Megan L. Killian

The role of mechanobiology in tendon healing.

Mechanical cues affect tendon healing, homeostasis, and development in a variety of settings. Alterations in the mechanical environment are known to result in changes in the expression of extracellular matrix proteins, growth factors, transcription factors, and cytokines that can alter tendon structure and cell viability. Loss of muscle force in utero or in the immediate postnatal period delays tendon and enthesis development. The response of healing tendons to mechanical load varies depending on anatomic location. Flexor tendons require motion to prevent adhesion formation, yet excessive force results in gap formation and subsequent weakening of the repair. Excessive motion in the setting of anterior cruciate ligament reconstruction causes accumulation of macrophages, which are detrimental to tendon graft healing. Complete removal of load is detrimental to rotator cuff healing; yet, large forces are also harmful. Controlled loading can enhance healing in most settings; however, a fine balance must be reached between loads that are too low (leading to a catabolic state) and too high (leading to microdamage). This review will summarize existing knowledge of the mechanobiology of tendon development, homeostasis, and healing.

Tendon-to-Bone Attachment: From Development to Maturity

The attachment between tendon and bone occurs across a complex transitional tissue that minimizes stress concentrations and allows for load transfer between muscles and skeleton. This unique tissue cannot be reconstructed following injury, leading to high incidence of recurrent failure and stressing the need for new clinical approaches. This review describes the current understanding of the development and function of the attachment site between tendon and bone. The embryonic attachment unit, namely, the tip of the tendon and the bone eminence into which it is inserted, was recently shown to develop modularly from a unique population of Sox9- and Scx-positive cells, which are distinct from tendon fibroblasts and chondrocytes. The fate and differentiation of these cells is regulated by transforming growth factor beta and bone morphogenetic protein signaling, respectively. Muscle loads are then necessary for the tissue to mature and mineralize. Mineralization of the attachment unit, which occurs postnatally at most sites, is largely controlled by an Indian hedgehog/parathyroid hormone-related protein feedback loop. A number of fundamental questions regarding the development of this remarkable attachment system require further study. These relate to the signaling mechanism that facilitates the formation of an interface with a gradient of cellular and extracellular phenotypes, as well as to the interactions between tendon and bone at the point of attachment.

Skeletal muscle fibrosis and stiffness increase after rotator cuff tendon injury and neuromuscular compromise in a rat model

Rotator cuff tears can cause irreversible changes (e.g., fibrosis) to the structure and function of the injured muscle(s). Fibrosis leads to increased muscle stiffness resulting in increased tension at the rotator cuff repair site. This tension influences repairability and healing potential in the clinical setting. However, the micro‐ and meso‐scale structural and molecular sources of these whole‐muscle mechanical changes are poorly understood. Here, single muscle fiber and fiber bundle passive mechanical testing was performed on rat supraspinatus and infraspinatus muscles with experimentally induced massive rotator cuff tears (Tenotomy) as well as massive tears with chemical denervation (Tenotomy + BTX) at 8 and 16 weeks post‐injury. Titin molecular weight, collagen content, and myosin heavy chain profiles were measured and correlated with mechanical variables. Single fiber stiffness was not different between controls and experimental groups. However, fiber bundle stiffness was significantly increased at 8 weeks in the Tenotomy + BTX group compared to Tenotomy or control groups. Many of the changes were resolved by 16 weeks. Only fiber bundle passive mechanics was weakly correlated with collagen content. These data suggest that tendon injury with concomitant neuromuscular compromise results in extra‐cellular matrix production and increases in stiffness of the muscle, potentially complicating subsequent attempts for surgical repair.

Traumatic Anterior Cruciate Ligament Tear and its Implications on Meniscal Degradation: A Preliminary Novel Lapine Osteoarthritis Model

BACKGROUND Injury patterns of the meniscus following impact trauma resulting in anterior cruciate ligament (ACL) rupture are not well understood. This study explored the spatial and temporal distribution of meniscal tears in a novel in vivo lapine model. METHODS Skeletally mature Flemish Giant rabbits were subjected to either tibiofemoral impaction resulting in ACL rupture or surgical ACL transection. Meniscal damage was assessed acutely and after 12 wk for traumatically torn, and after 12 wk in ACL transected animals. Morphological grading was assessed using previously established criteria, and descriptions of meniscal damage were diagnosed by a Board certified orthopedist. Histological assessment was also made on 12 wk traumatically torn and ACL transected animals using Fast-Green/Safranin-O staining. RESULTS Traumatic ACL rupture resulted in acute tears predominately in the lateral menisci. Animals subjected to both surgical transection and traumatic ACL rupture experienced degradation of the lateral and medial menisci 12 wk after injury. However, traumatic ACL rupture resulted in acute lateral damage and chronic degradation of the menisci, as well as more severe degradation of the menisci 12 wk after injury. CONCLUSIONS This study showed that unconstrained high-intensity impacts on the tibiofemoral joint lead to meniscal damage in conjunction with ACL ruptures. Both acute and chronic changes to the menisci following traumatic impaction were observed. This research has implications for the future use of lapine models for osteoarthritis, as it incorporates traumatic loading as a more realistic mode contributing to the progression of osteoarthritis (OA) compared to surgically transected models.

