The Achilles tendon enthesis (ATE) anchors the Achilles tendon into the calcaneus through fibrocartilaginous tissue. The latter is enriched in type II collagen and proteoglycans (PGs), both of which give the enthesis its capacity to withstand compressive stress. Because unloading and reloading induce remodeling of the ATE fibrocartilage (Camy et al., 2022), chronic changes in the mechanical load could modify the mechanical response under compressive stress. Therefore, we investigated the ATE fatigue behavior in mice, under cyclic compressive loading, after 14 days of hindlimb suspension and 6 days of reloading. In addition, we performed a qualitative histological study of PGs in ATE fibrocartilage. The mechanical behavior of ATE was impaired in unloaded mice. A significant loss of 27 % in Δd (difference between the maximum and minimum displacements) was observed at the end of the test. In addition, the hysteresis area decreased by approximately 27 % and the stiffness increased by over 45 %. The increased stiffness and loss of viscosity were thrice and almost twice those of the control, respectively. In the reloaded entheses, where the loss of Δd was not significant, we found a significant 28 % decrease in the hysteresis area and a 26 % increase in stiffness, both of which were higher regarding the control condition. These load-dependent changes in the mechanical response seem partly related to changes in PGs in the uncalficied part of the ATE. These findings highlight the importance of managing compressive loading on ATE when performing prophylactic and rehabilitation exercises.

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1 +/- menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1 +/- meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.

Fibrocartilaginous entheses consist of tendons, unmineralized and mineralized fibrocartilage, and subchondral bone, each exhibiting varying stiffness. Here we examined the functional role of sclerostin, expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of unmineralized fibrocartilage and concurrent replacement of epiphyseal hyaline cartilage by bone, unmineralized fibrocartilage reexpanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin at the base of mineralized fibrocartilage adjacent to subchondral bone. In Scx-deficient mice with less mechanical loading due to defects of the Achilles tendon, sclerostin+ fibrochondrocyte count significantly decreased in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, encoding sclerostin, elevated mineral density in mineralized zones of fibrocartilaginous entheses. Atomic force microscopy analysis revealed increased fibrocartilage stiffness. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of fibrocartilaginous entheses.

Entheses are classified into three types: fibrocartilaginous, fibrous, and periosteal insertions. However, the mechanism behind the development of fibrous entheses and periosteal insertions remains unclear. Since both entheses are part of the temporomandibular joint (TMJ), this study analyzes the TMJ entheses. Here, we show that SOX9 expression is negatively regulated during TMJ enthesis development, unlike fibrocartilage entheses which are modularly formed by SCX and SOX9 positive progenitors. The TMJ entheses was adjacent to the intramembranous bone rather than cartilage. SOX9 expression was diminished during TMJ enthesis development. To clarify the functional role of Sox9 in the development of TMJ entheses, we examined these structures in TMJ using Wnt1Cre;Sox9flox/+ reporter mice. Wnt1Cre;Sox9flox/+ mice showed enthesial deformation at the TMJ. Next, we also observed a diminished SOX9 expression area at the enthesis in contact with the clavicle\'s membranous bone portion, similar to the TMJ entheses. Together, these findings reveal that the timing of SOX9 expression varies with the ossification development mode.

Connective tissue attaches to bone across an insertion with spatial gradients in components, microstructure, and biomechanics. Due to regional stress concentrations between two mechanically dissimilar materials, the insertion is vulnerable to mechanical damage during joint movements and difficult to repair completely, which remains a significant clinical challenge. Despite interface stress concentrations, the native insertion physiologically functions as the effective load-transfer device between soft tissue and bone. This review summarizes tendon, ligament, and meniscus insertions cross-sectionally, which is novel in this field. Herein, the similarities and differences between the three kinds of insertions in terms of components, microstructure, and biomechanics are compared in great detail. This review begins with describing the basic components existing in the four zones (original soft tissue, uncalcified fibrocartilage, calcified fibrocartilage, and bone) of each kind of insertion, respectively. It then discusses the microstructure constructed from collagen, glycosaminoglycans (GAGs), minerals and others, which provides key support for the biomechanical properties and affects its physiological functions. Finally, the review continues by describing variations in mechanical properties at the millimeter, micrometer, and nanometer scale, which minimize stress concentrations and control stretch at the insertion. In summary, investigating the contrasts between the three has enlightening significance for future directions of repair strategies of insertion diseases and for bioinspired approaches to effective soft-hard interfaces and other tough and robust materials in medicine and engineering.

