Enthesis Formation Research Articles

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

ABSTRACTRestoring 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.

Enthesis fibrocartilage cells originate from a population of Hedgehog-responsive cells modulated by the loading environment.

Tendon attaches to bone across a specialized tissue called the enthesis. This tissue modulates the transfer of muscle forces between two materials, i.e. tendon and bone, with vastly different mechanical properties. The enthesis for many tendons consists of a mineralized graded fibrocartilage that develops postnatally, concurrent with epiphyseal mineralization. Although it is well described that the mineralization and development of functional maturity requires muscle loading, the biological factors that modulate enthesis development are poorly understood. By genetically demarcating cells expressing Gli1 in response to Hedgehog (Hh) signaling, we discovered a unique population of Hh-responsive cells in the developing murine enthesis that were distinct from tendon fibroblasts and epiphyseal chondrocytes. Lineage-tracing experiments revealed that the Gli1 lineage cells that originate in utero eventually populate the entire mature enthesis. Muscle paralysis increased the number of Hh-responsive cells in the enthesis, demonstrating that responsiveness to Hh is modulated in part by muscle loading. Ablation of the Hh-responsive cells during the first week of postnatal development resulted in a loss of mineralized fibrocartilage, with very little tissue remodeling 5 weeks after cell ablation. Conditional deletion of smoothened, a molecule necessary for responsiveness to Ihh, from the developing tendon and enthesis altered the differentiation of enthesis progenitor cells, resulting in significantly reduced fibrocartilage mineralization and decreased biomechanical function. Taken together, these results demonstrate that Hh signaling within developing enthesis fibrocartilage cells is required for enthesis formation.

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.

Mesenchymal Stem Cell-Dependent Formation of Heterotopic Tendon-Bone Insertions (Osteotendinous Junctions)

Ligament-to-bone and tendon-to-bone interfaces (entheses, osteotendinous junctions [OTJs]) serve to dissipate stress between soft tissue and bone. Surgical reconstruction of these interfaces is an issue of considerable importance as they are prone to injury and the integration of bone and tendon/ligament is in general not satisfactory. We report here the stem cell-dependent spontaneous formation of fibrocartilaginous and fibrous entheses in heterotopic locations of the mouse if progenitors possess a tenogenic and osteo-/chondrogenic capacity. This study followed the hypothesis that enhanced Bone Morphogenetic Protein (BMP)-signaling in adult mesenchymal stem cells that are induced for tendon formation may overcome the tendon-inherent interference with bone formation and may thus allow the stem cell-dependent formation of tendon-bone interfaces. The tenogenic and osteo-/chondrogenic competence was mediated by the adeno- and/or lentiviral expression of the biologically active Smad8 signaling mediator (Smad8ca) and of Bone Morphogenetic Protein 2 (BMP2). Modified mesenchymal progenitors were implanted in subcutaneous or intramuscular sites of the mouse. The stem cell-dependent enthesis formation was characterized histologically by immunohistological approaches and by in situ hybridization. Transplantation of modified murine stem cells resulted in the formation of tendinous and osseous structures exhibiting fibrocartilage-type OTJs, while, in contrast, the viral modification of primary human bone marrow-derived mesenchymal stromal/stem cells showed evidence of fibrous tendon-bone interface formation. Moreover, it could be demonstrated that Smad8ca expression alone was sufficient for the formation of tendon/ligament-like structures. These findings may contribute to the establishment of stem cell-dependent regenerative therapies involving tendon/ligaments and to the improvement of the insertion of tendon grafts at bony attachment sites, eventually.

Expression of intermediate filaments at muscle insertions in human fetuses

Desmin and vimentin are intermediate filaments that play crucial roles in the maturation, maintenance and recovery of muscle fibers and mesenchymal cells. The expression of these proteins has not been investigated extensively in human fetuses. In the present study, we examined the immunohistochemical expression of intermediate filaments in skeletal muscles of the head, neck and thorax in 12 mid-term human fetuses at 9-18 weeks of gestation. We also used immunohistochemistry to localize the expression of the myosin heavy chain and silver impregnation to identify the fetal endomysium. Expression of desmin and vimentin was already detectable in intercostal muscle at 9 weeks, especially at sites of muscle attachment to the perichondrium. At this stage, myosin heavy chain was expressed throughout the muscle fibers and the endomysium had already developed. Beginning with punctate expression, the positive areas became diffusely distributed in the muscle fibers. At 15-18 weeks, intermediate filament proteins were extensively expressed in all of the muscles examined. Expression at the bone-muscle interface was continuous with expression along the intramuscular tendon fibres. These results suggest that the development of intermediate filaments begins in areas of mechanical stress due to early muscle contraction. Their initially punctate distribution, as observed here, probably corresponds to the earliest stage of fetal enthesis formation.

