col3a1, and integrin β1 and downregulation of integrin α11 at 1‐day post‐loading and upregulation of Col1a1 at 7‐day post‐loading, consistent with a hypertrophic (adaptive) pattern. Lacerated tendons showed a typical acute wound response with upregulation of all examined remodeling genes. Differences found in tendon response to high‐ and low‐cycle loading are suggestive of the underlying mechanisms associated with a healthy or damaging response. Published by Wiley Periodicals, Inc. J Orthop Res 28:1380–1386, 2010     

CD44 deficiency improves healing tendon mechanics and increases matrix and cytokine expression in a mouse patellar tendon injury model

CD44 plays an important role in inflammation and healing. Previous studies investigated its role in inflammatory diseases and skin wounds; however, the role of CD44 in tendon healing is unknown. Therefore, we investigated the effect of CD44 in the healing of the patellar tendon in a knockout mouse model. We hypothesized that in comparison to wild‐type counterparts, CD44 knockout mice would have decreased material parameters, increased organization, decreased expression of proinflammatory cytokines, and increased expression of matrix components during healing. These hypotheses were tested through an in vivo surgical model and mechanical, organizational, and gene expression analyses. Material strength and tissue organization were significantly improved in the CD44 knockout mouse. This could be attributed to increased expression of cytokines and matrix components that are also elevated in regenerative healing. Our study showed that the absence of CD44 in a mouse patellar tendon injury creates an environment that is conducive to regenerative healing through altered gene expression, resulting in superior material properties and reduced cross‐sectional area. Therefore, limiting the role of CD44 may improve healing parameters in adult tendon injury. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1386–1391, 2009     

Impaired biomechanical properties correlate with neoangiogenesis as well as VEGF and MMP‐3 expression during rat patellar tendon healing

Recent studies reveal an important role of vascular endothelial growth factor (VEGF)‐induced angiogenesis in degenerative tendon diseases. The way how VEGF influences mechanical properties of the tendons is not well understood yet. We here hypothesized that tendinopathy results in a hypoxia‐mediated stimulation of VEGF and that the mechanical stability of the tendon is impaired in an angiogenic process by VEGF‐induced matrix metalloproteinases (MMPs). A modified in situ freezing model of patellar tendon was used to create a tendinopathy. 0, 7, 14, and 28 days post‐surgical animals were sacrificed and patellar tendons were dissected for biomechanical and immunohistochemical analysis. Native tendons were used as controls. Immunohistochemical staining revealed a peak in HIF‐1α stabilization immediately after surgery. Both VEGF and MMP‐3 were increased 7 days after surgery. Angiogenesis was also abundant 7 days after surgery. In contrast, biomechanical stability of the tendon was decreased 7 days after surgery. The current results reveal a time‐dependent correlation of HIF‐1/VEGF‐induced and MMP‐3‐supported angiogenesis with decreased biomechanical properties during tendon healing. The therapeutical modulation of neoangiogenesis by influencing the level of VEGF and MMP‐3 might be a promising target for new approaches in degenerative tendon diseases. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1952–1957, 2012     

The native cell population does not contribute to central‐third graft healing at 6, 12, or 26 weeks in the rabbit patellar tendon

Investigators do not yet understand the role of intrinsic tendon cells in healing at the tendon‐to‐bone enthesis. Therefore, our first objective was to understand how the native cell population influences tendon autograft incorporation in the central‐third patellar tendon (PT) defect site. To do this, we contrasted the histochemical and biomechanical properties of de‐cellularized patellar tendon autograft (dcPTA) and patellar tendon autograft (PTA) repairs in the skeletally mature New Zealand white rabbit. Recognizing that soft tissues in many animal models require up to 26 weeks to incorporate into bone, our second objective was to investigate how recovery time affects enthesis formation and graft tissue biomechanical properties. Thus, we examined graft structure and mechanics at 6, 12, and 26 weeks post‐surgery. Our results showed that maintaining the native cell population produced no histochemical or biomechanical benefit at 6, 12, or 26 weeks. These findings suggest that PTA healing is mediated more by extrinsic rather than intrinsic cellular mechanisms. Moreover, while repair tissue biomechanical properties generally increased from 6 to 12 weeks after surgery, no further improvements were noted up to 26 weeks. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 638–644, 2013     

Sodium cromolyn reduces expression of CTGF,ADAMTS1, and TIMP3 and modulates post‐injury patellar tendon morphology

The purpose of this study was to determine whether administration of a mast cell inhibitor (sodium cromolyn, SC) would influence tendon repair and extracellular matrix gene expression following acute injury. CD1 mouse patellar tendons were unilaterally injured and mast cell prevalence was determined. The effect of SC injection on tendon hypercellularity, cross‐sectional area, collagen organization, and expression of extracellular matrix‐related genes was examined. Mast cell prevalence was markedly increased in injured patellar tendons (p = 0.009), especially at 8 weeks post‐injury (p = 0.025). SC injection increased collagen organization compared to uninjected animals at 4 weeks and attenuated the development of tendon hypercellularity and tendon thickening post‐injury. Expression of CTGF, ADAMTS1, and TIMP3 in injured tendon was reduced in the SC group. SC injections moderated the structural alterations of healing tendon in association with downregulation of several genes associated with tendon fibrosis. This work corroborates previous findings pointing to a role of mast cells in tendon repair. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:678–683, 2011     

