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.

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.

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.

The early postnatal period represents a critical window for the maturation and development of orthopedic tissues, including those within the knee joint. To understand how mechanical loading impacts the maturational trajectory of the meniscus and other tissues of the hindlimb, perturbation of postnatal weight bearing was achieved through surgical resection of the sciatic nerve in neonatal mice at 1 or 14 days old. Sciatic nerve resection (SNR) produced significant and persistent disruptions in gait, leading to reduced tibial length and reductions in Achilles tendon mechanical properties. However, SNR resulted in minimal disruptions in morphometric parameters of the menisci and other structures in the knee joint, with no detectable differences in Col1a1-YFP or Col2a1-CFP expressing cells within the menisci. Furthermore, micromechanical properties of the meniscus and cartilage (as assessed by atomic force microscopy-based nanoindentation testing) were not different between experimental groups. In contrast to our initial hypothesis, reduced hindlimb weight bearing via neonatal SNR did not significantly impact the growth and development of the knee meniscus. This unexpected finding demonstrates that the input mechanical threshold required to sustain meniscus development may be lower than previously hypothesized, though future studies incorporating skeletal kinematic models coupled with force plate measurements will be required to calculate the loads passing through the affected hindlimb and precisely define these thresholds. Collectively, these results provide insight into the mechanobiological responses of the meniscus to alterations in load, and contribute to our understanding of the factors that influence normal postnatal development.

BACKGROUND: No single graft type has been shown to have a benefit in acetabular labral reconstruction. The native labrum and lateral meniscus share many similarities, suggesting that the meniscus may be a promising source of graft material in labral reconstruction.
OBJECTIVE: Using a pig model, we sought to evaluate the healing process of fresh-frozen meniscus allograft for acetabular reconstruction by assessing (1) MRI and macroscopic observations of the meniscus allograft; (2) histologic appearance and immunohistologic evaluation of the meniscus allograft, native meniscus, and labrum; (3) microscopic assessment of the native labrum and meniscus via scanning electron microscopy; and (4) biomechanical assessment of tensile properties.
METHODS: Twelve skeletally mature male miniature Bama pigs (24 hips) were randomly divided into two groups: labral defect group (control) and lateral meniscus allograft group. The selection of Bama pig specimens was based on the similarity of their acetabular labrum to that of the human acetabular labrum, characterized by the presence of fibrocartilage-like tissue lacking blood vessels. The pigs underwent bilateral hip surgery. Briefly, a 1.5-cm-long section was resected in the anterior dorsal labrum, which was left untreated or reconstructed using an allogeneic lateral meniscus. The pigs were euthanized at 12 and 24 weeks postoperatively, and then evaluated by macroscopic observations and MRI measurement to assess the extent of coverage of the labral defect. We also performed a histologic analysis and immunohistologic evaluation to assess the composition and structure of meniscus allograft, native labrum, and meniscus, as well as scanning electron microscopy assessment of the microstructure of the native labrum and meniscus and biomechanical assessment of tensile properties.
RESULTS: Imaging measurement and macroscopic observations revealed that the resected area of the labrum was fully filled in the lateral meniscus allograft group, whereas in the control group, the labral defect remained at 24 weeks. The macroscopic scores of the meniscus allograft group (8.2 ± 0.8) were higher than those of the control groups (4.8 ± 1.0) (mean difference 3.3 [95% CI 1.6 to 5.0]; p < 0.001). Moreover, in the meniscus allograft group, histologic assessment identified fibrocartilage-like cell cluster formation at the interface between the graft and acetabulum; cells and fibers arranged perpendicularly to the acetabulum and tideline structure that were similar to those of native labrum could be observed at 24 weeks. Immunohistochemical results showed that the average optical density value of Type II collagen at the graft-acetabulum interface was increased in the meniscus allograft group at 24 weeks compared with at 12 weeks (0.259 ± 0.031 versus 0.228 ± 0.023, mean difference 0.032 [95% CI 0.003 to 0.061]; p = 0.013). Furthermore, the tensile modulus of the lateral meniscus allograft was near that of the native labrum at 24 weeks (54.7 ± 9.9 MPa versus 63.2 ± 11.3 MPa, mean difference -8.4 MPa [95% CI -38.3 to 21.4]; p = 0.212).
CONCLUSIONS: In a pig model, lateral meniscus allografts fully filled labral defects in labral reconstruction. Regeneration of a fibrocartilage transition zone at the graft-acetabulum interface was observed at 24 weeks.
CONCLUSIONS: The use of an autograft meniscus for labral reconstruction may be a viable option when labral tears are deemed irreparable. Before its clinical implementation, it is imperative to conduct a comparative study involving tendon grafts, which are extensively used in current clinical practice.

The attachment site of the rotator cuff (RC) is a classic fibrocartilaginous enthesis, which is the junction between bone and tendon with typical characteristics of a fibrocartilage transition zone. Enthesis development has historically been studied with lineage tracing of individual genes selected a priori, which does not allow for the determination of single-cell landscapes yielding mature cell types and tissues. Here, in together with open-source GSE182997 datasets (three samples) provided by Fang et al., we applied Single-cell RNA sequencing (scRNA-seq) to delineate the comprehensive postnatal RC enthesis growth and the temporal atlas from as early as postnatal day 1 up to postnatal week 8. And, we furtherly performed single-cell spatial transcriptomic sequencing on postnatal day 1 mouse enthesis, in order to deconvolute bone-tendon junction (BTJ) chondrocytes onto spatial spots. In summary, we deciphered the cellular heterogeneity and the molecular dynamics during fibrocartilage differentiation. Combined with current spatial transcriptomic data, our results provide a transcriptional resource that will support future investigations of enthesis development at the mechanistic level and may shed light on the strategies for enhanced RC healing outcomes.