Osteochondral Tissue Research Articles

A natural composite hydrogel laden with mesenchymal stromal cells for osteochondral repair: Comparison between casting and 3D bioprinting

Synovial joints, and particularly the osteochondral unit, are prone to lesions, with high risk of degeneration towards osteoarthritis. Various treatment strategies have been developed, including surgical techniques and cellular therapies, but they all show limitations. In this context, tissue engineering approaches, particularly 3D bioprinting, are promising for generating osteochondral tissue substitutes for joint repair. In this work, two biofabrication techniques, casting and extrusion-based 3D bioprinting, of an optimized formulation of a gelatin/alginate/fibrinogen bioink loaded with murine mesenchymal stromal cells (MSCs) were compared for the generation of cartilage and bone substitutes. Cell viability, proliferation and differentiation were characterized. Both techniques showed similar results in terms of viability and proliferation, but only the 3D bioprinted constructs allowed for differentiation towards the chondrogenic or osteogenic lineage using specific culture media. Bioprinting of biphasic osteochondral constructs comprising a cartilage compartment on top of a bone compartment was also explored. The study highlights the potential of our natural composite hydrogel bioink and extrusion-based 3D bioprinting for the generation of osteochondral tissue substitutes. Although further optimizations are needed, the study laid the groundwork for future advancements in osteochondral tissue engineering.

Osteochondral Regeneration With Anatomical Scaffold 3D-Printing-Design Considerations for Interface Integration.

There is a clinical need for osteochondral scaffolds with complex geometries for restoring articulating joint surfaces. To address that need, 3D-printing has enabled scaffolds to be created with anatomically shaped geometries and interconnected internal architectures, going beyond simple plug-shaped scaffolds that are limited to small, cylindrical, focal defects. A key challenge for restoring articulating joint surfaces with 3D-printed constructs is the mechanical loading environment, particularly to withstand delamination or mechanical failure. Although the mechanical performance of interfacial scaffolds is essential, interface strength testing has rarely been emphasized in prior studies with stratified scaffolds. In the pioneering studies where interface strength was assessed, varying methods were employed, which has made direct comparisons difficult. Therefore, the current review focused on 3D-printed scaffolds for osteochondral applications with an emphasis on interface integration and biomechanical evaluation. This 3D-printing focus included both multiphasic cylindrical scaffolds and anatomically shaped scaffolds. Combinations of different 3D-printing methods (e.g., fused deposition modeling, stereolithography, bioprinting with pneumatic extrusion of cell-laden hydrogels) have been employed in a handful of studies to integrate osteoinductive and chondroinductive regions into a single scaffold. Most 3D-printed multiphasic structures utilized either an interdigitating or a mechanical interlocking design to strengthen the construct interface and to prevent delamination during function. The most effective approach to combine phases may be to infill a robust 3D-printed osteal polymer with an interlocking chondral phase hydrogel. Mechanical interlocking is therefore recommended for scaling up multiphasic scaffold applications to larger anatomically shaped joint surface regeneration. For the evaluation of layer integration, the interface shear test is recommended to avoid artifacts or variability that may be associated with alternative approaches that require adhesives or mechanical grips. The 3D-printing literature with interfacial scaffolds provides a compelling foundation for continued work toward successful regeneration of injured or diseased osteochondral tissues in load-bearing joints such as the knee, hip, or temporomandibular joint.

A modular approach to 3D-printed bilayer composite scaffolds for osteochondral tissue engineering

