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.

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.

Leach's storm-petrel (Hydrobates leucorhous), a long-lived seabird shows flexible, condition-dependent, feeding strategies in response to poor chick condition.

BACKGROUND: Parent-offspring conflict represents the sensitive balance of resource allocation between self-maintenance and reproduction. Two strategies have been proposed to better understand how species manage this conflict. In fixed-level feeding behavior, parents feed offspring consistent quantities of food; while flexible feeding shows plasticity in parental allocation based on offspring need. Life-history theory predicts that parents of long-lived species prioritize their survival and may favor the fixed-level hypothesis to maximize lifetime reproductive success. In this study, we highlight the natural variation of parent-offspring allocation strategies within a unique population of Leach's storm-petrels (Hydrobates leucorhous), and through month-long food supplementation and restriction manipulations, we investigate how chick condition affects parental provisioning during the chick-rearing period of reproduction. RESULTS: We show that the parents upregulated chick feeding frequency of nutritionally deprived chicks, resulting in a larger total amount of food delivered during the study period. Additionally, the proportion of nights when both parents fed was highest in restricted chicks, and the proportion of nights when neither parents fed was lowest in restricted chicks, suggesting that storm-petrel parents shorten their foraging bouts to deliver food more often when their chicks are in relatively poor condition. CONCLUSIONS: Our results support that Leach's storm-petrels use a flexible-level feeding strategy, suggesting that parents can assess offspring condition, and respond by feeding chicks at higher frequencies. These data provide insight on how a long-lived seabird balances its own energetic demands with that of their offspring during the reproductive period.

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.

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.

Synovium and infrapatellar fat pad share common mesenchymal progenitors and undergo coordinated changes in osteoarthritis.

Osteoarthritis (OA) affects multiple tissues in the knee joint, including the synovium and intra-articular adipose tissue (IAAT) that are attached to each other. However, whether these two tissues share the same progenitor cells and hence function as a single unit in joint homeostasis and diseases is largely unknown. Single-cell transcriptomic profiling of synovium and infrapatellar fat pad (IFP), the largest IAAT, from control and OA mice revealed five mesenchymal clusters and predicted mesenchymal progenitor cells (MPCs) as the common progenitors for other cells: synovial lining fibroblasts (SLFs), myofibroblasts (MFs), and preadipocytes 1 and 2. Histologic examination of joints in reporter mice having Dpp4-CreER and Prg4-CreER that label MPCs and SLFs, respectively, demonstrated that Dpp4+ MPCs reside in the synovial sublining layer and give rise to Prg4+ SLFs and Perilipin+ adipocytes during growth and OA progression. After OA injury, both MPCs and SLFs gave rise to MFs, which remained in the thickened synovium at later stages of OA. In culture, Dpp4+ MPCs possessed mesenchymal progenitor properties, such as proliferation and multilineage differentiation. In contrast, Prg4+ SLFs did not contribute to adipocytes in IFP and Prg4+ cells barely grew in vitro. Taken together, we demonstrate that the synovium and joint fat pad are one integrated functional tissue sharing common mesenchymal progenitors and undergoing coordinated changes during OA progression.

Both synovium and intra-articular adipose tissue (IAAT) in knee joint play a critical role in joint health and osteoarthritis (OA) progression. Recent single-cell RNA-sequencing studies have been performed on the mouse and human synovium. However, IAATs residing in close proximity to the synovium have not been studied yet. Our study reveals mesenchymal cell heterogeneity of synovium/infrapatellar fat pad (Syn/IFP) tissue and their OA responses. We identify Dpp4+ multipotent progenitors as a source that give rise to Prg4+ lining layer fibroblasts in the synovium, adipocytes in the IFP, and myofibroblasts in the OA Syn/IFP tissue. Our work demonstrates that Syn/IFP is a functionally connected tissue that shares common mesenchymal progenitors and undergoes coordinated OA changes. This novel insight advances our knowledge of previously understudied joint tissues and provides new directions for drug discovery to treat joint disorders.

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.

Modulating mechanobiology as a therapeutic target for synovial fibrosis to restore joint lubrication.

OBJECTIVES: Fibroses are disorders linked to persistence of myofibroblasts due to biochemical (e.g., Transforming growth factor-ß) and biophysical cues (e.g., a stiff microenvironment). In the context of osteoarthritis, fibrotic changes in the joint-lining synovium have been linked with disease progression. The objective of this study was to probe synovial fibroblast mechanobiology and how essential functions (i.e., lubrication) are altered in fibrotic environments. DESIGN: Both ex vivo and in vitro synovium models were assessed for fibrotic and lubrication biomarkers to better understand the role of mechanobiology and lubrication. Additionally, in vitro, work on small molecules targeting mechanobiology was assessed. RESULTS: Our results indicated that modulating mechanobiology could rescue the fibrotic phenotype instigated by stiffening microenvironment that resulted in altered lubricant expression. A small molecule therapeutic, fasudil, blocked ROCK-mediated contractility and this inhibition of the fibrotic mechano-response of synovial fibroblasts restored proper lubrication function, providing insight into mechanisms of disease progression as well as a new avenue for therapeutic development. CONCLUSION: This study identifies synovial fibrosis as a condition that potentially has joint-wide deficits through inhibiting lubrication. Additionally, modulating mechanobiology (i.e., ROCK-mediated contractility) may pose a potential target for small molecule therapies that can be delivered to the joint space. CLASSIFICATION: Applied Biological Sciences.

