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Varying degrees of hydroxyapatite (HA) surface functionalization have been implicated as the primary driver of differential osteogenesis observed in infiltrating cells. The ability to reliably create spatially controlled areas of mineralization in composite engineered tissues is of growing interest in the field, and the use of HA-functionalized biomaterials may provide a robust solution to this challenge. In this study, we successfully fabricated polycaprolactone salt-leached scaffolds with two levels of a biomimetic calcium phosphate coating to examine their effects on MSC osteogenesis. Longer duration coating in simulated body fluid (SBF) led to increased HA crystal nucleation within scaffold interiors as well as more robust HA crystal formation on scaffold surfaces. Ultimately, the increased surface stiffness of scaffolds coated in SBF for 7 days in comparison to scaffolds coated in SBF for 1 day led to more robust osteogenesis of MSCs in vitro without the assistance of osteogenic signaling molecules. This study also demonstrated that the use of SBF-based HA coatings can promote higher levels of osteogenesis in vivo. Finally, when incorporated as the endplate region of a larger tissue-engineered intervertebral disc replacement, HA coating did not induce mineralization in or promote cell migration out of neighboring biomaterials. Overall, these results verified tunable biomimetic HA coatings as a promising biomaterial modification to promote discrete regions of mineralization within composite engineered tissues.

The meniscus serves important load-bearing functions and protects the underlying articular cartilage. Unfortunately, meniscus tears are common and impair the ability of the meniscus to distribute loads, increasing the risk of developing osteoarthritis. Therefore, surgical repair of the meniscus is a frequently performed procedure; however, repair does not always prevent osteoarthritis. This is hypothesized to be due to altered joint loading post-injury and repair, where the functional deficit of the meniscus prevents it from performing its role of distributing forces. The objective of this study was to quantify joint kinematics in an intact joint, after a meniscus root tear, and after suture repair in cadaveric porcine knees, a frequently used in vivo model. We utilized an magnetic resonance images-compatible loading device and novel use of a T1 vibe sequence to measure meniscus and femur displacements under physiological axial loads. We found that anterior root tear led to large meniscus displacements under physiological axial loading and that suture anchor repair reduced these displacements but did not fully restore intact joint kinematics. After tear and repair, the anterior region of the meniscus moved posteriorly and medially as it was forced out of the joint space under loading, while the posterior region had small displacements as the posterior attachment acted as a hinge about which the meniscus pivoted in the axial plane. Methods from this study can be applied to assess altered joint kinematics following human knee injuries and evaluate repair strategies aimed to restore joint kinematics.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native nucleus pulposus cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived nucleus pulposus cells in the postnatal mouse disc. Specifically, we established the existence of early and late stage nucleus pulposus cells, corresponding to notochordal progenitor and mature cells, respectively. Late stage cells exhibited significantly higher expression levels of extracellular matrix genes including aggrecan, and collagens II and VI, along with elevated TGF-β and PI3K-Akt signaling. Additionally, we identified Cd9 as a novel surface marker of late stage nucleus pulposus cells, and demonstrated that these cells were localized to the nucleus pulposus periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show the Cd9+ nucleus pulposus cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy nucleus pulposus extracellular matrix. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

The zygapophyseal joints of the spine, also known as the facet joints, are paired diarthrodial joints posterior to the intervertebral disc and neural elements. The pathophysiology of facet osteoarthritis (OA), as well as crosstalk between the disc and facets, remains largely understudied compared to disc degeneration. The purpose of this study was to characterize alterations to human facet cartilage and subchondral bone across a spectrum of degeneration and to investigate correlations between disc and facet degeneration. Human lumbar facet articular surfaces from six independent donors were subject to creep indentation mechanical testing to quantify cartilage mechanical properties, followed by microcomputed tomography (µCT) analyses for subchondral bone morphometry. The degenerative state of each articular surface was assessed via macroscopic scoring and via Osteoarthritis Research Society International histopathology scoring. Our data suggest reduced facet cartilage compressive and tensile moduli and increased permeability with increasing degenerative grade, particularly at the lower levels of the spine. µCT analyses revealed spinal level-dependent alterations to the subchondral bone, with an increase in trabecular bone at the L4-L5 level, but a decrease at the upper levels of the lumbar spine with increasing degenerative grade. Cortical bone volume fraction was generally decreased with increasing degenerative grade across spinal levels. Correlation analysis revealed several associations between quantitative measures of disc degeneration and facet OA. This study showed that alterations in the mechanical properties of facet cartilage and in the structural properties of facet subchondral bone correlated with aspects of disc degeneration and were highly dependent on spinal level.

