Type V Collagen Exhibits Distinct Regulatory Activities in TMJ Articular Disc versus Condylar Cartilage During Postnatal Growth and Remodeling.

Understanding matrix molecular activities that regulate the postnatal growth and remodeling of temporomandibular joint (TMJ) condylar cartilage and articular disc will enable the development of effective regenerative strategies targeting TMJ disorder. This study elucidated the distinct roles of type V collagen (collagen V) in regulating these two units. Studying the TMJ of young adult Col5a1+/- mice, we found loss of collagen V resulted in substantial changes in the proliferation, clustering, and density of progenitors in condylar cartilage, but did not have a major impact on disc cells that are more fibroblast-like. Although loss of collagen V led to thickened collagen fibrils with increased heterogeneity in the disc, there were no significant changes in local micromodulus except for a reduction at the posterior end of the inferior side. Following the induction of aberrant occlusal loading by the unilateral anterior crossbite (UAC) procedure, both wild-type (WT) and Col5a1+/- condylar cartilage exhibited salient remodeling, and Col5a1+/- condyle developed more pronounced degeneration and hypertrophy at the posterior end than the WT. In contrast, neither UAC nor collagen V deficiency induced marked changes in the morphology or mechanical properties of the disc. Together, our findings highlight the distinct roles of collagen V in regulating these two units during postnatal growth and remodeling, emphasizing its more crucial role in condylar cartilage due to its impact on the highly mechanosensitive progenitors. Results thus provide the foundation for using collagen V to improve the regeneration of TMJ and the care of patients with TMJ disorder. STATEMENT OF SIGNIFICANCE: Successful regeneration of temporomandibular joint (TMJ) condylar cartilage and articular disc remains a significant challenge due to the limited understanding of matrix molecular activities that regulate the formation and remodeling of these tissues. This study demonstrates that collagen V plays distinct and critical roles in these processes. In condylar cartilage, collagen V is essential for regulating progenitor cell fate and maintaining matrix integrity. In the disc, collagen V also regulates fibril structure and local micromechanics, but has a limited impact on cell phenotype or its remodeling response. Our findings establish collagen V as a key component in maintaining the integrity of these two units, with a more crucial role in condylar cartilage due to its impact on progenitor cell activities.

Focal adhesion kinase regulates tendon cell mechanoresponse and physiological tendon development.

Tendons enable locomotion by transmitting high tensile mechanical forces between muscle and bone via their dense extracellular matrix (ECM). The application of extrinsic mechanical stimuli via muscle contraction is necessary to regulate healthy tendon function. Specifically, applied physiological levels of mechanical loading elicit an anabolic tendon cell response, while decreased mechanical loading evokes a degradative tendon state. Although the tendon response to mechanical stimuli has implications in disease pathogenesis and clinical treatment strategies, the cell signaling mechanisms by which tendon cells sense and respond to mechanical stimuli within the native tendon ECM remain largely unknown. Therefore, we explored the role of cell-ECM adhesions in regulating tendon cell mechanotransduction by perturbing the genetic expression and signaling activity of focal adhesion kinase (FAK) through both in vitro and in vivo approaches. We determined that FAK regulates tendon cell spreading behavior and focal adhesion morphology, nuclear deformation in response to applied mechanical strain, and mechanosensitive gene expression. In addition, our data reveal that FAK signaling plays an essential role in in vivo tendon development and postnatal growth, as FAK-knockout mouse tendons demonstrated reduced tendon size, altered mechanical properties, differences in cellular composition, and reduced maturity of the deposited ECM. These data provide a foundational understanding of the role of FAK signaling as a critical regulator of in situ tendon cell mechanotransduction. Importantly, an increased understanding of tendon cell mechanotransductive mechanisms may inform clinical practice as well as lead to the discovery of diagnostic and/or therapeutic molecular targets.

Characterization of TGFß1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system.

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-ß (TGFß) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Developmental morphogens direct human induced pluripotent stem cells toward an annulus fibrosus-like cell phenotype.