Prolonged osteochondral tissue engineering damage can result in osteoarthritis and decreased quality of life. Multiphasic scaffolds, where different layers model different microenvironments, are a promising treatment approach, yet stable joining between layers during fabrication remains challenging. To overcome this problem, in this study, a bilayer scaffold for osteochondral tissue regeneration was fabricated using 3D printing technology which containing a layer of PCL/hydroxyapatite (HA) nanoparticles and another layer of PCL/gelatin with various concentrations of fibrin (10, 20 and 30 wt.%). These printed scaffolds were evaluated with SEM (Scanning Electron Microscopy), FTIR (Fourier Transform Infrared Spectroscopy) and mechanical properties. The results showed that the porous scaffolds fabricated with pore size of 210–255 µm. Following, the ductility increased with the further addition of fibrin in bilayer composites which showed these composites scaffolds are suitable for the cartilage part of osteochondral. Also, the contact angle results demonstrated the incorporation of fibrin in bilayer scaffolds based on PCL matrix, can lead to a decrease in contact angle and result in the improvement of hydrophilicity that confirmed by increasing the degradation rate of scaffolds containing further fibrin percentage. The bioactivity study of bilayer scaffolds indicated that both fibrin and hydroxyapatite can significantly improve the cell attachment on fabricated scaffolds. The MTT assay, DAPI and Alizarin red tests of bilayer composite scaffolds showed that samples containing 30% fibrin have the more biocompatibility than that of samples with 10 and 20% fibrin which indicated the potential of this bilayer scaffold for osteochondral tissue regeneration.Graphical

Gelatin-based hydrogels with tunable network structure and mechanical property for promoting osteogenic differentiation

Osteoarthritis (OA) is a joint disease involving all joint components, including cartilage, calcified cartilage, and subchondral bone. The repair of osteochondral defects remains a significant challenge in orthopedics. Development of new strategies is essential for effective osteochondral injury repair. In this study, gelatin (Gel), polyethylene glycol diglycidyl ether (PEGDGE), hydroxyethyl cellulose (HEC) and chitosan (CS) were used to prepare semi-IPNs and IPNs hydrogels. Mechanical properties of Gel based hydrogels significantly improved with the semi-IPN and IPN structures. Tensile strength ranges from 238.7 KPa to 479.5 KPa, and its compressive strength ranges from 35.6 KPa to 112.7 KPa. Additionally, the stress relaxation rate increased with higher CS concentrations, ranging from 25 % to 35 %. The network structure of Gel-based hydrogels was a key factor in regulating stress relaxation. Viscoelasticity was adjusted by its network structures. Swelling and degradation behaviors of Gel based hydrogels were systematically investigated. Gel based hydrogels had good cytocompatibility. Both semi-IPN and IPN structures Gel based hydrogels could promote cell spreading and osteogenic differentiation. G10HEC1 and G10CS1 hydrogels show promise as candidates for osteochondral tissue regeneration, offering a new strategy for osteochondral tissue engineering.

Dual cross-linked polyurethane-alginate biomimetic hydrogel for elastic gradient simulation in osteochondral structures: Microenvironment modulation and tissue regeneration

The distinctive composition and functions of osteochondral structures result in constrained regeneration. Insufficient healing processes may precipitate the emergence of tissue growth disorders or excessive subchondral bone formation, which can culminate in the deterioration and failure of osteochondral tissue repair. To overcome these limitations, materials designed for osteochondral repair must provide region-specific modulation of the microenvironment and mechanical compatibility. To address these challenges, we propose a method to create continuous hydrogels with distinct structural and functional properties by a precise cross-linking method. We have developed an innovative polyurethane enriched with dimethylglyoxime, facilitating the coordinated loading and precise release of Zn2+. This strategy enables the meticulous control of alginate cross-linking, resulting in an elastic gradient hydrogel that closely resembles the osteochondral interface. The SeSe within the hydrogel effectively modulates the inflammatory microenvironment and fosters the M2 polarization of macrophages. The hydrogel's lower layer is designed to rapidly release Zn2+, thereby enhancing bone regeneration. The upper layer is intended to prevent bone overgrowth and stimulate chondrogenic differentiation. This dual-layer strategy allows targeted stimuli to each region, promoting the seamless integration of neoosteochondral tissue. Our study demonstrates the potential of this stratified hydrogel in achieving uniform and smooth osteochondral tissue regeneration.