Tendon to bone tunnel healing—A study on the time‐dependent changes in biomechanics,bone remodeling,and histology in a rat model

Tendons and ligaments attach to bone through a transitional connective tissue with complex biomechanical properties. This unique tissue is not regenerated during healing, and surgical reattachment therefore often fails. The present study was designed to evaluate tendon healing in a bone tunnel and to evaluate the utilized rat model. Wistar rats (n = 61) were operated with the Achilles tendon through a bone tunnel in the distal tibia. Healing was evaluated at 2, 3, 4, and 12 weeks by biomechanical testing, bone mineral density and histology. After 2 weeks median (interquartile range) pull‐out force was 2.2 N (1.9). The pull‐out force increased chronologically, by 12 weeks fivefold to 11.2 N (11.4). Energy absorption, stiffness, and bone mineral density increased similarly. The histological analyses showed inflammation at early stages with increasing callus by time. Our data showed a slow healing response the first 4 weeks followed by an accelerated healing period, favoring that most of the gain in mechanical strength occurred later than 4 weeks postoperatively. These findings support the concern of a vulnerable tendon bone tunnel interface in the early stages of healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:216–223, 2015.     

Ablating hedgehog signaling in tenocytes during development impairs biomechanics and matrix organization of the adult murine patellar tendon enthesis

Restoring the native structure of the tendon enthesis, where collagen fibers of the midsubstance are integrated within a fibrocartilaginous structure, is problematic following injury. As current surgical methods fail to restore this region adequately, engineers, biologists, and clinicians are working to understand how this structure forms as a prerequisite to improving repair outcomes. We recently reported on the role of Indian hedgehog (Ihh), a novel enthesis marker, in regulating early postnatal enthesis formation. Here, we investigate how inactivating the Hh pathway in tendon cells affects adult (12‐week) murine patellar tendon (PT) enthesis mechanics, fibrocartilage morphology, and collagen fiber organization. We show that ablating Hh signaling resulted in greater than 100% increased failure insertion strain (0.10 v. 0.05 mm/mm, p<0.01) as well as sub‐failure biomechanical deficiencies. Although collagen fiber orientation appears overtly normal in the midsubstance, ablating Hh signaling reduces mineralized fibrocartilage by 32%, leading to less collagen embedded within mineralized tissue. Ablating Hh signaling also caused collagen fibers to coalesce at the insertion, which may explain in part the increased strains. These results indicate that Ihh signaling plays a critical role in the mineralization process of fibrocartilaginous entheses and may be a novel therapeutic to promote tendon‐to‐bone healing. © 2015 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 33:1142–1151, 2015.     

Microarray analysis of healing rat Achilles tendon: evidence for glutamate signaling mechanisms and embryonic gene expression in healing tendon tissue.

Tendon healing is a complex process consisting of a large number of intricate pathways roughly divided into the phases of inflammation, proliferation, and remodeling. Although these processes have been extensively studied at a variety of levels in recent years, there is still much that remains unknown. This study used microarray analyses to investigate the process at a genetic level in healing rat Achilles tendon at 1, 7, and 21 days postinjury, roughly representing the inflammation, proliferation, and remodeling phases. An interesting temporal expression profile was demonstrated, identifying both known and novel genes and pathways involved in the progression of tendon healing. Both inflammatory response and pro-proliferative genes were shown to be significantly upregulated from 24 h postinjury through to 21 days. Day 7 showed the largest increase in genetic activity, particularly with the expression of collagens and other extracellular matrix genes. Interestingly, there was also evidence of central nervous system-like glutamate-based signaling machinery present in tendon cells, as has recently been shown in bone. This type of signaling mechanism has not previously been shown to exist in tendon. Another novel finding from these analyses is that there appears to be several genes upregulated during healing which have exclusively or primarily been characterized as key modulators of proliferation and patterning during embryonic development. This may suggest that similar pathways are employed in wound healing as in the tightly regulated progression of growth and development in the embryo. These results could be of use in designing novel gene-based therapies to increase the efficacy and efficiency of tendon healing.     