Introduction: Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs toward the AF fate remain unknown. Methods: iPSC-derived sclerotome cells were treated with various combinations of developmental signals including transforming growth factor beta 3 (TGF-ß3), connective tissue growth factor (CTGF), platelet derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), or the Hedgehog pathway activator, Purmorphamine, and gene expression changes in major AF-associated ECM genes were assessed. The top performing combination treatments were further validated by using three distinct iPSC lines and by assessing the production of upregulated ECM proteins of interest. To conduct a broader analysis of the transcriptomic shifts elicited by each factor combination, and to compare genetic profiles of treated cells to mature human AF cells, a 96.96 Fluidigm gene expression array was applied, and principal component analysis was employed to identify the transcriptional signatures of each cell population and treatment group in comparison to native AF cells. Results: TGF-ß3, in combination with PDGF-BB, CTGF, or IGF-1, induced an upregulation of key AF ECM genes in iPSC-derived sclerotome cells. In particular, treatment with a combination of TGF-ß3 with PDGF-BB for 14 days significantly increased gene expression of collagen II and aggrecan and increased protein deposition of collagen I and elastin compared to other treatment groups. Assessment of genes uniquely highly expressed by AF cells or SCL cells, respectively, revealed a shift toward the genetic profile of AF cells with the addition of TGF-ß3 and PDGF-BB for 14 days. Discussion: These findings represent an initial approach to guide human induced pluripotent stem cells toward an AF-like fate for cellular delivery strategies.

Reduction in postnatal weight-bearing does not alter the trajectory of murine meniscus growth and maturation.

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.

YAP and TAZ couple osteoblast precursor mobilization to angiogenesis and mechanoregulation in murine bone development.

Fetal bone development occurs through the conversion of avascular cartilage to vascularized bone at the growth plate. This requires coordinated mobilization of osteoblast precursors with blood vessels. In adult bone, vessel-adjacent osteoblast precursors are maintained by mechanical stimuli; however, the mechanisms by which these cells mobilize and respond to mechanical cues during embryonic development are unknown. Here, we show that the mechanoresponsive transcriptional regulators Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) spatially couple osteoblast precursor mobilization to angiogenesis, regulate vascular morphogenesis to control cartilage remodeling, and mediate mechanoregulation of embryonic murine osteogenesis. Mechanistically, YAP and TAZ regulate a subset of osteoblast-lineage cells, identified by single-cell RNA sequencing as vessel-associated osteoblast precursors, which regulate transcriptional programs that direct blood vessel invasion through collagen-integrin interactions and Cxcl12. Functionally, in 3D human cell co-culture, CXCL12 treatment rescues angiogenesis impaired by stromal cell YAP/TAZ depletion. Together, these data establish functions of the vessel-associated osteoblast precursors in bone development.

Preclinical tendon and ligament models: Beyond the 3Rs (replacement, reduction, and refinement) to 5W1H (why, who, what, where, when, how).

Several tendon and ligament animal models were presented at the 2022 Orthopaedic Research Society Tendon Section Conference held at the University of Pennsylvania, May 5 to 7, 2022. A key objective of the breakout sessions at this meeting was to develop guidelines for the field, including for preclinical tendon and ligament animal models. This review summarizes the perspectives of experts for eight surgical small and large animal models of rotator cuff tear, flexor tendon transection, anterior cruciate ligament tear, and Achilles tendon injury using the framework: "Why, Who, What, Where, When, and How" (5W1H). A notable conclusion is that the perfect tendon model does not exist; there is no single gold standard animal model that represents the totality of tendon and ligament disease. Each model has advantages and disadvantages and should be carefully considered in light of the specific research question. There are also circumstances when an animal model is not the best approach. The wide variety of tendon and ligament pathologies necessitates choices between small and large animal models, different anatomic sites, and a range of factors associated with each model during the planning phase. Attendees agreed on some guiding principles including: providing clear justification for the model selected, providing animal model details at publication, encouraging sharing of protocols and expertise, improving training of research personnel, and considering greater collaboration with veterinarians. A clear path for translating from animal models to clinical practice was also considered as a critical next step for accelerating progress in the tendon and ligament field.