Meniscal tissue explants response depends on level of dynamic compressive strain

OBJECTIVE Following partial meniscectomy, the remaining meniscus is exposed to an altered loading environment. In vitro 20% dynamic compressive strains on meniscal tissue explants has been shown to lead to an increase in release of glycosaminoglycans from the tissue and increased expression of interleukin-1alpha (IL-1alpha). The goal of this study was to determine if compressive loading which induces endogenously expressed IL-1 results in downstream changes in gene expression of anabolic and catabolic molecules in meniscal tissue, such as MMP expression. METHOD Relative changes in gene expression of MMP-1, MMP-3, MMP-9, MMP-13, A Disintegrin and Metalloproteinase with ThromboSpondin 4 (ADAMTS4), ADAMTS5, TNFalpha, TGFbeta, COX-2, Type I collagen (COL-1) and aggrecan and subsequent changes in the concentration of prostaglandin E(2) released by meniscal tissue in response to varying levels of dynamic compression (0%, 10%, and 20%) were measured. Porcine meniscal explants were dynamically compressed for 2h at 1Hz. RESULTS 20% dynamic compressive strains upregulated MMP-1, MMP-3, MMP-13 and ADAMTS4 compared to no dynamic loading. Aggrecan, COX-2, and ADAMTS5 gene expression were upregulated under 10% strain compared to no dynamic loading while COL-1, TIMP-1, and TGFbeta gene expression were not dependent on the magnitude of loading. CONCLUSION This data suggests that changes in mechanical loading of the knee joint meniscus from 10% to 20% dynamic strain can increase the catabolic activity of the meniscus.

The effect of unloading on gene expression of healthy and injured rotator cuffs.

Tendon unloading following rupture of one of the rotator cuff tendons can induce alterations in muscle physiology and tendon structure, which can subsequently affect reparability and healing potential. Yet little is known about the effects of muscle and tendon unloading on the molecular response of the rotator cuff. We determined the effect of mechanical unloading on gene expression and morphology of healthy supraspinatus tendons and muscles, and the same muscles after acute injury and repair. Mechanical unloading was achieved by tenotomy and/or botulinum toxin A (BTX) chemical denervation in a rat rotator cuff model of injury and repair. Gene expression profiles varied across regions of the muscle, with the greatest changes seen in the distal aspect of the muscle for most genes. Myogenic and adipogenic genes were upregulated in muscle when unloaded (tenotomy and BTX). Tendon injury, with and without repair, resulted in upregulation of fibrosis‐ and tendon‐specific gene expression. The expression of scleraxis, a transcription factor necessary for tendon development, was upregulated in response to injury and repair. In summary, tendon detachment and repair had the greatest effect on tendon gene expression, while unloading had the greatest effect on muscle gene expression.

Regional and zonal histo-morphological characteristics of the lapine menisci

The menisci have crucial weight‐bearing roles in the knee. Regional variations in structure and cellularity of the meniscus have only been minimally investigated. Therefore, the goal of this study was to illustrate the regional cell density, tissue area, and structure of healthy lapine menisci. Skeletally mature Flemish Giant rabbits were used for this study. Upon sacrifice, menisci were removed, fixed in formalin, and cryosectioned. Histological analysis was performed for the detection of sulfated glycosaminoglycans (GAG), collagen Types I and II, cellular density, and tissue area. ANOVA and paired t tests were used for testing of statistical significance. Glycosaminoglycan coverage of the medial meniscus significantly varied between regions, with the anterior region demonstrating significantly more GAG coverage than the posterior region. Inter‐ and intra‐meniscal comparisons revealed variations between zones, with trends that outer zones of the medial menisci had less GAG coverage. Collagen Types I and II had marked characteristics and varying degrees of coverage across regions. Tissue area varied between regions for both medial and lateral menisci. Cellular density was dependent on region in the lateral meniscus. This is the first study to illustrate regional and zonal variation in glycosaminoglycan coverage, size, and cellular density for healthy lapine meniscal tissue. This data provides baseline information for future investigations in meniscal injury models in rabbits. Anat Rec, 2010.

