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.

Learning on a Limb: An outreach module to engage high school students in orthopaedics.

Orthopaedic researchers need new strategies for engaging diverse students. Our field has demonstrated noticeable gaps in racial, ethnic, and gender diversity, which inhibit our ability to innovate and combat the severe socioeconomic burden of musculoskeletal disorders. Towards this goal, we designed, implemented, and evaluated Learning on a Limb, an orthopaedic research outreach module to teach diverse high school students about orthopaedic research. During the 4-hr module, students completed hands-on activities to learn how biomechanical testing, microcomputed tomography, cell culture, and histology are used in orthopaedic research. Over three years, we recruited 32 high school students from the Greater Philadelphia Area to participate in Learning on a Limb. Most participants identified as racial/ethnic or gender minorities in orthopaedic research. Using pre/post-tests, we found that students experienced significant learning gains of 51 percentage points from completing Learning on a Limb. In addition to teaching students about orthopaedic research, post-survey data demonstrated that participating in Learning on a Limb strongly influenced students' interest in orthopaedic research. Several students acted on this interest by completing summer research experiences in the McKay Orthopaedic Research Laboratory at the University of Pennsylvania. Learning on a Limb instructors also benefited by having the opportunity to "pay it forward" to the next generation of students and build community within their department. Empowering institutions to host modules like Learning on a Limb would synergistically inspire diverse high school students and strengthen community within orthopaedic departments to ultimately enhance orthopaedic research innovations.

TYPE III COLLAGEN REGULATES MATRIX ARCHITECTURE AND MECHANOSENSING DURING WOUND HEALING.

Post-natal cutaneous wound healing is characterized by development of collagen-rich scar lacking the architecture and functional integrity of unwounded tissue. Directing cell behaviors to efficiently heal wounds while minimizing scar formation remains a major wound management goal. Herein, we demonstrate type III collagen (Col3) as a critical regulator of re-epithelialization and scar formation during healing of Col3-enriched, regenerative (Acomys), scar-permissive (CD-1 Mus and wild-type Col3B6/B6 mice), and Col3-deficient, scar-promoting (Col3F/F, a murine conditional knockdown model) cutaneous wound models. We define a scar-permissive fibrillar collagen architecture signature characterized by elongated and anisotropically-aligned collagen fibers that is dose-dependently suppressed by Col3. Further, loss of Col3 alters how cells interpret their microenvironment - their mechanoperception - such that Col3-deficient cells display mechanically-active phenotypes in the absence of increased microenvironmental stiffness via upregulation and engagement of the profibrotic integrin α11. Further understanding Col3's role in regulating matrix architecture and mechanoresponses may inform clinical strategies that harness pro-regenerative mechanisms.

Precision Repair of Zone-Specific Meniscal Injuries Using a Tunable Extracellular Matrix-Based Hydrogel System.

Meniscus injuries present significant therapeutic challenges due to their limited self-healing capacity and diverse biological and mechanical properties across meniscal tissue. Conventional repair strategies neglect to replicate the complex zonal characteristics within the meniscus, resulting in suboptimal outcomes. In this study, we introduce an innovative, age- and stiffness-tunable meniscus decellularized extracellular matrix (DEM)-based hydrogel system designed for precision repair of heterogeneous, zonal-dependent meniscus injuries. By synthesizing age-dependent DEM hydrogels, we identified distinct cellular responses: fetal bovine meniscus-derived DEM promoted chondrogenic differentiation, while adult meniscus-derived DEM supported fibrochondrogenic phenotypes. The incorporation of methacrylate hyaluronic acid (MeHA) further refined the mechanical properties and injectability of the DEM-based hydrogels. The combination of age-dependent DEM with MeHA allowed for precise stiffness tuning, influencing cell differentiation and closely mimicking native tissue environments. In vivo tests confirmed the biocompatibility of hydrogels and their integration with native meniscus tissues. Furthermore, advanced 3D bioprinting techniques enabled the fabrication of hybrid hydrogels with biomaterial and mechanical gradients, effectively emulating the zonal properties of meniscus tissue and enhancing cell integration. This study represents a significant advancement in meniscus tissue engineering, providing a promising platform for customized regenerative therapies across a range of heterogeneous fibrous connective tissues.

Revealing the Biophysics of Lamina-Associated Domain Formation by Integrating Theoretical Modeling and High-Resolution Imaging.

