Activating Mitochondrial Sirtuin 3 in Chondrocytes Alleviates Aging-Induced Fibrocartilage Layer Degeneration and Promotes Healing of Degenerative Rotator Cuff Injury.

The present study aimed to examine the impact of mitochondrial sirtuin 3 (SIRT3) on the degenerative rotator cuff injury, which is a prevalent issue among the elderly population primarily due to aging-related tissue degradation. The study hypothesized that SIRT3, as a major deacetylase in mitochondria, is a significant factor in controlling the quality of mitochondria and the deterioration of fibrocartilage, a crucial component of the rotator cuff. Results showed that the aging process led to weakened biomechanical properties and degeneration of the fibrocartilage layer in mice, accompanied by a decrease in SIRT3 expression. SIRT3 activation ameliorated the aging-related disruption of chondrocyte phenotype and fibrocartilage degradation. SIRT3 activator honokiol improved the phenotype of senescent chondrocytes and promoted rotator cuff healing in aged mice through SIRT3 activation. In conclusion, the findings suggested that the decline in SIRT3 levels with age contributes to rotator cuff degeneration and chondrocyte senescence, and that SIRT3 activation through the use of honokiol is an effective approach for promoting rotator cuff healing in the elderly population.

Spatial Heterogeneity Directs Energy Dissipation in Condylar Fibrocartilage.

Condylar fibrocartilage with structural and compositional heterogeneity can efficiently orchestrate load-bearing and energy dissipation, making the temporomandibular joint (TMJ) survive high occlusion loads for a prolonged lifetime. How the thin condylar fibrocartilage can achieve efficient energy dissipation to cushion enormous stresses remains an open question in biology and tissue engineering. Here, three distinct zones in the condylar fibrocartilage are identified by analyzing the components and structure from the macro-and microscale to the nanoscale. Specific proteins are highly expressed in each zone related to its mechanics. The heterogeneity of condylar fibrocartilage can direct energy dissipation through the nano-micron-macro gradient spatial scale, by atomic force microscope (AFM), nanoindentation, dynamic mechanical analyzer assay (DMA), and the corresponding energy dissipation mechanisms are exclusive for each distinct zone. This study reveals the significance of the heterogeneity of condylar fibrocartilage in mechanical behavior and provides new insights into the research methods for cartilage biomechanics and the design of energy-dissipative materials.

Applied Compressive Strain Governs Hyaline-like Cartilage versus Fibrocartilage-like ECM Produced within Hydrogel Constructs.

The goal of cartilage tissue engineering (CTE) is to regenerate new hyaline cartilage in joints and treat osteoarthritis (OA) using cell-impregnated hydrogel constructs. However, the production of an extracellular matrix (ECM) made of fibrocartilage is a potential outcome within hydrogel constructs when in vivo. Unfortunately, this fibrocartilage ECM has inferior biological and mechanical properties when compared to native hyaline cartilage. It was hypothesized that compressive forces stimulate fibrocartilage development by increasing production of collagen type 1 (Col1), an ECM protein found in fibrocartilage. To test the hypothesis, 3-dimensional (3D)-bioprinted hydrogel constructs were fabricated from alginate hydrogel impregnated with ATDC5 cells (a chondrogenic cell line). A bioreactor was used to simulate different in vivo joint movements by varying the magnitude of compressive strains and compare them with a control group that was not loaded. Chondrogenic differentiation of the cells in loaded and unloaded conditions was confirmed by deposition of cartilage specific molecules including glycosaminoglycans (GAGs) and collagen type 2 (Col2). By performing biochemical assays, the production of GAGs and total collagen was also confirmed, and their contents were quantitated in unloaded and loaded conditions. Furthermore, Col1 vs. Col2 depositions were assessed at different compressive strains, and hyaline-like cartilage vs. fibrocartilage-like ECM production was analyzed to investigate how applied compressive strain affects the type of cartilage formed. These assessments showed that fibrocartilage-like ECM production tended to reduce with increasing compressive strain, though its production peaked at a higher compressive strain. According to these results, the magnitude of applied compressive strain governs the production of hyaline-like cartilage vs. fibrocartilage-like ECM and a high compressive strain stimulates fibrocartilage-like ECM formation rather than hyaline cartilage, which needs to be addressed by CTE approaches.