Biphasic ECM assembled graphene oxide-collagen/nHap composites mimicking articular microenvironment for in situ osteochondral defect repair

Both bone and cartilage microenvironments play a crucial role in the regeneration of osteochondral defects caused by osteoarthritis (OA). A major challenge is how to simulate gradient changing mechanical properties and inducing abilities of regenerative microenvironments on the integrated scaffold to achieve simultaneous regeneration. In this work, a concept of biphasic ECM assembled graphene oxide-collagen (GO-COL)/ Nano-hydroxyapatite (nHap) composite is proposed. GO-COL/nHap bilayer scaffold with gradient concentration was formed by crosslinking and freeze-drying in stages to mimic the gradient distribution of mechanical properties of articular cartilage and bone. Then we prepared chondrogenic/osteogenic ECM membranes (CgECM/OgECM) with chondrocytes from rat knee respectively, and assembled them onto corresponding layers. The morphology, physical properties, biocompatibility, and osteochondrogenic capability of the scaffold were evaluated in vitro and in vivo. Mechanically, gene expression and protein level assessments showed greater osteochondrogenic activity in the biphasic ECM scaffold group compared to the GO-COL bilayer scaffold group. In vivo studies using a rat knee osteochondral defect model further confirmed the superior repair effect of the biphasic ECM-scaffold group compared to the blank group. Taken together, the proposed ECM assembled GO-COL/nHap strategy open a window to realize different microenvironment mimicking in a whole scaffold, making it a potential off-the-shelf method for osteochondral tissue engineering.

Advances in Additive Manufactured Scaffolds Mimicking the Osteochondral Interface

Architectural, compositional, and mechanical gradients are present in many interfacial tissues in the body. Yet desired for regeneration, the recreation of these complex natural gradients in porous scaffolds remains a challenging task. Additive manufacturing (AM) has been highlighted as a technology to fabricate constructs to regenerate interfacial tissues. Integration of different types of gradients, which can be physical, mechanical, and/or biochemical, shows promise to control cell fate and the regeneration process in a spatial controlled manner. One of the most studied tissue interfaces is the osteochondral unit which connects cartilage to bone. This tissue is often damaged because of trauma or ageing, leading to osteoarthritis; a degenerative disease and a major cause of disability worldwide. Therefore, in view of osteochondral (OC) regeneration, a state‐of‐the‐art overview of current approaches is presented to manufacture gradient scaffolds prepared by AM techniques. The focus is on thermoplastic, hydrogel, and hybrid scaffolds comprising gradients that induce biomimicry by their physical and biological properties. The effect of these different systems on OC tissue formation in‐vitro and in‐vivo is addressed. Finally, an outlook on current trends of dynamic materials is provided, including proposals on how these materials could improve the mimicry of scaffolds applied for OC regeneration.

Biofabrication of engineered tissues by 3D bioprinting of tissue specific high cell-density bioinks.

Bioprinting of high cell-density bioinks is a promising technique for cellular condensation-based tissue engineering and regeneration medicine. However, it remains difficult to create precisely controlled complex structures and organization of tissues with high cell-density bioink-based bioprinting for tissue specific condensation. In this study, we present newly biofabricated tissues from directly assembled, tissue specific, high cell-density bioinks which have been three-dimensionally printed into a photocrosslinkable and biodegradable hydrogel microparticle supporting bath. Three types of tissue specific high cell-density bioinks have been prepared with individual stem cells or stem cell aggregates by incorporation of growth factor-loaded gelatin microparticles. The bioprinted tissue specific high cell-density bioinks in the photocrosslinked microgel supporting bath condense together and differentiate down tissue-specific lineages to form multi-phase tissues (e.g., osteochondral tissues). By changing the growth factors and cell types, these tissue specific high cell-density bioinks enable engineering of various functional tissues with controlled architecture and organization of cells.

Osteochondral Tissue-On-a-Chip: A Novel Model for Osteoarthritis Research.