Ectopic chondro‐ossification and erroneous extracellular matrix deposition in a tendon window injury model

The acquisition of chondro‐osteogenic phenotypes and erroneous matrix deposition may account for poor tissue quality after acute tendon injury. We investigated the presence of chondrocyte phenotype, ossification, and the changes in the expression of major collagens and proteoglycans in the window wound in a rat patellar tendon window injury model using histology, von Kossa staining and immunohistochemistry of Sox 9, major collagens, and proteoglycans. Our results showed that the repair tissue did not restore to normal after acute injury. Ectopic chondrogenesis was observed in 33% of samples inside wound at week 4 while ectopic ossification surrounded by chondrocyte‐like cells were observed in the window wound in 50% of samples at week 12. There was sustained expression of biglycan and reduced expression of aggrecan and decorin in the tendon matrix in the repair tissue. The erroneous deposition of extracellular matrix and ectopic chondro‐ossification in the repair tissue, both might influence each other, might account for the poor tissue quality after acute injury. Higher expression of biglycan and aggrecan were observed in the ectopic chondro‐ossification sites in the repair tissue, suggesting that they might have roles in ectopic chondro‐osteogenesis. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:37–46, 2012     

Mechanical,compositional, and structural properties of the mouse patellar tendon with changes in biglycan gene expression

Tendons have complex mechanical properties that depend on their structure and composition. Some studies have assessed the role of small leucine‐rich proteoglycans (SLRPs) in the mechanical response of tendon, but the relationships between sophisticated mechanics, assembly of collagen and SLRPs have not been well characterized. In this study, biglycan gene expression was varied in a dose dependent manner using biglycan null, biglycan heterozygote and wild type mice. Measures of mechanical (tension and compression), compositional and structural changes of the mouse patellar tendon were evaluated. Viscoelastic, tensile dynamic modulus was found to be increased in the biglycan heterozygous and biglycan null tendons compared to wild type. Gene expression analyses revealed biglycan gene expression was closely associated in a dose‐dependent allelic manner. No differences were seen between genotypes in elastic or compressive properties or quantitative measures of collagen structure. These results suggest that biglycan, a member of the SLRP family, plays a role in tendon viscoelasticity that cannot be completely explained by its role in collagen fibrillogenesis. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1430–1437, 2013     

Effects of age and platelet‐rich plasma on ACL cell viability and collagen gene expression

Platelet‐rich plasma (PRP) has shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, in animal models, the healing potential of the ACL is dependent on animal age. In this study, we hypothesized that there are age‐dependent differences in ACL cell metabolism, collagen gene expression, and the ability of the cells to respond to growth factors in PRP. To test this hypothesis, ACL cells were obtained from skeletally immature, adolescent and adult pigs, and cultured in a collagen type I hydrogel with or without PRP for 14 days. When cultured in collagen‐only hydrogel, ACL cells from adult pigs had a 19% lower apoptotic rate as compared to immature pigs (p = 0.001) and a 25% higher cellular metabolic activity as compared to adolescent pigs (p = 0.006). The addition of PRP to the collagen hydrogel resulted in a significantly increased cellular metabolic activity, reduced apoptotic rate, and stimulation of collagen production in the cells from the immature and adolescent animals (p < 0.05 for all comparisons) but had less effect on adult cells. These findings suggest that skeletal maturity may influence ACL cells' metabolic activity, apoptosis, collagen production, and response to PRP. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:79–85, 2012     

Mouse treadmill running enhances tendons by expanding the pool of tendon stem cells (TSCs) and TSC‐related cellular production of collagen

Exercise is known to enhance tendon size and strength, but the stem cell‐based mechanisms for such exercise‐induced effects are largely unknown. This study aims to explore these mechanisms by using a mouse treadmill running model to examine the effects of exercise on newly discovered tendon stem cells (TSCs). After treadmill running, patellar TSCs (PTSCs) and Achilles TSCs (ATSCs) were isolated from the mice, and their proliferation was measured in vitro. We found that treadmill running nearly doubled proliferation rates of both PTSCs and ATSCs compared to cage control mice. Moreover, using a mixed tendon cell culture consisting of TSCs and tenocytes, cellular production of collagen was found to increase by 70% and 200% in PTSCs and ATSCs, respectively, from the treadmill running group over cells from the cage control group. These findings suggest that exercise exerts its anabolic effects on tendons at least in part by increasing proliferation to expand the pool of TSCs and also by increasing TSC‐related cellular production of collagen, the predominant component of tendons. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1178–1183, 2010     

The early inflammatory response after flexor tendon healing: A gene expression and histological analysis

Despite advances in surgical techniques over the past three decades, tendon repairs remain prone to poor clinical outcomes. Previous attempts to improve tendon healing have focused on the later stages of healing (i.e., proliferation and matrix synthesis). The early inflammatory phase of tendon healing, however, is not fully understood and its modulation during healing has not yet been studied. Therefore, the purpose of this work was to characterize the early inflammatory phase of flexor tendon healing with the goal of identifying inflammation‐related targets for future treatments. Canine flexor tendons were transected and repaired using techniques identical to those used clinically. The inflammatory response was monitored for 9 days. Temporal changes in immune cell populations and gene expression of inflammation‐, matrix degradation‐, and extracellular matrix‐related factors were examined. Gene expression patterns paralleled changes in repair‐site cell populations. Of the observed changes, the most dramatic effect was a greater than 4,000‐fold up‐regulation in the expression of the pro‐inflammatory factor IL‐1β. While an inflammatory response is likely necessary for healing to occur, high levels of pro‐inflammatory cytokines may result in collateral tissue damage and impaired tendon healing. These findings suggest that future tendon treatment approaches consider modulation of the inflammatory phase of healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:645–652, 2014.