Chronic Degeneration Leads to Poor Healing of Repaired Massive Rotator Cuff Tears in Rats

Background: Chronic rotator cuff tears present a clinical challenge, often with poor outcomes after surgical repair. Degenerative changes to the muscle, tendon, and bone are thought to hinder healing after surgical repair; additionally, the ability to overcome degenerative changes after surgical repair remains unclear. Purpose/Hypothesis: The purpose of this study was to evaluate healing outcomes of muscle, tendon, and bone after tendon repair in a model of chronic rotator cuff disease and to compare these outcomes to those of acute rotator cuff injuries and repair. The hypothesis was that degenerative rotator cuff changes associated with chronic multitendon tears and muscle unloading would lead to poor structural and mechanical outcomes after repair compared with acute injuries and repair. Study Design: Controlled laboratory study. Methods: Chronic rotator cuff injuries, induced via detachment of the supraspinatus (SS) and infraspinatus (IS) tendons and injection of botulinum toxin A into the SS and IS muscle bellies, were created in the shoulders of rats. After 8 weeks of injury, tendons were surgically reattached to the humeral head, and an acute, dual-tendon injury and repair was performed on the contralateral side. After 8 weeks of healing, muscles were examined histologically, and tendon-to-bone samples were examined microscopically, histologically, and biomechanically and via micro–computed tomography. Results: All repairs were intact at the time of dissection, with no evidence of gapping or ruptures. Tendon-to-bone healing after repair in our chronic injury model led to reduced bone quality and morphological disorganization at the repair site compared with acute injuries and repair. SS and IS muscles were atrophic at 8 weeks after repair of chronic injuries, indicating incomplete recovery after repair, whereas SS and IS muscles exhibited less atrophy and degeneration in the acute injury group at 8 weeks after repair. After chronic injuries and repair, humeral heads had decreased total mineral density and an altered trabecular structure, and the repair had decreased strength, stiffness, and toughness, compared with the acute injury and repair group. Conclusion: Chronic degenerative changes in rotator cuff muscles, tendons, and bone led to inferior healing characteristics after repair compared with acute injuries and repair. The changes were not reversible after repair in the time course studied, consistent with clinical impressions. Clinical Relevance: High retear rates after rotator cuff repair are associated with tear size and chronicity. Understanding the mechanisms behind this association may allow for targeted tissue therapy for tissue degeneration that occurs in the setting of chronic tears.

Recent advances in shoulder research

Shoulder pathology is a growing concern for the aging population, athletes, and laborers. Shoulder osteoarthritis and rotator cuff disease represent the two most common disorders of the shoulder leading to pain, disability, and degeneration. While research in cartilage regeneration has not yet been translated clinically, the field of shoulder arthroplasty has advanced to the point that joint replacement is an excellent and viable option for a number of pathologic conditions in the shoulder. Rotator cuff disease has been a significant focus of research activity in recent years, as clinicians face the challenge of poor tendon healing and irreversible changes associated with rotator cuff arthropathy. Future treatment modalities involving biologics and tissue engineering hold further promise to improve outcomes for patients suffering from shoulder pathologies.

Scleraxis is required for the development of a functional tendon enthesis

The attachment of dissimilar materials is a major engineering challenge, yet this challenge is seemingly overcome in biology. This study aimed to determine how the transcription factor Scleraxis (Scx) influences the development and maturation of the tendon‐to‐bone attachment (enthesis). Mice with conditional knockout (cKO) for Scx (Scxflx/‐, Prx1Cre+) and wild‐type [(WT) Scxflx/+ or Scxflx/flx] littermates were killed at postnatal days 7‐56 (P7‐P56). Enthesis morphometry, histology, and collagen alignment were investigated throughout postnatal growth. Enthesis tensile mechanical properties were also assessed. Laser microdissection of distinct musculoskeletal tissues was performed at P7 for WT, cKO, and muscle‐unloaded (botulinum toxin A treated) attachments for quantitative PCR. cKO mice were smaller, with altered bone shape and impaired enthesis morphology, morphometry, and organization. Structural alterations led to altered mechanical properties; cKO entheses demonstrated reduced strength and stiffness. In P7 attachments, cKO mice had reduced expression of transforming growth factor (TGF) superfamily genes in brocartilage compared with WT mice. In conclusion, deletion of Scx led to impairments in enthesis structure, which translated into impaired functional (i. e., mechanical) outcomes. These changes may be driven by transient signaling cues from mechanical loading and growth factors.—Killian, M. L., Thomopoulos, S. Scleraxis is required for the development of a functional tendon enthesis. FASEB J. 30, 301‐311 (2016). www.fasebj.org