The existing in vitro and in vivo models for studying osteoarthritis have significant limitations in replicating the complexity of joint tissues. This research aims to validate a Tissue-On-a-Chip system for osteoarthritis research. Osteochondral tissues obtained from knee replacement surgeries of patients with osteoarthritis were cultured in an Organ-On-a-Chip system. This system was designed to supply oxygen and glucose to the cartilage from the bone. The distribution of oxygen and glucose was evaluated by fluorescence using Image-iT Green Hypoxia and 2-NBDG, respectively. Cytotoxicity was measured using lactate dehydrogenase (LDH) levels in chip cultures compared to plate cultures (12 tissues per method). Glycosaminoglycans (GAGs), alkaline phosphatase (ALP), Coll2-1, and procollagen type II N-terminal propeptide (PIINP) were measured in the perfused medium of the Tissue-On-a-Chip over a period of 70 days. Fluorescence of Image-iT Green Hypoxia was observed only in the cartilage area, while 2-NBDG was distributed throughout the tissue. An increase in LDH levels was noted in the plate cultures on day 24 and in the Tissue-On-a-Chip cultures on day 63. Compared to the start of the culture, GAG content increased on day 52, while ALP showed variations. A notable increase in GAG, ALP, and Coll2-1 levels was observed on day 59. PIINP levels remained stable throughout the experiment. The validated osteochondral Tissue-On-a-Chip system can replicate the joint microenvironment, with hypoxic conditions in cartilage and normoxic conditions in bone. Tissue survival and component stability were maintained for approximately two months. This platform is a useful tool for evaluating new drugs and represents a viable alternative to animal models.

Development of novel osteochondral scaffolds and related in vitro environment with the aid of chemical engineering principles

In tissue engineering, collaboration among experts from different fields is needed to design appropriate cell scaffolds and the required three-dimensional environment. Osteochondral tissue engineering is particularly challenging due to the need to provide scaffolds that imitate structural and compositional differences between two neighboring tissues, articular cartilage and bone, and the required complex biophysical environments for cultivating such scaffolds. This work focuses on two key objectives: first, to develop bilayered osteochondral scaffolds based on gellan gum and bioactive glass and, second, to create a biomimetic environment for scaffold characterization by designing and utilizing novel dual-medium cultivation bioreactor chambers. Basic chemical engineering principles were utilized to help achieve both aims. First, a simple heat transport model based on one-dimensional conduction was applied as a guideline for bilayer scaffold preparation, leading to the formation of a gelatinous upper part and a macroporous lower part with a thin, well-integrated interfacial zone. Second, a novel cultivation chamber was developed to be used in a dynamic compression bioreactor to provide possibilities for flow of two different media, such as chondrogenic and osteogenic. These chambers were utilized for characterization of the novel scaffolds with regard to bioactivity and stability under dynamic compression and fluid perfusion over 14 d, while flow distribution under different conditions was analyzed by a tracer method and residence time distribution analysis.

Applications of type I and II collagen in osteochondral tissue engineering: Respective features and future perspectives

At present, osteoarthritis stands as one of the most prevalent degenerative musculoskeletal diseases globally. The inherent lack of self-healing capacity in articular cartilage presents a considerable obstacle in treating osteoarthritis. Tissue engineering emerged as a promising strategy for the regeneration of osteochondral defects. Collagen, a primary component of bone and cartilage, stands out as a widely utilized natural material in osteochondral tissue engineering. Among various collagen types, Type I and type II collagen are notably prominent in this field. This review initially summarizes the advantageous and disadvantageous properties of type I/II collagen in osteochondral tissue engineering, proceeds to introduce their applications in this context, and concludes with a prospective outlook on the future development of type I/II collagen's applications.

Drug-Loaded Bioscaffolds for Osteochondral Regeneration.

Osteochondral defect is a complex tissue loss disease caused by arthritis, high-energy trauma, and many other reasons. Due to the unique structural characteristics of osteochondral tissue, the repair process is sophisticated and involves the regeneration of both hyaline cartilage and subchondral bone. However, the current clinical treatments often fall short of achieving the desired outcomes. Tissue engineering bioscaffolds, especially those created via three-dimensional (3D) printing, offer promising solutions for osteochondral defects due to their precisely controllable 3D structures. The microstructure of 3D-printed bioscaffolds provides an excellent physical environment for cell adhesion and proliferation, as well as nutrient transport. Traditional 3D-printed bioscaffolds offer mere physical stimulation, while drug-loaded 3D bioscaffolds accelerate the tissue repair process by synergistically combining drug therapy with physical stimulation. In this review, the physiological characteristics of osteochondral tissue and current treatments of osteochondral defect were reviewed. Subsequently, the latest progress in drug-loaded bioscaffolds was discussed and highlighted in terms of classification, characteristics, and applications. The perspectives of scaffold design, drug control release, and biosafety were also discussed. We hope this article will serve as a valuable reference for the design and development of osteochondral regenerative bioscaffolds and pave the way for the use of drug-loaded bioscaffolds in clinical therapy.