Chondrocyte phenotype is preserved when cells are round and the actin cytoskeleton is cortical. Conversely, these cells rapidly dedifferentiate in vitro with increased mechanoactive Rho signaling, which increases cell size and causes large actin stress fiber to form. While the effects of Rho on chondrocyte phenotype are well established, the molecular mechanism is not yet fully elucidated. Yap, a transcriptional co-regulator, is regulated by Rho in a mechanotransductive manner and can suppress chondrogenesis in vivo. Here, we sought to elucidate the relationship between mechanoactive Rho and Yap on chondrogenic gene expression. We first show that decreasing mechanoactive state through Rho inhibition results in a broad increase in chondrogenic gene expression. Next, we show that Yap and its co-regulator Taz, are negative regulators of chondrogenic gene expression, and removal of these factors promotes chondrogenesis even in environments that promote cell spreading. Finally, we establish that Yap/Taz is essential for translating Rho-mediated signals to negatively regulate chondrogenic gene expression, and that its removal negates the effects of increased Rho signaling. Together, these data indicate that Rho is a mechanoregulator of chondrogenic differentiation, and that its impact on chondrogenic expression is exerted principally through mechanically-induced translocation and activity of Yap and Taz.

The meniscus serves important load-bearing functions and protects the underlying articular cartilage. Unfortunately, meniscus tears are common and impair the ability of the meniscus to distribute loads, greatly increasing the risk for developing osteoarthritis. Therefore, surgical repair of the meniscus is a frequently performed procedure; however, this repair does not always prevent osteoarthritis. This is hypothesized to be due to altered joint loading post injury and repair, where the functional deficit of the meniscus prevents it from performing its role of distributing forces. However, many studies of meniscus function required opening the joint, which alters kinematics. The objective of this study was to use novel MRI methods to image the intact joint under axial load and measure the acute meniscus and femur displacements in an intact joint, after a meniscus root tear, and after suture repair in the porcine knee, a frequently used in vivo model. We found that anterior root tear led to large meniscus and femur displacements under physiological axial loading, and that suture anchor repair reduced these displacements, but did not fully restore intact joint kinematics. After tear and repair, the anterior region of the meniscus moved posteriorly and medially as it was forced out of the joint space under loading, while the posterior region had small displacements as the posterior attachment acted as a hinge about which the meniscus rotated in the axial plane. This technique can be applied to evaluate the effect of knee injuries and to develop improved repair strategies to restore joint kinematics.

The dense extracellular matrix of connective tissues impedes cell migration and subsequent matrix formation at sites of injury. We recently employed transient nuclear softening via histone deacetylase inhibition with trichostatin A (TSA) treatment to overcome the stiff nuclear impediments to cell migration through dense tissues and electrospun matrices. Despite these positive findings, the long-term implications of transient nuclear softening on cell transcriptional phenotype and matrix formation capacity are unknown. To address this, we investigated the influence of transient TSA treatment on porcine meniscal cell behavior, beginning with the efficacy and reproducibility of transient TSA treatment on histone acetylation and chromatin remodeling in vitro and cell migration through native meniscus tissue. Within 3 days after cessation of transient TSA treatment, histone acetylation and chromatin remodeling returned to control levels. Following TSA treatment, endogenous cell migration through native meniscus tissue increased greater than 3-fold compared to controls. Importantly, meniscal cells completely restored their transcriptional phenotype and maintained their capacity to respond transcriptionally and functionally to a secondary pro-matrix stimuli (i.e., transforming growth factor β3) within 7 days after cessation of TSA treatment. Towards translation, we also showed the feasibility of biomaterial-delivered TSA to increase endogenous cell migration to a wound edge ex vivo. Together, this work defines the efficacy, reproducibility, safety, and feasibility of future translational approaches for nuclear softening to treat dense connective tissue injuries.