Common cellular origin and diverging developmental programs for different sesamoid bones.

Sesamoid bones are small auxiliary bones that form near joints and contribute to their stability and function. Thus far, providing a comprehensive developmental model or classification system for this highly diverse group of bones has been challenging. Here, we compare our previously reported mechanisms of patella development in the mouse with those of two anatomically different sesamoids, namely lateral fabella and digit sesamoids. We show that all three types of sesamoid bones originate from Sox9+ /Scx+ progenitors under the regulation of TGFß and independently of mechanical stimuli from muscles. Whereas BMP2 regulates the growth of all examined sesamoids, the differentiation of lateral fabella or digit sesamoids is regulated redundantly by BMP4 and BMP2. Next, we show that whereas patella and digit sesamoids initially form in juxtaposition to long bones, lateral fabella forms independently and at a distance. Finally, our evidence suggests that, unlike the synovial joint that separates patella from femur, digit sesamoids detach from the phalanx by formation of a fibrocartilaginous joint. These findings highlight both common and divergent molecular and mechanical features of sesamoid bone development, which underscores their evolutionary plasticity.

The role of SLRPs and large aggregating proteoglycans in collagen fibrillogenesis, extracellular matrix assembly, and mechanical function of fibrocartilage.

PURPOSE: Proteoglycans, especially small leucine rich proteoglycans (SLRPs), play major roles in facilitating the development and regulation of collagen fibers and other extracellular matrix components. However, their roles in fibrocartilage have not been widely reviewed. Here, we discuss both SLRP and large aggregating proteoglycan's roles in collagen fibrillogenesis and extracellular matrix assembly in fibrocartilage tissues such as the meniscus, annulus fibrosus (AF), and TMJ disc. We also discuss their expression levels throughout development, aging and degeneration, as well as repair. METHODS: A review of literature discussing proteoglycans and collagen fibrillogenesis in fibrocartilage was conducted and data from these manuscripts were analyzed and grouped to discuss trends throughout the tissue's architectural zones and developmental stage. RESULTS: The spatial collagen architecture of these fibrocartilaginous tissues is reflected in the distribution of proteoglycans expressed, suggesting that each proteoglycan plays an important role in the type of architecture presented and associated mechanical function. CONCLUSION: The unique structure-function relationship of fibrocartilage makes the varied architectures throughout the tissues imperative for their success and understanding the functions of these proteoglycans in developing and maintaining the fiber structure could inform future work in fibrocartilage replacement using tissue engineered constructs.

Strain-Dependent Diffusivity of Small and Large Molecules in Meniscus.

Due to lack of full vascularization, the meniscus relies on diffusion through the extracellular matrix to deliver small (e.g., nutrients) and large (e.g., proteins) to resident cells. Under normal physiological conditions, the meniscus undergoes up to 20% compressive strains. While previous studies characterized solute diffusivity in the uncompressed meniscus, to date, little is known about the diffusive transport under physiological strain levels. This information is crucial to fully understand the pathophysiology of the meniscus. The objective of this study was to investigate strain-dependent diffusive properties of the meniscus fibrocartilage. Tissue samples were harvested from the central portion of porcine medial menisci and tested via fluorescence recovery after photobleaching to measure diffusivity of fluorescein (332 Da) and 40 K Da dextran (D40K) under 0%, 10%, and 20% compressive strain. Specifically, average diffusion coefficient and anisotropic ratio, defined as the ratio of the diffusion coefficient in the direction of the tissue collagen fibers to that orthogonal, were determined. For all the experimental conditions investigated, fluorescein diffusivity was statistically faster than that of D40K. Also, for both molecules, diffusion coefficients significantly decreased, up to ∼45%, as the strain increased. In contrast, the anisotropic ratios of both molecules were similar and not affected by the strain applied to the tissue. This suggests that compressive strains used in this study did not alter the diffusive pathways in the meniscus. Our findings provide new knowledge on the transport properties of the meniscus fibrocartilage that can be leveraged to further understand tissue pathophysiology and approaches to tissue restoration.