Lack of skeletal muscle contraction disrupts fibrous tissue morphogenesis in the developing murine knee.

Externally applied forces, such as those generated through skeletal muscle contraction, are important to embryonic joint formation, and their loss can result in gross morphologic defects including joint fusion. While the absence of muscle contraction in the developing chick embryo leads to dissociation of dense connective tissue structures of the knee and ultimately joint fusion, the central knee joint cavitates whereas the patellofemoral joint does not in murine models lacking skeletal muscle contraction, suggesting a milder phenotype. These differential results suggest that muscle contraction may not have as prominent of a role in the growth and development of dense connective tissues of the knee. To explore this question, we investigated the formation of the menisci, tendon, and ligaments of the developing knee in two murine models that lack muscle contraction. We found that while the knee joint does cavitate, there were multiple abnormalities in the menisci, patellar tendon, and cruciate ligaments. The initial cellular condensation of the menisci was disrupted and dissociation was observed at later embryonic stages. The initial cell condensation of the tendon and ligaments were less affected than the meniscus, but these tissues contained cells with hyper-elongated nuclei and displayed diminished growth. Interestingly, lack of muscle contraction led to the formation of an ectopic ligamentous structure in the anterior region of the joint as well. These results indicate that muscle forces are essential for the continued growth and maturation of these structures during this embryonic period.

Rapid specialization and stiffening of the primitive matrix in developing articular cartilage and meniscus.

Understanding early patterning events in the extracellular matrix (ECM) formation can provide a blueprint for regenerative strategies to better recapitulate the function of native tissues. Currently, there is little knowledge on the initial, incipient ECM of articular cartilage and meniscus, two load-bearing counterparts of the knee joint. This study elucidated distinctive traits of their developing ECMs by studying the composition and biomechanics of these two tissues in mice from mid-gestation (embryonic day 15.5) to neo-natal (post-natal day 7) stages. We show that articular cartilage initiates with the formation of a pericellular matrix (PCM)-like primitive matrix, followed by the separation into distinct PCM and territorial/interterritorial (T/IT)-ECM domains, and then, further expansion of the T/IT-ECM through maturity. In this process, the primitive matrix undergoes a rapid, exponential stiffening, with a daily modulus increase rate of 35.7% [31.9 39.6]% (mean [95% CI]). Meanwhile, the matrix becomes more heterogeneous in the spatial distribution of properties, with concurrent exponential increases in the standard deviation of micromodulus and the slope correlating local micromodulus with the distance from cell surface. In comparison to articular cartilage, the primitive matrix of meniscus also exhibits exponential stiffening and an increase in heterogeneity, albeit with a much slower daily stiffening rate of 19.8% [14.9 24.9]% and a delayed separation of PCM and T/IT-ECM. These contrasts underscore distinct development paths of hyaline versus fibrocartilage. Collectively, these findings provide new insights into how knee joint tissues form to better guide cell- and biomaterial-based repair of articular cartilage, meniscus and potentially other load-bearing cartilaginous tissues. STATEMENT OF SIGNIFICANCE: Successful regeneration of articular cartilage and meniscus is challenged by incomplete knowledge of early events that drive the initial formation of the tissues' extracellular matrix in vivo. This study shows that articular cartilage initiates with a pericellular matrix (PCM)-like primitive matrix during embryonic development. This primitive matrix then separates into distinct PCM and territorial/interterritorial domains, undergoes an exponential daily stiffening of ≈36% and an increase in micromechanical heterogeneity. At this early stage, the meniscus primitive matrix shows differential molecular traits and exhibits a slower daily stiffening of ≈20%, underscoring distinct matrix development between these two tissues. Our findings thus establish a new blueprint to guide the design of regenerative strategies to recapitulate the key developmental steps in vivo.

Histological and immunohistochemical guide to tendon tissue.