Single cell RNA sequencing reveals emergent notochord-derived cell subpopulations in the postnatal nucleus pulposus.

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 (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP 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 NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-beta and phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. 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.

Mechanical crosstalk between the intervertebral disc, facet joints, and vertebral endplate following acute disc injury in a rabbit model.

Background: Vertebral endplate sclerosis and facet osteoarthritis have been documented in animals and humans. However, it is unclear how these adjacent pathologies engage in crosstalk with the intervertebral disc. This study sought to elucidate this crosstalk by assessing each compartment individually in response to acute disc injury. Methods: Eleven New Zealand White rabbits underwent annular disc puncture using a 16G or 21G needle. At 4 and 10 weeks, individual compartments of the motion segment were analyzed. Discs underwent T 1 relaxation mapping with MRI contrast agent gadodiamide as well T 2 mapping. Both discs and facets underwent mechanical testing via vertebra-disc-vertebra tension-compression creep testing and indentation testing, respectively. Endplate bone density was quantified via µCT. Discs and facets were sectioned and stained for histology scoring. Results: Intervertebral discs became more degenerative with increasing needle diameter and time post-puncture. Bone density also increased in endplates adjacent to both 21G and 16G punctured discs leading to reduced gadodiamide transport at 10 weeks. The facet joints, however, did not follow this same trend. Facets adjacent to 16G punctured discs were less degenerative than facets adjacent to 21G punctured discs at 10 weeks. 16G facets were more degenerative at 4 weeks than at 10, suggesting the cartilage had recovered. The formation of severe disc osteophytes in 16G punctured discs between 4 and 10 weeks likely offloaded the facet cartilage, leading to the recovery observed. Conclusions: Overall, this study supports that degeneration spans the whole spinal motion segment following disc injury. Vertebral endplate thickening occurred in response to disc injury, which limited the diffusion of small molecules into the disc. This work also suggests that altered disc mechanics can induce facet degeneration, and that extreme bony remodeling adjacent to the disc may promote facet cartilage recovery through offloading of the articular cartilage.

Injectable Radiopaque Hyaluronic Acid Granular Hydrogels for Intervertebral Disc Repair.

Injectable hydrogels offer minimally-invasive treatment options for degenerative disc disease, a prevalent condition affecting millions annually. Many hydrogels explored for intervertebral disc (IVD) repair suffer from weak mechanical integrity, migration issues, and expulsion. To overcome these limitations, an injectable and radiopaque hyaluronic acid granular hydrogel is developed. The granular structure provides easy injectability and low extrusion forces, while the radiopacity enables direct visualization during injection into the disc and non-invasive monitoring after injection. The radiopaque granular hydrogel is injected into rabbit disc explants to investigate restoration of healthy disc mechanics following needle puncture injury ex vivo and then delivered in a minimally-invasive manner into the intradiscal space in a clinically-relevant in vivo large animal goat model of IVD degeneration initiated through degradation by chondroitinase. The radiopaque granular hydrogel successfully halted loss of disc height due to degeneration. Further, the hydrogel not only enhanced proteoglycan content and reduced collagen content in the nucleus pulposus (NP) region compared to degenerative discs, but also helped to maintain the structural integrity of the disc and promote healthy segregation of the NP and annulus fibrosus regions. Overall, this study demonstrates the great potential of an injectable radiopaque granular hydrogel for treatment of degenerative disc disease.

Tension-activated nanofiber patches delivering an anti-inflammatory drug improve repair in a goat intervertebral disc herniation model.

Conventional microdiscectomy treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus, resulting in a high incidence of recurrent herniation and persistent dysfunction. The lack of repair and the acute inflammation that arise after injury can further compromise the disc and result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annulus fibrosus repair and local delivery of the anti-inflammatory factor anakinra (a recombinant interleukin-1 receptor antagonist). TARPs transmit physiologic strain to mechanically activated microcapsules embedded within the patch, which release encapsulated bioactive molecules in direct response to spinal loading. Mechanically activated microcapsules carrying anakinra were loaded into TARPs, and the effects of TARP-mediated annular repair and anakinra delivery were evaluated in a goat model of annular injury in the cervical spine. TARPs integrated with native tissue and provided structural reinforcement at the injury site that prevented aberrant disc-wide remodeling resulting from detensioning of the annular fibrosus. The delivery of anakinra by TARP implantation increased matrix deposition and retention at the injury site and improved maintenance of disc extracellular matrix. Anakinra delivery additionally attenuated the inflammatory response associated with TARP implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the annulus fibrosus. These results demonstrate the therapeutic potential of TARPs for the treatment of intervertebral disc herniation.

Microinterfaces in 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 may instead utilize existing ECM microstructures or microtracks as innate pathways for migration. While hydrogels in general are valuable tools for probing the extracellular regulators of 3D migration, few have recapitulated these natural migration paths. Here, we developed a biopolymer-based (i.e., gelatin and hyaluronic acid) bicontinuous hydrogel system formed through controlled solution immiscibility whose continuous subdomains and high micro-interfacial surface area enabled rapid 3D migration, particularly when compared to homogeneous hydrogels. Migratory behavior was mesenchymal in nature and regulated by biochemical and biophysical signals from the hydrogel, which was shown across various cell types and physiologically relevant contexts (e.g., cell spheroids, ex vivo tissues, in vivo tissues). Our findings introduce a new design that leverages important local interfaces to guide rapid cell migration.

Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects.

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage.

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.