Conventional treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus (AF), resulting in a high incidence of recurrent herniation and persistent disfunction. The lack of repair and the acute inflammation that arise after injury further compromises the disc and can result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annular repair and the local delivery of bioactive anti-inflammatory factors. TARPs transmit physiologic strains to mechanically-activated microcapsules (MAMCs) embedded within, which activate and release encapsulated biomolecules in response to physiologic loading. Here, we demonstrate that the TARP design modulates implant biomechanical properties and regulates MAMC mechano-activation. Next, the FDA-approved anti-inflammatory molecule, interleukin 1 receptor antagonist, Anakinra, was loaded in TARPs and the effects of TARP-mediated annular repair and Anakinra delivery was evaluated in a model of annular injury in the goat cervical spine. TARPs showed robust integration with the native tissue and provided structural reinforcement at the injury site that prevented disc-wide aberrant remodeling resulting from AF detensioning. The delivery of Anakinra via TARP implantation improved the retention of disc biochemical composition through increased matrix deposition and retention at the site of annular injury. Anakinra delivery additionally attenuated the inflammatory response associated by scaffold implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the AF. These results demonstrate the translational and therapeutic potential of this novel TARP system for the treatment of intervertebral disc herniations.One Sentence SummaryTension-activated repair patches delivering bioactive anti-inflammatory factors improve healing in an in vivo goat cervical disc injury model.

Changes in the micro-environment of fibrous connective tissue can lead to alterations in the phenotypes of tissue-resident cells, yet the underlying mechanisms are poorly understood. Here, by visualizing the dynamics of histone spatial reorganization in tenocytes and mesenchymal stromal cells from fibrous tissue of human donors via super-resolution microscopy, we show that physiological and pathological chemomechanical cues can directly regulate the spatial nanoscale organization and density of chromatin in these tissue-resident cell populations. Specifically, changes in substrate stiffness, altered oxygen tension and the presence of inflammatory signals drive chromatin relocalization and compaction into the nuclear boundary, mediated by the activity of the histone methyltransferase EZH2 and an intact cytoskeleton. In healthy cells, chemomechanically triggered changes in the spatial organization and density of chromatin are reversible and can be attenuated by dynamically stiffening the substrate. In diseased human cells, however, the link between mechanical or chemical inputs and chromatin remodelling is abrogated. Our findings suggest that aberrant chromatin organization in fibrous connective tissue may be a hallmark of disease progression that could be leveraged for therapeutic intervention.

Given its complex shape and relatively small size, the trapezium surface at the trapeziometacarpal (TMC) joint is a particularly attractive target for anatomic biologic joint resurfacing, especially given its propensity to develop osteoarthritis, and the limited and sub-optimal treatment options available. For this to advance to clinical translation, however, an appropriate large animal model is required. In this study, we explored the porcine accessory carpal bone (ACB) as a model for the human trapezium. We characterized ACB anatomy, geometry, joint and tissue-scale mechanics, and composition across multiple donors. We showed that the ACB is similar both in size, and in the saddle shape of the main articulating surface to the human trapezium, and that loads experienced across each joint are similar. Using this information, we then devised a fabrication method and workflow to produce patient-specific tissue-engineered replicas based on CT scans, and showed that when such replicas are implanted orthotopically in an ex vivo model, normal loading is restored. Data from this study establish the porcine ACB as a model system in which to evaluate function of engineered living joint resurfacing strategies. STATEMENT OF SIGNIFICANCE: Biologic joint resurfacing, or the replacement of a joint with living tissue as opposed to metal and plastic, is the holy grail of orthopaedic tissue engineering. However, despite marked advances in engineering native-like osteochondral tissues and in matching patient-specific anatomy, these technologies have not yet reached clinical translation. Given its propensity for developing osteoarthritis, as well as its small size and complex shape, the trapezial surface of the trapeziometacarpal joint at the base of the thumb presents a unique opportunity for pursuing a biologic joint resurfacing strategy. This work establishes the porcine accessory carpal bone as an animal model for the human trapezium and presents a viable test-bed for evaluating the function of engineered living joint resurfacing strategies.