Tendons are unique dense connective tissues with discrete zones having specific structure and function. They are juxtaposed with other tissues (e.g., bone, muscle, and fat) with different compositional, structural, and mechanical properties. Additionally, tendon properties change drastically with growth and development, disease, aging, and injury. Consequently, there are unique challenges to performing high quality histological assessment of this tissue. To address this need, histological assessment was one of the breakout session topics at the 2022 Orthopaedic Research Society (ORS) Tendon Conference hosted at the University of Pennsylvania. The purpose of the breakout session was to discuss needs from members of the ORS Tendon Section related to histological procedures, data presentation, knowledge dissemination, and guidelines for future work. Therefore, this review provides a brief overview of the outcomes of this discussion and provides a set of guidelines, based on the perspectives from our laboratories, for histological assessment to assist researchers in their quest to utilize these techniques to enhance the outcomes and interpretations of their studies.

Investigating the temporal roles of decorin and biglycan in tendon healing.

The small leucine-rich proteoglycans, decorin and biglycan, are minor components of the tendon extracellular matrix that regulate fibrillogenesis and matrix assembly. Our study objective was to define the temporal roles of decorin and biglycan during tendon healing using inducible knockout mice to include genetic knockdown at specific phases of healing: time of injury, the proliferative phase, and the remodeling phase. We hypothesized that knockdown of decorin or biglycan would adversely affect tendon healing, and that by prescribing the timing of knockdown, we could elucidate the temporal roles of these proteins during healing. Contrary to our hypothesis, decorin knockdown did not affect tendon healing. However, when biglycan was knocked down, either alone or coupled with decorin, tendon modulus was increased relative to wild-type mice, and this finding was consistent among all induction timepoints. At 6 weeks postinjury, we observed increased expression of genes associated with the extracellular matrix and growth factor signaling in the biglycan knockdown and compound decorin-biglycan knockdown tendons. Interestingly, these groups demonstrated opposing trends in gene expression as a function of knockdown-induction timepoint, highlighting distinct temporal roles for decorin and biglycan. In summary, this study finds that biglycan plays multiple functions throughout tendon healing, with the most impactful, detrimental role likely occurring during late-stage healing. Statement of clinical importance: This study helps to define the molecular factors that regulate tendon healing, which may aid in the development of new clinical therapies.

Targeting the hedgehog signaling pathway to improve tendon-to-bone integration.

OBJECTIVE: While the role of hedgehog (Hh) signaling in promoting zonal fibrocartilage production during development is well-established, whether this pathway can be leveraged to improve tendon-to-bone repair in adults is unknown. Our objective was to genetically and pharmacologically stimulate the Hh pathway in cells that give rise to zonal fibrocartilaginous attachments to promote tendon-to-bone integration. DESIGN: Hh signaling was stimulated genetically via constitutive Smo (SmoM2 construct) activation of bone marrow stromal cells or pharmacologically via systemic agonist delivery to mice following anterior cruciate ligament reconstruction (ACLR). To assess tunnel integration, we measured mineralized fibrocartilage (MFC) formation in these mice 28 days post-surgery and performed tunnel pullout testing. RESULTS: Hh pathway-related genes increased in cells forming the zonal attachments in wild-type mice. Both genetic and pharmacologic stimulation of the Hh pathway increased MFC formation and integration strength 28 days post-surgery. We next conducted studies to define the role of Hh in specific stages of the tunnel integration process. We found Hh agonist treatment increased the proliferation of the progenitor pool in the first week post-surgery. Additionally, genetic stimulation led to continued MFC production in the later stages of the integration process. These results indicate that Hh signaling plays an important biphasic role in cell proliferation and differentiation towards fibrochondrocytes following ACLR. CONCLUSION: This study reveals a biphasic role for Hh signaling during the tendon-to-bone integration process after ACLR. In addition, the Hh pathway is a promising therapeutic target to improve tendon-to-bone repair outcomes.

Mechanoepigenetic regulation of extracellular matrix homeostasis via Yap and Taz.