Characterization of biological and mechanical properties of 3D-bioprinted osteochondral plugs

Three-dimensional (3D) bioprinting offers significant potential for the repair of articular cartilage by engineering functional osteochondral tissue. However, progress has been hindered by a lack of printable bioinks that promote the development of bone and chondral tissue while also maintaining sufficient cytocompatibility and mechanical strength. Herein, we designed a biphasic osteochondral plug with distinct chondral and bone regions and developed suitable bioinks for each tissue using photorheology and compression testing. The chondral region consisted of human bone marrow-derived mesenchymal stem cells (hbMSCs) encapsulated in a chondral bioink composed of methacrylated hyaluronic acid and high molecular weight hyaluronic acid. The bone region was 3D bioprinted from an hbMSC-laden methacrylated gelatin (GelMA) bioink and a biodegradable thermoplastic and ceramic lattice that provided mechanical strength. The viability and functionality of hbMSC encapsulated in the bioinks were confirmed through live/dead assays, histology, biochemical assays, and fluorescence microscopy. Over 56 days of culture in a chondrogenic medium, hbMSCs encapsulated in chondral bioink deposited cartilage-like extracellular matrix components, such as type II collagen and glycosaminoglycans. Similarly, cells encapsulated in the bone bioink and cultured in osteogenic medium deposited hydroxyapatite, a key component of bone. These findings provide promising initial results for using 3D-bioprinted plugs to repair osteochondral defects in articular cartilage.  

Bioprinting of osteochondral tissues: A perspective on current gaps and future trends.

Osteochondral tissue regeneration has remained a critical challenge in orthopaedic surgery, especially due to complications of arthritic degeneration arising out of mechanical dislocations of joints. The common gold standard of autografting has several limitations in presenting tissue engineering strategies to solve the unmet clinical need. However, due to the complexity of joint anatomy, and tissue heterogeneity at the interface, the conventional tissue engineering strategies have certain limitations. The advent of bioprinting has now provided new opportunities for osteochondral tissue engineering. Bioprinting can uniquely mimic the heterogeneous cellular composition and anisotropic extra-cellular matrix (ECM) organization, while allowing for targeted gene delivery to achieve heterotypic differentiation. In this perspective, we discuss the current advances made towards bioprinting of composite osteochondral tissues and present an account of challenges—in terms of tissue integration, long-term survival, and mechanical strength at the time of implantation—required to be addressed for effective clinical translation of bioprinted tissues. Finally, we highlight some of the future trends related to osteochondral bioprinting with the hope of in-clinical translation.

Nanoclay-Composite Hydrogels for Bone Tissue Engineering.

Nanoclay-composite hydrogels represent a promising avenue for advancing bone tissue engineering. Traditional hydrogels face challenges in providing mechanical strength, biocompatibility, and bioactivity necessary for successful bone regeneration. The incorporation of nanoclay into hydrogel matrices offers a potential unique solution to these challenges. This review provides a comprehensive overview of the fabrication, physico-chemical/biological performance, and applications of nanoclay-composite hydrogels in bone tissue engineering. Various fabrication techniques, including in situ polymerization, physical blending, and 3D printing, are discussed. In vitro and in vivo studies evaluating biocompatibility and bioactivity have demonstrated the potential of these hydrogels for promoting cell adhesion, proliferation, and differentiation. Their applications in bone defect repair, osteochondral tissue engineering and drug delivery are also explored. Despite their potential in bone tissue engineering, nanoclay-composite hydrogels face challenges such as optimal dispersion, scalability, biocompatibility, long-term stability, regulatory approval, and integration with emerging technologies to achieve clinical application. Future research directions need to focus on refining fabrication techniques, enhancing understanding of biological interactions, and advancing towards clinical translation and commercialization. Overall, nanoclay-composite hydrogels offer exciting opportunities for improving bone regeneration strategies.