The meniscus plays a critical role in knee mechanical function but is commonly injured given its central load bearing role. In the adult, meniscus repair is limited, given the low number of endogenous cells, the density of the matrix, and the limited vascularity. Menisci are fibrocartilaginous tissues composed of a micro-/nano-fibrous extracellular matrix (ECM) and a mixture of chondrocyte-like and fibroblast-like cells. Here, we developed a fibrous scaffold system that consists of bioactive components (decellularized meniscus ECM (dME) within a poly(e-caprolactone) material) fashioned into a biomimetic morphology (via electrospinning) to support and enhance meniscus cell function and matrix production. This work supports that the incorporation of dME into synthetic nanofibers increased hydrophilicity of the scaffold, leading to enhanced meniscus cell spreading, proliferation, and fibrochondrogenic gene expression. This work identifies a new biomimetic scaffold for therapeutic strategies to substitute or replace injured meniscus tissue.STATEMENT OF SIGNIFICANCEIn this study, we show that a scaffold electrospun from a combination of synthetic materials and bovine decellularized meniscus ECM provides appropriate signals and a suitable template for meniscus fibrochondrocyte spreading, proliferation, and secretion of collagen and proteoglycans. Material characterization andin vitrocell studies support that this new bioactive material is susceptible to enzymatic digestion and supports meniscus-like tissue formation.

Aging is the most prominent risk factor for osteoarthritis onset, but the etiology of aging-associated cartilage degeneration is not fully understood. Recent studies by Guilak and colleagues have highlighted the crucial roles of cell-matrix interactions in cartilage homeostasis and disease. This study thus quantified aging-associated changes in cartilage biomechanics and chondrocyte intracellular calcium signaling, [Ca

Tendons transmit mechanical forces between muscle and bone, and their biomechanical function requires high tensile strength, which is provided by highly organized collagen fibers. Tenocytes mainly drive tendon growth via extracellular matrix (ECM) production and organization. The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well established, partly due to the lack of a reliable in vitro system. Recent scaffold-free, cell-based tissue engineering approaches developed several unique in vitro tendon-like constructs. However, the application of these constructs is limited to the study of embryonic tendon development due to high cell density and immature matrix organization. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells and a differentially adherent growth channel. The 3D tendon constructs exhibited tissue maturation similar to the postnatal mouse tendon, including decreased cell density, increased thickness, and elongated cells between highly aligned extracellular matrix. The 3D tendon culture system is also feasible for genetic manipulation using adenovirus. Overall, the results suggest that the 3D tendon culture system using mouse tendon cells is a reliable in vitro system to study underlying biological mechanisms that regulate cellular and matrix maturation in postnatal tendon development.

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 orthopaedic 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 towards the annulus fibrosus fate remain unknown. Here, we screened a number of candidate factors (and their combinations) and assessed the transcriptomic signatures of key signaling factors involved in embryonic AF development and differentiated function. The transcriptional signatures of treated cells were compared to those of mature human AF cells, and conditions that promoted expression of annulus fibrosus extracellular matrix genes and key transcription factors involved in embryonic AF development were identified. These findings represent an initial approach to guide human induced pluripotent stem cells towards an annulus fibrosus-like fate for cellular delivery strategies.

Articular cartilage injury can lead to joint-wide erosion and the early onset of osteoarthritis. To address this, we recently developed a rapid fabrication method to produce patient-specific engineered cartilage tissues to replace an entire articular surface. Here, we extended that work by coupling a mesenchymal stromal cell-laden hydrogel (methacrylated hyaluronic acid) with the porous polycaprolactone (PCL) bone integrating phase and assessed the composition and mechanical performance of these constructs over time. To improve initial construct stability, PCL/hydrogel interface parameters were first optimized by varying PCL pretreatment (with sodium hydroxide before ethanol) before hydrogel infusion. Next, cylindrical osteochondral constructs were formed and cultured in media containing transforming growth factor β3 for up to 8 weeks, with constructs evaluated for viability, histological features, and biochemical content. Mechanical properties were also assessed in axial compression and via an interface shear strength assay. Results showed that the fabrication process was compatible with cell viability, and that construct biochemical content and mechanical properties increased with time. Interestingly, compressive properties peaked at 5 weeks, while interfacial shear properties continued to improve beyond this time point. Finally, these fabrication methods were combined with a custom mold developed from limb-specific computed tomography imaging data to create an anatomic implantable cell-seeded biologic joint surface, which showedmaturation similar to the osteochondral cylinders. Future work will apply these advances in large animal models of critically sized osteochondral defects to study repair and whole joint resurfacing.