Cells integrate mechanical cues to direct fate specification to maintain tissue function and homeostasis. While disruption of these cues is known to lead to aberrant cell behavior and chronic diseases, such as tendinopathies, the underlying mechanisms by which mechanical signals maintain cell function are not well understood. Here, we show using a model of tendon de-tensioning that loss of tensile cues in vivo acutely changes nuclear morphology, positioning, and expression of catabolic gene programs, resulting in subsequent weakening of the tendon. In vitro studies using paired ATAC/RNAseq demonstrate that the loss of cellular tension rapidly reduces chromatin accessibility in the vicinity of Yap/Taz genomic targets while also increasing expression of genes involved in matrix catabolism. Concordantly, the depletion of Yap/Taz elevates matrix catabolic expression. Conversely, overexpression of Yap results in a reduction of chromatin accessibility at matrix catabolic gene loci, while also reducing transcriptional levels. The overexpression of Yap not only prevents the induction of this broad catabolic program following a loss of cellular tension, but also preserves the underlying chromatin state from force-induced alterations. Taken together, these results provide novel mechanistic details by which mechanoepigenetic signals regulate tendon cell function through a Yap/Taz axis.

CD206+ tendon resident macrophages and their potential crosstalk with fibroblasts and the ECM during tendon growth and maturation.

Resident macrophages exist in a variety of tissues, including tendon, and play context-specific roles in their tissue of residence. In this study, we define the spatiotemporal distribution and phenotypic profile of tendon resident macrophages and their crosstalk with neighboring tendon fibroblasts and the extracellular matrix (ECM) during murine tendon development, growth, and homeostasis. Fluorescent imaging of cryosections revealed that F4/80+ tendon resident macrophages reside adjacent to Col1a1-CFP+ Scx-GFP+ fibroblasts within the tendon fascicle from embryonic development (E15.5) into adulthood (P56). Through flow cytometry and qPCR, we found that these tendon resident macrophages express several well-known macrophage markers, including Adgre1 (F4/80), Mrc1 (CD206), Lyve1, and Folr2, but not Ly-6C, and express the Csf1r-EGFP ("MacGreen") reporter. The proportion of Csf1r-EGFP+ resident macrophages in relation to the total cell number increases markedly during early postnatal growth, while the density of macrophages per mm2 remains constant during this same time frame. Interestingly, proliferation of resident macrophages is higher than adjacent fibroblasts, which likely contributes to this increase in macrophage proportion. The expression profile of tendon resident macrophages also changes with age, with increased pro-inflammatory and anti-inflammatory cytokine expression in P56 compared to P14 macrophages. In addition, the expression profile of limb tendon resident macrophages diverges from that of tail tendon resident macrophages, suggesting differential phenotypes across anatomically and functionally different tendons. As macrophages are known to communicate with adjacent fibroblasts in other tissues, we conducted ligand-receptor analysis and found potential two-way signaling between tendon fibroblasts and resident macrophages. Tendon fibroblasts express high levels of Csf1, which encodes macrophage colony stimulating factor (M-CSF) that acts on the CSF1 receptor (CSF1R) on macrophages. Importantly, Csf1r-expressing resident macrophages preferentially localize to Csf1-expressing fibroblasts, supporting the "nurturing scaffold" model for tendon macrophage patterning. Lastly, we found that tendon resident macrophages express high levels of ECM-related genes, including Mrc1 (mannose receptor), Lyve1 (hyaluronan receptor), Lair1 (type I collagen receptor), Ctss (elastase), and Mmp13 (collagenase), and internalize DQ Collagen in explant cultures. Overall, our study provides insights into the potential roles of tendon resident macrophages in regulating fibroblast phenotype and the ECM during tendon growth.

Multiplexed tape-stabilized cryohistology of mineralized large animal specimens.

Tape-stabilized cryohistology is a powerful histological method to reinforce tissue samples during and after sectioning, enhancing the overall image quality. This technique has widely been applied to section mineralized small animal (i.e., mice, rat, rabbit) specimens, but has only been sparsely implemented for large animal samples that have a greater tendency to tear due to their increased surface area. Here, we present an optimized protocol for tape-stabilized cryohistology of undecalcified minipig vertebral body, femoral head, and temporomandibular joint samples. This protocol further develops a pipeline for sequential staining and imaging of the tape-stabilized cryosections. Images from multiple rounds of staining (endogenous bone mineral labels, aligned collagen (polarized light), tartrate resistant phosphatase (TRAP), alkaline phosphatase (AP), and toluidine blue) are overlaid to provide insight into dynamic bone remodeling. Overall, the established multiplexed tape-stabilized cryohistology protocol provides step-by-step instructions and guidance to cryosection large, mineralized tissues, and maximize data output from a single histological section.