3D-printed gradient scaffolds for osteochondral defects: Current status and perspectives.

Articular osteochondral defects are quite common in clinical practice, and tissue engineering techniques can offer a promising therapeutic option to address this issue.The articular osteochondral unit comprises hyaline cartilage, calcified cartilage zone (CCZ), and subchondral bone.As the interface layer of articular cartilage and bone, the CCZ plays an essentialpart in stress transmission and microenvironmental regulation.Osteochondral scaffolds with the interface structure for defect repair are the future direction of tissue engineering. Three-dimensional (3D) printing has the advantages of speed, precision, and personalized customization, which can satisfy the requirements of irregular geometry, differentiated composition, and multilayered structure of articular osteochondral scaffolds with boundary layer structure. This paper summarizes the anatomy, physiology, pathology, and restoration mechanisms of the articular osteochondral unit, and reviews the necessity for a boundary layer structure in osteochondral tissue engineering scaffolds and the strategy for constructing the scaffolds using 3D printing. In the future, we should not only strengthen the basic research on osteochondral structural units, but also actively explore the application of 3D printing technology in osteochondral tissue engineering. This will enable better functional and structural bionics of the scaffold, which ultimately improve the repair of osteochondral defects caused by various diseases.

Increased nerve growth factor expression and osteoclast density are associated with subchondral bone marrow lesions in osteoarthritic knees

ObjectivesSubchondral bone marrow lesions (BMLs) detected on magnetic resonance imaging in knee osteoarthritis (OA) are associated with knee pain, though the mechanisms remain unknown. Increased nerve growth factor (NGF) expression and osteoclast density in subchondral bone appear to be the key features associated with bone pain in knee OA. Therefore, we aimed to identify associations among NGF, osteoclasts, and BMLs in knee OA. MethodsTwenty tibial plateaus were obtained from patients undergoing total knee arthroplasty for medial knee OA with BMLs at the medial tibial plateau (MTP). Osteochondral tissue samples from the weight-bearing part of the MTP, with and without BML, and from the weight-bearing part of the lateral tibial plateau (LTP), without BML, were collected. NGF expression and density of osteoclasts were compared among the three osteochondral tissue types. ResultsMTP bone with BMLs exhibited significantly higher NGF expression in bone marrow space and osteochondral channel, and higher osteoclast density than MTP bone without BML and LTP bone. The mean differences in NGF-positive area in the bone marrow space and the percentage of NGF-positive channels between MTP bones with and without BML were 9.0% (95% confidence interval [CI]: 5.9–12.1%) and 23.1% (95% CI: 11.3–35.0%), respectively. The difference in osteoclast density between MTP bones with and without BML was 0.6 osteoclasts per mm (95% CI: 0.3–0.9 osteoclasts per mm). ConclusionsIncreased NGF expression and osteoclast density are associated with subchondral BMLs in knee OA, contribute to understanding the mechanisms underlying BML-related bone pain in knee OA.

Spatiotemporal delivery of BMP-2 and FGF-18 in 3D-bioprinted tri-phasic osteochondral scaffolds enhanced compartmentalized osteogenic and chondrogenic differentiation of mesenchymal stem cells isolated from rats with varied organizational morphologies