Objective Cartilage repair strategies have seen improvement in recent years, especially with the use of scaffolds that serve as a template for cartilage formation. However, current fixation strategies are inconsistent with regards to retention, may be technically challenging, or may damage adjacent tissues or the implant itself. Therefore, the goal of this study was to evaluate the retention and repair potential of cartilage scaffolds fixed with an easy-to-implement bioresorbable pin. Design Electrospun hyaluronic acid scaffolds were implanted into trochlear groove defects in 3 juvenile and 3 adult pigs to evaluate short-term retention (2 weeks; pin fixation vs. press-fit and fibrin fixation) and long-term repair (8 months; scaffold vs. microfracture), respectively. Results For the retention study, press-fit and fibrin fixation resulted in short-term scaffold dislodgment ( n = 2 each), whereas pin fixation retained all scaffolds that were implanted ( n = 6). Pin fixation did not cause any damage to the opposing patellar surface, and only minor changes in the subchondral bone were observed. For long-term repair, no differences were observed between microfracture and scaffold groups, in terms of second-look arthroscopy and indentation testing. On closer visualization with micro computed tomography and histology, a high degree of variability was observed between animals with regard to subchondral bone changes and cartilage repair quality, yet each Scaffold repair displayed similar properties to its matched microfracture control. Conclusions In this study, pin fixation did not cause adverse events in either the short- or the long-term relative to controls, indicating that pin fixation successfully retained scaffolds within defects without inhibiting repair.

OBJECTIVE The aim was to identify the source of cells within the center of the abnormal fibrocartilage tissue of the degenerative intervertebral disc after injury. DESIGN Cross-breeding of mice with an inducible type II promoter collagen construct (Col2CreER) to Rosa26-TdTomato mice has been shown to result in Cre-recombinase activity and Tomato expression in inner annulus fibrosus cells after tamoxifen injection. To investigate the role of the inner annulus fibrosus in the intervertebral disc injury response, tail intervertebral discs of Col2CreER/tdTomato mice were punctured with a needle and examined 1-4 wks after injury. N-cadherin was examined by immunostaining. RESULTS After the injury, the fibrocartilage in the degenerative intervertebral disc consisted of residual diseased nucleus pulposus cells and encroaching inner annulus fibrosus cells. The residual nucleus pulposus cells had lost their epithelial cell-like morphology and instead became oval shaped, with reduced adhesion to neighboring nucleus pulposus cells. This change in cellular morphology coincided with a loss of N-cadherin, which contributes to maintenance of healthy nucleus pulposus cell morphology. As expected, injured tail intervertebral discs showed reduced compressive properties as determined by biomechanical assessments. CONCLUSIONS The cellular composition of the degenerative intervertebral disc has been defined here, which is an important step in developing future treatments.

OBJECTIVE To elucidate the role of decorin, a small leucine-rich proteoglycan, in the degradation of cartilage matrix during the progression of post-traumatic osteoarthritis (OA). METHODS Three-month-old decorin-null (Dcn-/- ) and inducible decorin-knockout (DcniKO ) mice were subjected to surgical destabilization of the medial meniscus (DMM) to induce post-traumatic OA. The OA phenotype that resulted was evaluated by assessing joint morphology and sulfated glycosaminoglycan (sGAG) staining via histological analysis (n = 6 mice per group), surface collagen fibril nanostructure via scanning electron microscopy (n = 4 mice per group), tissue modulus via atomic force microscopy-nanoindentation (n = 5 or more mice per group) and subchondral bone structure via micro-computed tomography (n = 5 mice per group). Femoral head cartilage explants from wild-type and Dcn-/- mice were stimulated with the inflammatory cytokine interleukin-1β (IL-1β) in vitro (n = 6 mice per group). The resulting chondrocyte response to IL-1β and release of sGAGs were quantified. RESULTS In both Dcn-/- and DcniKO mice, the absence of decorin resulted in accelerated sGAG loss and formation of highly aligned collagen fibrils on the cartilage surface relative to the control (P < 0.05). Also, Dcn-/- mice developed more salient osteophytes, illustrating more severe OA. In cartilage explants treated with IL-1β, loss of decorin did not alter the expression of either anabolic or catabolic genes. However, a greater proportion of sGAGs was released to the media from Dcn-/- mouse explants, in both live and devitalized conditions (P < 0.05). CONCLUSION In post-traumatic OA, decorin delays the loss of fragmented aggrecan and fibrillation of cartilage surface, and thus, plays a protective role in ameliorating cartilage degeneration.