Type V collagen regulates the structure and biomechanics of TMJ condylar cartilage: A fibrous-hyaline hybrid.

This study queried the role of type V collagen in the post-natal growth of temporomandibular joint (TMJ) condylar cartilage, a hybrid tissue with a fibrocartilage layer covering a secondary hyaline cartilage layer. Integrating outcomes from histology, immunofluorescence imaging, electron microscopy and atomic force microscopy-based nanomechanical tests, we elucidated the impact of type V collagen reduction on TMJ condylar cartilage growth in the type V collagen haploinsufficiency and inducible knockout mice. Reduction of type V collagen led to significantly thickened collagen fibrils, decreased tissue modulus, reduced cell density and aberrant cell clustering in both the fibrous and hyaline layers. Post-natal growth of condylar cartilage involves the chondrogenesis of progenitor cells residing in the fibrous layer, which gives rise to the secondary hyaline layer. Loss of type V collagen resulted in reduced proliferation of these cells, suggesting a possible role of type V collagen in mediating the progenitor cell niche. When the knockout of type V collagen was induced in post-weaning mice after the start of physiologic TMJ loading, the hyaline layer exhibited pronounced thinning, supporting an interplay between type V collagen and occlusal loading in condylar cartilage growth. The phenotype in hyaline layer can thus be attributed to the impact of type V collagen on the mechanically regulated progenitor cell activities. In contrast, knee cartilage does not contain the progenitor cell population at post-natal stages, and develops normal structure and biomechanical properties with the loss of type V collagen. Therefore, in the TMJ, in addition to its established role in regulating the assembly of collagen I fibrils, type V collagen also impacts the mechanoregulation of progenitor cell activities in the fibrous layer. We expect such knowledge to establish a foundation for understanding condylar cartilage matrix development and regeneration, and to yield new insights into the TMJ symptoms in patients with classic Ehlers-Danlos syndrome, a genetic disease due to autosomal mutation of type V collagen.

Intrinsic and growth-mediated cell and matrix specialization during murine meniscus tissue assembly.

The incredible mechanical strength and durability of mature fibrous tissues and their extremely limited turnover and regenerative capacity underscores the importance of proper matrix assembly during early postnatal growth. In tissues with composite extracellular matrix (ECM) structures, such as the adult knee meniscus, fibrous (Collagen-I rich), and cartilaginous (Collagen-II, proteoglycan-rich) matrix components are regionally segregated to the outer and inner portions of the tissue, respectively. While this spatial variation in composition is appreciated to be functionally important for resisting complex mechanical loads associated with gait, the establishment of these specialized zones is poorly understood. To address this issue, the following study tracked the growth of the murine meniscus from its embryonic formation through its first month of growth, encompassing the critical time-window during which animals begin to ambulate and weight bear. Using histological analysis, region specific high-throughput qPCR, and Col-1, and Col-2 fluorescent reporter mice, we found that matrix and cellular features defining specific tissue zones were already present at birth, before continuous weight-bearing had occurred. These differences in meniscus zones were further refined with postnatal growth and maturation, resulting in specialization of mature tissue regions. Taken together, this work establishes a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level throughout meniscus maturation. The findings of this study provide a framework for investigating the reciprocal feedback between cells and their evolving microenvironments during assembly of a mechanically robust fibrocartilage tissue, thus providing insight into mechanisms of tissue degeneration and effective regenerative strategies.

The critical role of Hedgehog-responsive mesenchymal progenitors in meniscus development and injury repair.

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1-lineage cells. Normally, meniscal tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh agonist right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.

Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation.

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.