Replicating the heterogeneous structure and promoting compartmentalized osteogenesis/chondrogenesis are critical considerations in designing scaffolds for osteochondral tissue regeneration. However, desirable osteochondral regeneration cannot be achieved mainly due to the absence of effective delivery strategies for growth factors (GFs) and the insufficiency of desirable organizational morphologies for seed cells. Herein, we developed a tri-phasic osteochondral scaffold consisting of bone morphogenetic protein-2 (BMP-2)-loaded subchondral layer, fibroblast growth factor-18 (FGF-18)-loaded cartilage layer, and an interface layer that acted as a barrier to reduce the mutual interference of GFs, via cryogenic 3D bioprinting. BMP-2 could exert osteogenic effects for 14 days, and FGF-18 could exert chondrogenic effects for 21 days, demonstrating the time-controlled release function of BMP-2 and FGF-18. By further seeding discrete rat bone marrow mesenchymal stem cells (rBMSCs) and rBMSC microspheres, respectively, onto the subchondral layer and cartilage layer, the engineered cell-laden osteochondral tissue was constructed. The spatiotemporal release of BMP-2 and FGF-18 in the subchondral layer and cartilage layer promoted the osteogenic differentiation of discrete rBMSCs and chondrogenic differentiation of rBMSC microspheres in the subchondral layer and cartilage layer, respectively. In summary, by seeding rBMSCs with varied organizational morphologies in 3D-printed osteochondral scaffolds with a spatiotemporally controlled strategy, engineered osteochondral tissue with compartmentalized osteogenic/chondrogenic differentiation potent can be formed, displaying a facile and promising way to achieve desirable osteochondral tissue regeneration.

Aging alters the subchondral bone response 7 days after noninvasive traumatic joint injury in C57BL/6JN mice.

Posttraumatic osteoarthritis (PTOA) commonly develops following anterior cruciate ligament (ACL) injuries, affecting around 50% of individuals within 10-20 years. Recent studies have highlighted early changes in subchondral bone structure after ACL injury in adolescent or young adult mice, which could contribute to the development of PTOA. However, ACL injuries do not only occur early in life. Middle-aged and older patients also experience ACL injuries and PTOA, but whether the aged subchondral bone also responds rapidly to injury is unknown. This study utilized a noninvasive, single overload mouse injury model to assess subchondral bone microarchitecture, turnover, and material properties in both young adults (5 months) and early old age (22 months) female C57BL/6JN mice at 7 days after injury. Mice underwent either joint injury (i.e., produces ACL tears) or sham injury procedures on both the loaded and contralateral limbs, allowing evaluation of the impacts of injury versus loading. The subchondral bone response to ACL injury is distinct for young adult and aged mice. While 5-month mice show subchondral bone loss and increased bone resorption postinjury, 22-month mice did not show loss of bone structure and had lower bone resorption. Subchondral bone plate modulus increased with age, but not with injury. Both ages of mice showed several bone measures were altered in the contralateral limb, demonstrating the systemic skeletal response to joint injury. These data motivate further investigation to discern how osteochondral tissues differently respond to injury in aging, such that diagnostics and treatments can be refined for these demographics.

Bioceramic-mediated chondrocyte hypertrophy promotes calcified cartilage formation for rabbit osteochondral defect repair

Osteochondral tissue is a highly specialized and complex tissue composed of articular cartilage and subchondral bone that are separated by a calcified cartilage interface. Multilayered or gradient scaffolds, often in conjunction with stem cells and growth factors, have been developed to mimic the respective layers for osteochondral defect repair. In this study, we designed a hyaline cartilage-hypertrophic cartilage bilayer graft (RGD/RGDW) with chondrocytes. Previously, we demonstrated that RGD peptide-modified chondroitin sulfate cryogel (RGD group) is chondro-conductive and capable of hyaline cartilage formation. Here, we incorporated whitlockite (WH), a Mg2+-containing calcium phosphate, into RGD cryogel (RGDW group) to induce chondrocyte hypertrophy and form collagen X-rich hypertrophic cartilage. This is the first study to use WH to produce hypertrophic cartilage. Chondrocytes-laden RGDW cryogel exhibited significantly upregulated expression of hypertrophy markers in vitro and formed ectopic hypertrophic cartilage in vivo, which mineralized into calcified cartilage in bone microenvironment. Subsequently, RGD cryogel and RGDW cryogel were combined into bilayer (RGD/RGDW group) and implanted into rabbit osteochondral defect, where RGD layer supports hyaline cartilage regeneration and bioceramic-containing RGDW layer promotes calcified cartilage formation. While the RGD group (monolayer) formed hyaline-like neotissue that extends into the subchondral bone, the RGD/RGDW group (bilayer) regenerated hyaline cartilage tissue confined to its respective layer and promoted osseointegration for integrative defect repair.