Diffusion within extracellular matrix is essential to deliver nutrients and larger metabolites to the avascular region of the meniscus. It is well known that both structure and composition of the meniscus vary across its regions; therefore, it is crucial to fully understand how the heterogenous meniscal architecture affects its diffusive properties. The objective of this study was to investigate the effect of meniscal region (core tissue, femoral, and tibial surface layers) and molecular weight on the diffusivity of several molecules in porcine meniscus. Tissue samples were harvested from the central area of porcine lateral menisci. Diffusivity of fluorescein (MW 332 Da) and three fluorescence-labeled dextrans (MW 3k, 40k, and 150k Da) was measured via fluorescence recovery after photobleaching. Diffusivity was affected by molecular size, decreasing as the Stokes\' radius of the solute increased. There was no significant effect of meniscal region on diffusivity for fluorescein, 3k and 40k dextrans (p>0.05). However, region did significantly affect the diffusivity of 150k Dextran, with that in the tibial surface layer being larger than in the core region (p = 0.001). Our findings contribute novel knowledge concerning the transport properties of the meniscus fibrocartilage. This data can be used to advance the understanding of tissue pathophysiology and explore effective approaches for tissue restoration.

The healing of tendon-bone injuries is very difficult, often resulting in poor biomechanical performance and unsatisfactory functional recovery. The tendon-bone insertion has a complex four distinct layers structure, and previous studies have often focused on promoting the regeneration of the fibrocartilage layer, neglecting the role of its bone end repair in tendon-bone healing. This study focuses on the role of treadmill training in promoting bone regeneration at the tendon-bone insertion and its related mechanisms.
After establishing the tendon-bone insertion injury model, the effect of treadmill training on tendon-bone healing was verified by Micro CT and HE staining; then the effect of CX3CL1 on osteoclast differentiation was verified by TRAP staining and cell culture; and finally the functional recovery of the mice was verified by biomechanical testing and behavioral test.
Treadmill training suppresses the secretion of CX3CL1 and inhibits the differentiation of local osteoclasts after tendon-bone injury, ultimately reducing osteolysis and promoting tendon bone healing.
Our research has found the interaction between treadmill training and the CX3CL1-C3CR1 axis, providing a certain theoretical basis for rehabilitation training.

The fibrocartilaginous enthesis is a highly specialised tissue interface that ensures a smooth mechanical transfer between tendon or ligament and bone through a fibrocartilage area. This tissue is prone to injury and often does not heal, even after surgical intervention. Enthesis augmentation approaches are challenging due to the complexity of the tissue that is characterised by the coexistence of a range of cellular and extracellular components, architectural features and mechanical properties within only hundreds of micrometres. Herein, we discuss enthesis repair and regeneration strategies, with particular focus on elegant interfacial and functionalised scaffold-based designs.

Meniscus is a complex and crucial fibrocartilaginous tissue within the knee joint. Meniscal regeneration remains to be a scientific and translational challenge. We clarified that mesenchymal stem cells (MSCs) participated in meniscal maturation and regeneration using MSC-tracing transgenic mice model. Here, inspired by meniscal natural maturational and regenerative process, we developed an effective and translational strategy to facilitate meniscal regeneration by three-dimensionally printing biomimetic meniscal scaffold combining autologous synovium transplant, which contained abundant intrinsic MSCs. We verified that this facilitated anisotropic meniscus-like tissue regeneration and protected cartilage from degeneration in large animal model. Mechanistically, the biomechanics and matrix stiffness up-regulated Piezo1 expression, facilitating concerted activation of calcineurin and NFATc1, further activated YAP-pSmad2/3-SOX9 axis, and consequently facilitated fibrochondrogenesis of MSCs during meniscal regeneration. In addition, Piezo1 induced by biomechanics and matrix stiffness up-regulated collagen cross-link enzyme expression, which catalyzed collagen cross-link and thereby enhanced mechanical properties of regenerated tissue.

Current tendon and ligament reconstruction surgeries rely on scar tissue healing which differs from native bone-to-tendon interface (BTI) tissue. We aimed to engineer Synovium-derived mesenchymal stem cells (Sy-MSCs) based scaffold-free fibrocartilage constructs and investigate in vivo bone-tendon interface (BTI) healing efficacy in a rat anterior cruciate ligament (ACL) reconstruction model.
Sy-MSCs were isolated from knee joint of rats. Scaffold-free sy-MSC constructs were fabricated and cultured in differentiation media including  TGF-β-only, CTGF-only, and TGF-β + CTGF. Collagenase treatment on tendon grafts was optimized to improve cell-to-graft integration. The effects of fibrocartilage differentiation and collagenase treatment on BTI integration was assessed by conducting histological staining, cell adhesion assay, and tensile testing. Finally, histological and biomechanical analyses were used to evaluate in vivo efficacy of fibrocartilage construct in a rat ACL reconstruction model.
Fibrocartilage-like features were observed with in the scaffold-free sy-MSC constructs when applying TGF-β and CTGF concurrently. Fifteen minutes collagenase treatment increased cellular attachment 1.9-fold compared to the Control group without affecting tensile strength. The failure stress was highest in the Col + D + group (22.494 ± 13.74 Kpa) compared to other groups at integration analysis in vitro. The ACL Recon + FC group exhibited a significant 88% increase in estimated stiffness (p = 0.0102) compared to the ACL Recon group at the 4-week postoperative period.
Scaffold-free, fibrocartilage engineering together with tendon collagenase treatment enhanced fibrocartilaginous BTI healing in ACL reconstruction.