Part I: Development and Physiology of the Temporomandibular Joint.

PURPOSE OF REVIEW: Investigate the developmental physiology of the temporomandibular joint (TMJ), a unique articulation between the cranium and the mandible. RECENT FINDINGS: Principal regulatory factors for TMJ and disc development are Indian hedgehog (IHH) and bone morphogenetic protein (BMP-2). The mechanism is closely associated with ear morphogenesis. Secondary condylar cartilage emerges as a subperiosteal blastema on the medial surface of the posterior mandible. The condylar articular surface is immunoreactive for tenascin-C, so it is a modified fibrous periosteum with an underlying proliferative zone (cambrium layer) that differentiates into fibrocartilage. The latter cushions high loads and subsequently produces endochondral bone. The TMJ is a heavily loaded joint with three cushioning layers of fibrocartilage in the disc, as well as in subarticular zones in the fossa and mandibular condyle. The periosteal articular surface produces fibrocartilage to resist heavy loads, and has unique healing and adaptive properties for maintaining life support functions under adverse environmental conditions.

SR-FTIR as a tool for quantitative mapping of the content and distribution of extracellular matrix in decellularized book-shape bioscaffolds.

BACKGROUND: To evaluate synchrotron radiation-based Fourier transform infrared microspectroscopy (SR-FTIR) as a tool for quantitative mapping of the content and distribution of the extracellular matrix in decellularized fibrocartilage bioscaffolds, and to provide a new platform for quantitatively characterizing bioscaffolds for tissue engineering. METHODS: Fibrocartilage was harvested and cut into book-shape bioscaffolds (N = 54), which were then decellularized. The structures and distribution of collagen fibrous and intrinsic ultrastructure in decellularized fibrocartilage bioscaffolds were evaluated by histological staining and scanning electron microscopy (SEM), respectively. The content of collagen and proteoglycan in the cellularized or decellularized bioscaffolds were also measured by SR-FTIR and biochemical assay. RESULTS: Book-shape fibrocartilage decellularized bioscaffolds were successfully obtained. Histological examination revealed that the structure of extracellular matrix endured during decellularization. Histology and DNA quantification analysis confirmed substantial removal of cells during decellularization. SEM demonstrated that intrinsic ultrastructure of the fibrocartilage bioscaffold was also well preserved. SR-FTIR quantitative analysis confirmed that decellularization had a significant effect on the content and distribution of collagen and proteoglycan in fibrocartilage bioscaffolds, these results are confirmed with the biochemical assay results. CONCLUSION: SR-FTIR imaging can capture the histological morphology of decellularized bioscaffolds. Moreover, it can be used for quantitative mapping of the content and distribution of collagen in the bioscaffolds.

Microstructural heterogeneity directs micromechanics and mechanobiology in native and engineered fibrocartilage.

Treatment strategies to address pathologies of fibrocartilaginous tissue are in part limited by an incomplete understanding of structure-function relationships in these load-bearing tissues. There is therefore a pressing need to develop micro-engineered tissue platforms that can recreate the highly inhomogeneous tissue microstructures that are known to influence mechanotransductive processes in normal and diseased tissue. Here, we report the quantification of proteoglycan-rich microdomains in developing, ageing and diseased fibrocartilaginous tissues, and the impact of these microdomains on endogenous cell responses to physiologic deformation within a native-tissue context. We also developed a method to generate heterogeneous tissue-engineered constructs (hetTECs) with non-fibrous proteoglycan-rich microdomains engineered into the fibrous structure, and show that these hetTECs match the microstructural, micromechanical and mechanobiological benchmarks of native tissue. Our tissue-engineered platform should facilitate the study of the mechanobiology of developing, homeostatic, degenerating and regenerating fibrous tissues.