The interactions between chromatin and the nuclear lamina orchestrate cell type-specific gene activity by forming lamina-associated domains (LADs) which preserve cellular characteristics through gene repression. However, unlike the interactions between chromatin segments, the strength of chromatin-lamina interactions and their dependence on cellular environment are not well understood. Here, we develop a theory to predict the size and shape of peripheral heterochromatin domains by considering the energetics of chromatin-chromatin interactions, the affinity between chromatin and the nuclear lamina and the kinetics of methylation and acetylation9in human mesenchymal stem cells (hMSCs). Through the analysis of super-resolution images of peripheral heterochromatin domains using this theoretical framework, we determine the nuclear lamina-wide distribution of chromatin-lamina affinities. We find that the extracted affinity is highly spatially heterogeneous and shows a bimodal distribution, indicating regions along the lamina with strong chromatin binding and those exhibiting vanishing chromatin affinity interspersed with some regions exhibiting a relatively diminished chromatin interactions, in line with the presence of structures such as nuclear pores. Exploring the role of environmental cues on peripheral chromatin, we find that LAD thickness increases when hMSCs are cultured on a softer substrate, in correlation with contractility-dependent translocation of histone deacetylase 3 (HDAC3) from the cytosol to the nucleus. In soft microenvironments, chromatin becomes sequestered at the nuclear lamina, likely due to the interactions of HDAC3 with the chromatin anchoring protein LAP2 ß ,increasing chromatin-lamina affinity, as well as elevated levels of the intranuclear histone methylation. Our findings are further corroborated by pharmacological interventions that inhibit contractility, as well as by manipulating methylation levels using epigenetic drugs. Notably, in the context of tendinosis, a chronic condition characterized by collagen degeneration, we observed a similar increase in the thickness of peripheral chromatin akin to that of cells cultured on soft substrates consistent with theoretical predictions. Our findings underscore the pivotal role of the microenvironment in shaping genome organization and highlight its relevance in pathological conditions.

Structure-Mechanics Principles and Mechanobiology of Fibrocartilage Pericellular Matrix: A Pivotal Role of Type V Collagen.

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.

Interleukin receptor therapeutics attenuate inflammation in canine synovium following cruciate ligament injury.

OBJECTIVE: In the knee, synovial fibrosis after ligamentous injury is linked to progressive joint pain and stiffness. The objective of this study was to evaluate changes in synovial architecture, mechanical properties, and transcriptional profiles following naturally occurring cruciate ligament injury in canines and to test potential therapeutics that target drivers of synovial inflammation and fibrosis. DESIGN: Synovia from canines with spontaneous cruciate ligament tears and from healthy knees were assessed via histology (n = 10/group) and micromechanical testing (n = 5/group) to identify changes in tissue architecture and stiffness. Additional samples (n = 5/group) were subjected to RNA-sequencing to define the transcriptional response to injury. Finally, synovial tissue samples from injured animals (n = 6 (IL1) or n = 8 (IL6)/group) were assessed in vitro for response to therapeutic molecules directed against interleukin (IL) signaling (IL1 or IL6). RESULTS: Cruciate injury resulted in increased synovial fibrosis, vascularity, inflammatory cell infiltration, and intimal hyperplasia. Additionally, the stiffness of both the intima and subintima regions were higher in diseased compared to healthy tissue. Differential gene expression analysis showed that diseased synovium had an upregulation of immune response and cell adhesion pathways and a downregulation of Rho protein transduction pathways. In vitro application of small molecule therapeutics targeting IL1 (anakinra) or IL6 (tocilizumab) dampened expression of inflammatory and matrix deposition mediators. CONCLUSION: Spontaneous cruciate ligament injury in canines is associated with synovial inflammation and fibrosis in a relevant model for testing emerging intra-articular treatments. Small molecule therapeutics targeting IL pathways may be ideal interventions for delivery to the joint space after injury.

Physical-property-based patterning: simply engineering complex tissues.

The field of biofabrication is rapidly expanding with the advent of new technologies and material systems to engineer complex tissues. In this opinion article, we introduce an emerging tissue patterning method, physical-property-based patterning, that has strong translational potential given its simplicity and limited dependence on external hardware. Physical-property-based patterning relies solely on the intrinsic density, magnetic susceptibility, or compressibility of an object, its surrounding solution, and the noncontact application of a remote field. We discuss how physical properties can be exploited to pattern objects and design a variety of biologic tissues. Finally, we pose several open questions that, if addressed, could transform the status quo of biofabrication, pushing us one step closer to patterning tissues in situ.

Microinterfaces in biopolymer-based bicontinuous hydrogels guide rapid 3D cell migration.

Cell migration is critical for tissue development and regeneration but requires extracellular environments that are conducive to motion. Cells may actively generate migratory routes in vivo by degrading or remodeling their environments or instead utilize existing extracellular matrix microstructures or microtracks as innate pathways for migration. While hydrogels in general are valuable tools for probing the extracellular regulators of 3-dimensional migration, few recapitulate these natural migration paths. Here, we develop a biopolymer-based bicontinuous hydrogel system that comprises a covalent hydrogel of enzymatically crosslinked gelatin and a physical hydrogel of guest and host moieties bonded to hyaluronic acid. Bicontinuous hydrogels form through controlled solution immiscibility, and their continuous subdomains and high micro-interfacial surface area enable rapid 3D migration, particularly when compared to homogeneous hydrogels. Migratory behavior is mesenchymal in nature and regulated by biochemical and biophysical signals from the hydrogel, which is shown across various cell types and physiologically relevant contexts (e.g., cell spheroids, ex vivo tissues, in vivo tissues). Our findings introduce a design that leverages important local interfaces to guide rapid cell migration.

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.