Tissue-engineered replacement discs are an area of intense investigation for the treatment of end-stage intervertebral disc (IVD) degeneration. These living implants can integrate into the IVD space and recapitulate native motion segment function. We recently developed a multiphasic tissue-engineered disc-like angle-ply structure (DAPS) that models the micro-architectural and functional features of native tissue. While these implants resulted in functional restoration of the motion segment in rat and caprine models, we also noted deficiencies in cell infiltration and homogeneity of matrix deposition in the electrospun poly(ε-caprolactone) outer region (annulus fibrosus, AF) of the DAPS. To address this limitation, here, we incorporated a sacrificial water-soluble polymer, polyethylene oxide (PEO), as a second fiber fraction within the AF region to increase porosity of the implant. Maturation of these PEO-modified DAPS were evaluated after 5 and 10 weeks of in vitro culture in terms of AF biochemical content, MRI T2 values, overall construct mechanical properties, AF micromechanical properties and cell and matrix distribution. To assess the performance of the PEO-modified DAPS in vivo, precultured constructs were implanted into the rat caudal IVD space for 10 weeks. Results showed that matrix distribution was more homogenous in PCL/PEO DAPS, as evidenced by more robust histological staining, organized collagen deposition and micromechanical properties, compared to standard PCL-only DAPS in vitro. Cell and matrix infiltration were also improved in vivo, but no differences in macromechanical properties and a trend towards improved micromechanical properties were observed. These findings demonstrate that the inclusion of a sacrificial PEO fiber fraction in the DAPS AF region improves cellular colonization, matrix elaboration, and in vitro and in vivo function of an engineered IVD implant. STATEMENT OF SIGNIFICANCE: This work establishes a method for improving cell infiltration and matrix distribution within tissue-engineered dense fibrous scaffolds for intervertebral disc replacement. Tissue-engineered whole disc replacements are an attractive alternative to the current gold standard (mechanical disc arthroplasty or vertebral fusion) for the clinical treatment of patients with advanced disc degeneration.

The knee menisci are comprised of two orthogonal collagenous networks – circumferential and radial – that combine to enable efficient load bearing by the tissue in adults. Here, we assessed how the structural and functional characteristics of these networks developed over the course of skeletal maturation and determined the role of these fiber networks in defect tolerance with tissue injury. Imaging of the radial tie fiber (RTF) collagen structure in medial bovine menisci from fetal, juvenile, and adult specimens showed increasing heterogeneity, anisotropy, thickness, and density with skeletal development. Mechanical analysis showed that the tensile modulus in the radial direction did not change with skeletal development, though the resilience (in the radial direction) increased and the tolerance to defects in the circumferential direction decreased, in adult compared to fetal tissues. This loss of defect tolerance correlated with increased order in the RTF network in adult tissue. These data provide new insights into the role of the radial fiber network in meniscus function, will lead to improved clinical decision-making in the presence of a tear, and may improve engineering efforts to reproduce this critical load-bearing structure in the knee.

The meniscus plays a central load bearing role in the knee joint. Unfortunately, meniscus injury is common and can lead to joint degeneration and osteoarthritis. In small animal models, progressive degenerative changes occur with unloading of the meniscus via destabilization of the medial meniscus (DMM). However, few large animal models of DMM exist and the joint-wide initiation of disease has not yet been defined in these models. Thus, the goal of this study was to develop and validate a large-animal model of surgically-induced destabilization of the medial meniscus and to use multi-modal (mechanical, histological, and MRI) and multi-scale (joint to tissue level) quantitative measures to evaluate degeneration in both the meniscus and cartilage. DMM was achieved using an arthroscopic approach in thirteen Yucatan minipigs. One month after DMM, joint contact area decreased and peak pressure increased, indicating altered load transmission as a result of meniscus destabilization. By three months, the joint had adapted to the injury and load transmission patterns were restored to baseline, likely due to the formation and maturation of a fibrovascular scar at the anterior aspect of the meniscus. Despite this, we found a decrease in the indentation modulus of the tibial cartilage and an increase in cartilage histopathology scores at one month compared to Sham operated animals; these deleterious changes persisted through three months. Over this same time course, meniscus remodeling was evident through decreased proteoglycan staining in DMM compared to Sham menisci at both one and three months. These findings support that arthroscopic DMM results in joint degeneration in the Yucatan minipig and provides a new large animal test bed in which to evaluate therapeutics and interventions to treat post-traumatic osteoarthritis (PTOA) that originates from meniscal injury.