Effect of Strain, Region, and Tissue Composition on Glucose Partitioning in Meniscus Fibrocartilage.

A nearly avascular tissue, the knee meniscus relies on diffusive transport for nutritional supply to cells. Nutrient transport depends on solute partitioning in the tissue, which governs the amount of nutrients that can enter a tissue. The purpose of the present study was to investigate the effects of mechanical strain, tissue region, and tissue composition on the partition coefficient of glucose in meniscus fibrocartilage. A simple partitioning experiment was employed to measure glucose partitioning in porcine meniscus tissues from two regions (horn and central), from both meniscal components (medial and lateral), and at three levels of compression (0%, 10%, and 20%). Partition coefficient values were correlated to strain level, water volume fraction, and glycosaminoglycan (GAG) content of tissue specimens. Partition coefficient values ranged from 0.47 to 0.91 (n = 48). Results show that glucose partition coefficient is significantly (p < 0.001) affected by compression, decreasing with increasing strain. Furthermore, we did not find a statistically significant effect of tissue when comparing medial versus lateral (p = 0.181) or when comparing central and horn regions (p = 0.837). There were significant positive correlations between tissue water volume fraction and glucose partitioning for all groups. However, the correlation between GAG content and partitioning was only significant in the lateral horn group. Determining how glucose partitioning is affected by tissue composition and loading is necessary for understanding nutrient availability and related tissue health and/or degeneration. Therefore, this study is important for better understanding the transport and nutrition-related mechanisms of meniscal degeneration.

Cell and matrix response of temporomandibular cartilage to mechanical loading.

OBJECTIVES: The generation of transgenic mice expressing green fluorescent proteins (GFPs) has greatly aided our understanding of the development of connective tissues such as bone and cartilage. Perturbation of a biological system such as the temporomandibular joint (TMJ) within its adaptive remodeling capacity is particularly useful in analyzing cellular lineage progression. The objectives of this study were to determine: (i) if GFP reporters expressed in the TMJ indicate the different stages of cell maturation in fibrocartilage and (ii) how mechanical loading affects cellular response in different regions of the cartilage. DESIGN/METHODS: Four-week-old transgenic mice harboring combinations of fluorescent reporters (Dkk3-eGFP, Col1a1(3.6 kb)-GFPcyan, Col1a1(3.6 kb)-GFPtpz, Col2a1-GFPcyan, and Col10a1-RFPcherry) were used to analyze the expression pattern of transgenes in the mandibular condylar cartilage (MCC). To study the effect of TMJ loading, animals were subjected to forced mouth opening with custom springs exerting 50 g force for 1 h/day for 5 days. Dynamic mineralization and cellular proliferation (EdU-labeling) were assessed in loaded vs control mice. RESULTS: Dkk3 expression was seen in the superficial zone of the MCC, followed by Col1 in the cartilage zone, Col2 in the prehypertrophic zone, and Col10 in the hypertrophic zone at and below the tidemark. TMJ loading increased expression of the GFP reporters and EdU-labeling of cells in the cartilage, resulting in a thickness increase of all layers of the cartilage. In addition, mineral apposition increased resulting in Col10 expression by unmineralized cells above the tidemark. CONCLUSION: The TMJ responded to static loading by forming thicker cartilage through adaptive remodeling.

Identification of a progenitor cell population destined to form fracture fibrocartilage callus in Dickkopf-related protein 3-green fluorescent protein reporter mice.