Contributions of the different rabbit models to our understanding of rotator cuff pathology

Animal models designed to investigate the biologic aspects of rotator cuff pathology are being used and being developed in the rat, rabbit, dog, and other mammals. The current models have provided valuable information regarding the etiology, pathogenesis, and healing potential of the tendon, as well as effective surgical repair techniques for rotator cuff tears. Rabbit models have primarily been used to describe the muscular changes, rotator cuff enthesis formation, and growth factor expression after rotator cuff injury. This article will serve to review the data obtained from previously described rabbit models and a newly developed rabbit model for studying rotator cuff pathology.

Delay of supraspinatus repair by up to 12 weeks does not impair enthesis formation: A quantitative histologic study in rabbits

The purpose of this study was to find out whether supraspinatus repair delayed by up to12 weeks affects the formation of a new enthesis when compared to an immediate repair. In 67 rabbits, the supraspinatus fibrocartilaginous enthesis of one shoulder was resected. The tendon was attached to the greater tuberosity either immediately, after a 6-week, or after a 12-week delay. Five histologic variables were used to assess enthesis formation: number of non-chondrocytes, number of chondrocytes, alignment of chondrocytes in rows, area of metachromasia on toluidine blue (TB)-stained sections indicating proteoglycan content, and area of diffracted polarized light indicating spatial alignment of collagen fibers. For every variable, progressive enthesis formation was observed. Again, for every variable and at every time point studied, no statistically significant difference was observed between tendons repaired immediately, after 6, or after 12 weeks (all p>0.05). Supraspinatus tendon repairs delayed by 6 and 12 weeks constituted an enthesis which proceeded identically to one immediately repaired. Formation of a fibrocartilaginous enthesis depended on the elapsed time after repair and not on the duration between detachment and repair. Despite stated limitations, these results support both a trial of conservative treatment after a rotator cuff tear and a positive outcome of rotator cuff repair even if delayed by up to 12 weeks.

Formation of a new enthesis after attachment of the supraspinatus tendon: A quantitative histologic study in rabbits

To quantify in a longitudinal study non-chondrocytic cells and chondrocytes, tissular architecture as well as extracellular matrix restoration during the formation of an enthesis following supraspinatus tendon attachment to the humerus. In 89 rabbits, one supraspinatus fibrocartilaginous enthesis was resected and the tendon either attached to the greater tuberosity (n=75) or not attached (n=14). The animals were sacrificed after 2, 6, 8, 12 or 24 weeks. The operated and contralateral shoulders were processed for histologic sections. Number of non-chondrocytes, chondrocytes and alignment of chondrocytes in rows were assessed histologically. Extracellular matrix restoration was measured based on (1) area of toluidine blue metachromasia indicating proteoglycan content and (2) on area of diffracted polarized light indicating spatial collagen fiber alignment. In the attached tendon, the number of non-chondrocytic cells sharply increased at 2 weeks, progressively decreased thereafter but remained higher than controls at all time points. Chondrocytes appeared at 2 weeks and their number reached control levels by 6 weeks (136+/-14 vs 144+/-15 controls, p>.05). The percentage of chondrocytes aligned in rows increased from 19+/-4% at 2 weeks to reach near normal values at 24 weeks (71+/-3% vs 78+/-2% controls, p>.05). Area of metachromasia increased from 0.1+/-0.1 mm(2) at 2 weeks to 3.8+/-0.3 mm(2) at 24 weeks, still below contralateral enthesis levels (4.6+/-0.1 mm(2), p<.05). Area of diffracted polarized light enlarged from 12+/-2 x 10(3) microm(2) at 2 weeks to 151+/-19 x 10(3) microm(2) at 24 weeks, still significantly smaller than contralateral levels (177+/-13 x 10(3) microm(2), p<.05). Neither chondrocytes nor metachromasia were observed in the non-attached tendons. A new enthesis was formed after attachment of the supraspinatus tendon into bony trough. Histomorphometry allowed to document extensive non-chondrocytic proliferation that was followed by appearance of chondrocytes and their spatial organization, a process was complete by 24 weeks. Extracellular matrix formation as well as spatial alignment of collagen fibers were delayed and not complete by 24 weeks. This first longitudinal investigation on the formation of the supraspinatus enthesis using quantitative outcome measures cautions against too early and too aggressive a rehabilitation program.