The incorporation of a secondary network into traditional single-network hydrogels can enhance mechanical properties, such as toughness and loading to failure. These features are important for many applications, including as biomedical materials; however, the processing of interpenetrating polymer network (IPN) hydrogels is often limited by their multistep fabrication procedures. Here, a one-pot scheme for the synthesis of biopolymer IPN hydrogels mediated by the simultaneous crosslinking of two independent networks with light, namely: i) free-radical crosslinking of methacrylate-modified hyaluronic acid (HA) to form the primary network and ii) thiol-ene crosslinking of norbornene-modified HA with thiolated guest-host assemblies of adamantane and β-cyclodextrin to form the secondary network, is reported. The mechanical properties of the IPN hydrogels are tuned by changing the network composition, with high water content (≈94%) hydrogels exhibiting excellent work of fracture, tensile strength, and low hysteresis. As proof-of-concept, the IPN hydrogels are implemented as low-viscosity Digital Light Processing resins to fabricate complex structures that recover shape upon loading, as well as in microfluidic devices to form deformable microparticles. Further, the IPNs are cytocompatible with cell adhesion dependent on the inclusion of adhesive peptides. Overall, the enhanced processing of these IPN hydrogels will expand their utility across applications.

Re-creating features of the native extracellular matrix (ECM) with engineered biomaterials has become a valuable tool to probe the influence of ECM properties on cellular functions (e.g., differentiation) and toward the engineering of tissues. However, characterization of newly secreted (nascent) matrix and turnover, which are important in the context of cells interacting with these biomaterials, has been limited by a lack of tools. We developed a protocol to visualize and quantify the spatiotemporal evolution of newly synthesized and deposited matrix by cells that are either cultured atop (2D) or embedded within (3D) biomaterial systems (e.g., hydrogels, fibrous matrices). This technique relies on the incorporation of a noncanonical amino acid (azidohomoalanine) into proteins as they are synthesized. Deposited nascent ECM components are then visualized with fluorescent cyclooctynes via copper-free cycloaddition for spatiotemporal analysis or modified with cleavable biotin probes for identification. Here we describe the preparation of hyaluronic acid hydrogels through ultraviolet or visible light induced cross-linking for 2D and 3D cell culture, as well as the fluorescent labeling of nascent ECM deposited by cells during culture. We also provide protocols for secondary immunofluorescence of specific ECM components and ImageJ-based ECM quantification methods. Hyaluronic acid polymer synthesis takes 2 weeks to complete, and hydrogel formation for 2D or 3D cell culture is performed in 2-3 h. Lastly, we detail the identification of nascent proteins, including enrichment, preparation and analysis with mass spectrometry, which can be completed in 10 d.

Due to limited intrinsic healing capacity of the meniscus, meniscal injuries pose a significant clinical challenge. The most common method for treatment of damaged meniscal tissues, meniscectomy, leads to improper loading within the knee joint, which can increase the risk of osteoarthritis. Thus, there is a clinical need for the development of constructs for meniscal repair that better replicate meniscal tissue organization to improve load distributions and function over time. Advanced three-dimensional bioprinting technologies such as suspension bath bioprinting provide some key advantages, such as the ability to support the fabrication of complex structures using non-viscous bioinks. In this work, the suspension bath printing process is utilized to print anisotropic constructs with a unique bioink that contains embedded hydrogel fibers that align via shear stresses during printing. Constructs with and without fibers are printed and then cultured for up to 56 d in vitro in a custom clamping system. Printed constructs with fibers demonstrate increased cell and collagen alignment, as well as enhanced tensile moduli when compared to constructs printed without fibers. This work advances the use of biofabrication to develop anisotropic constructs that can be utilized for the repair of meniscal tissue.

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.