Fracture healing is a complex biological process involving the proliferation of mesenchymal progenitor cells, and chondrogenic, osteogenic, and angiogenic differentiation. The mechanisms underlying the proliferation and differentiation of mesenchymal progenitor cells remain unclear. Here, we demonstrate Dickkopf-related protein 3 (Dkk3) expression in periosteal cells using Dkk3-green fluorescent protein reporter mice. We found that proliferation of mesenchymal progenitor cells began in the periosteum, involving Dkk3-positive cell proliferation near the fracture site. In addition, Dkk3 was expressed in fibrocartilage cells together with smooth muscle α-actin and Col3.6 in the early phase of fracture healing as a cell marker of fibrocartilage cells. Dkk3 was not expressed in mature chondrogenic cells or osteogenic cells. Transient expression of Dkk3 disappeared in the late phase of fracture healing, except in the superficial periosteal area of fracture callus. The Dkk3 expression pattern differed in newly formed type IV collagen positive blood vessels and the related avascular tissue. This is the first report that shows Dkk3 expression in the periosteum at a resting state and in fibrocartilage cells during the fracture healing process, which was associated with smooth muscle α-actin and Col3.6 expression in mesenchymal progenitor cells. These fluorescent mesenchymal lineage cells may be useful for future studies to better understand fracture healing.

Engineering anisotropic biomimetic fibrocartilage microenvironment by bioprinting mesenchymal stem cells in nanoliter gel droplets.

Over the past decade, bioprinting has emerged as a promising patterning strategy to organize cells and extracellular components both in two and three dimensions (2D and 3D) to engineer functional tissue mimicking constructs. So far, tissue printing has neither been used for 3D patterning of mesenchymal stem cells (MSCs) in multiphase growth factor embedded 3D hydrogels nor been investigated phenotypically in terms of simultaneous differentiation into different cell types within the same micropatterned 3D tissue constructs. Accordingly, we demonstrated a biochemical gradient by bioprinting nanoliter droplets encapsulating human MSCs, bone morphogenetic protein 2 (BMP-2), and transforming growth factor ß1 (TGF- ß1), engineering an anisotropic biomimetic fibrocartilage microenvironment. Assessment of the model tissue construct displayed multiphasic anisotropy of the incorporated biochemical factors after patterning. Quantitative real time polymerase chain reaction (qRT-PCR) results suggested genomic expression patterns leading to simultaneous differentiation of MSC populations into osteogenic and chondrogenic phenotype within the multiphasic construct, evidenced by upregulation of osteogenesis and condrogenesis related genes during in vitro culture. Comprehensive phenotypic network and pathway analysis results, which were based on genomic expression data, indicated activation of differentiation related mechanisms, via signaling pathways, including TGF, BMP, and vascular endothelial growth factor.

Assessing the role of surface layer and molecular probe size in diffusion within meniscus tissue.

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.

Compositional, Structural, and Biomechanical Properties of Three Different Soft Tissue-Hard Tissue Insertions: A Comparative Review.

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.

Downregulation of SOX9 expression in developing entheses adjacent to intramembranous bone.

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.

Paracrine signals influence patterns of fibrocartilage differentiation in a lyophilized gelatin hydrogel for applications in rotator cuff repair.

Rotator cuff injuries present a clinical challenge for repair due to current limitations in functional regeneration of the native tendon-to-bone enthesis. A biomaterial that can regionally instruct unique tissue-specific phenotypes offers potential to promote enthesis repair. We have recently demonstrated the mechanical benefits of a stratified triphasic biomaterial made up of tendon- and bone-mimetic collagen scaffold compartments connected via a continuous hydrogel, and we now explore the potential of a biologically favorable enthesis hydrogel for this application. Here we report in vitro behavior of human mesenchymal stem cells (hMSCs) within thiolated gelatin (Gel-SH) hydrogels in response to chondrogenic stimuli as well as paracrine signals derived from MSC-seeded bone and tendon scaffold compartments. Chondrogenic differentiation media promoted upregulation of cartilage and entheseal fibrocartilage matrix markers COL2, COLX, and ACAN as well as the enthesis-associated transcription factors SCX, SOX9, and RUNX2 in hMSCs within Gel-SH. Similar effects were observed in response to TGF-ß3 and BMP-4, enthesis-associated growth factors known to play a role in entheseal development and maintenance. Conditioned media generated by hMSCs seeded in tendon- and bone-mimetic collagen scaffolds influenced patterns of gene expression regarding enthesis-relevant growth factors, matrix markers, and tendon-to-bone transcription factors for hMSCs within the material. Together, these findings demonstrate that a Gel-SH hydrogel provides a permissive environment for enthesis tissue engineering and highlights the significance of cellular crosstalk between adjacent compartments within a spatially graded biomaterial.