Formation of a new enthesis after attachment of the supraspinatus tendon: A quantitative histologic study in rabbits

Purpose. To quantify in a longitudinal study non-chondrocytic cells and chondrocytes, tissular architecture as well as extracellular matrix restoration during the formation of an enthesis following supraspinatus tendon attachment to the humerus. Methods. In 89 rabbits, one supraspinatus fibrocartilaginous enthesis was resected and the tendon either attached to the greater tuberosity ( n = 75) or not attached ( n = 14). The animals were sacrificed after 2, 6, 8, 12 or 24 weeks. The operated and contralateral shoulders were processed for histologic sections. Number of non-chondrocytes, chondrocytes and alignment of chondrocytes in rows were assessed histologically. Extracellular matrix restoration was measured based on (1) area of toluidine blue metachromasia indicating proteoglycan content and (2) on area of diffracted polarized light indicating spatial collagen fiber alignment. Results. In the attached tendon, the number of non-chondrocytic cells sharply increased at 2 weeks, progressively decreased thereafter but remained higher than controls at all time points. Chondrocytes appeared at 2 weeks and their number reached control levels by 6 weeks (136 ± 14 vs 144 ± 15 controls, p > .05). The percentage of chondrocytes aligned in rows increased from 19 ± 4% at 2 weeks to reach near normal values at 24 weeks (71 ± 3% vs 78 ± 2% controls, p > .05). Area of metachromasia increased from 0.1 ± 0.1 mm 2 at 2 weeks to 3.8 ± 0.3 mm 2 at 24 weeks, still below contralateral enthesis levels (4.6 ± 0.1 mm 2, p < .05). Area of diffracted polarized light enlarged from 12 ± 2 × 10 3 μm 2 at 2 weeks to 151 ± 19 × 10 3 μm 2 at 24 weeks, still significantly smaller than contralateral levels (177 ± 13 × 10 3 μm 2, p < .05). Neither chondrocytes nor metachromasia were observed in the non-attached tendons. Conclusion. A new enthesis was formed after attachment of the supraspinatus tendon into bony trough. Histomorphometry allowed to document extensive non-chondrocytic proliferation that was followed by appearance of chondrocytes and their spatial organization, a process was complete by 24 weeks. Extracellular matrix formation as well as spatial alignment of collagen fibers were delayed and not complete by 24 weeks. This first longitudinal investigation on the formation of the supraspinatus enthesis using quantitative outcome measures cautions against too early and too aggressive a rehabilitation program.

A histological study of the augmented reconstruction of the anterior cruciate ligament in rabbits

An experimental study was carried out to investigate the histological properties of the anterior cruciate ligament (ACL) reconstructed by the augmentation procedure. After the resection of 42 knees in 42 adult New Zealand white rabbits, 21 of the knees were reconstructed with a patellar tendon alone (nonaugmented group) and 21 with an augmented patellar tendon using a Leeds-Keio artificial ligament (augmented group). The rabbits were destroyed 4, 8, 12, 16 and 24 weeks, respectively, after the operation. After 4 weeks, a remarkable proliferation of fibrous tissue was present in the augmented group ligaments, while tissue necrosis was dominant, with slight granulation, in the nonaugmented group. After 8 weeks, the fibrous tissue was regularly arranged in a longitudinal direction in the augmented group, while this took 12 weeks in the nonaugmented group. Furthermore, 16 weeks postoperatively, the collagen fibers had matured and resembled normal ligaments in the augmented group, but this took 24 weeks in the nonaugmented group. After 16 weeks, columnar formations and some mineralization of the fibrocartilage were observed in the bone-ligament junctions in the nonaugmented group, but not until after 24 weeks in the augmented group. After 24 weeks, there were no histological differences between the two groups, either in the reconstructed ligaments or in the bone-ligament junctions. It is suggested that augmentation had an effect on the early histological maturation of collagen fibers, and an adverse effect on the formation of enthesis.