Arguments for an increasing differentiation towards fibrocartilaginous components in midportion Achilles tendinopathy.

PURPOSE: The objective of this study was to investigate the fibrocartilaginous differentiation occurring in midportion Achilles tendinopathy. METHODS: Tendon samples were retrospectively collected from 23 patients, who had undergone surgery for midportion Achilles tendinopathy resistant to conservative treatment. Based on histological scores, the biopts were subdivided into three categories: a light, moderate and severe histopathological stage. Throughout these stages, immunohistochemical staining was performed against biglycan, aggrecan and collagen type II, components characteristic for fibrocartilage. Staining of these components was evaluated using a semi-quantitative scoring method. RESULTS: The immunohistochemical scores of biglycan and aggrecan were statistically significant between the histopathological stages (P < 0.001). The immunohistochemical scores were positively correlated with the increasing histopathological stages [Spearman's correlation coefficient = 0.93 for biglycan and 0.78 for aggrecan (P < 0.001)]. Staining for collagen type II remained negative throughout these stages. CONCLUSION: Immunohistochemical staining of the fibrocartilaginous components biglycan and aggrecan showed a progressive increase, correlated with a further evolved histopathological stage. This observation gave arguments for an increased differentiation towards fibrocartilaginous components at protein level in midportion Achilles tendinopathy.

Reconstruction of Fibrocartilage with Fibrous Alignment of Type I Collagen in Scaffold-Free Manner.

For functional reconstruction of fibrocartilage, it is necessary to reproduce the essential mechanical property exhibited by natural fibrocartilage. The distinctive mechanical property of fibrocartilage is originated from the specific histological features of fibrocartilage composed of highly aligned type I collagen (Col I) and an abundant cartilaginous matrix. While the application of tensile stimulation induces highly aligned Col I, our study reveals that it also exerts an antichondrogenic effect on scaffold-free tissues constructed with meniscal chondrocytes (MCs) and induces downregulation of Sox-9 expression and attenuated glycosaminoglycan production. Modulation of mechanotransduction by blocking nuclear translocation of Yes-associated protein (YAP) ameliorated the antichondrogenic effect in the presence of tensile stimulation. Since MCs subjected to mechanical doses either by surface stiffness or tensile stimulation showed reversibility of YAP status even after a long-term exposure to mechanotransduction, fibrocartilage tissue was constructed by sequentially inducing tissue alignment by tensile stimulation followed by inducing cartilaginous matrix production in a tension-released state. The minimal tensile dose to constitute durable tissue alignment was screened by investigating the alignment of cytoskeleton and Col I after culturing the scaffold-free tissue constructs with various tensile doses (10% static tension for 1, 3, 7, and 10 days) followed by maintaining in a released state for 5 days. Fluorescence-conjugated phalloidin binding and immunofluorescence of Col I indicated that the duration of static tension for more than 7 days resulted in durable tissue alignment for at least 5 days in the tension-released state. The tissues subjected to tensile stimulation for 7 days followed by 14 days in a released state in chondrogenic media resulted in abundant cartilaginous matrix as well as uniaxial anisotropic alignment. Our results show that the optimized tensile dose can facilitate the successful reconstruction of fibrocartilage by modulating the characteristics of matrix production by MCs.

Meniscal fibrocartilage regeneration inspired by meniscal maturational and regenerative process.

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.