Subchondral Bone Regeneration Research Articles

Multilayered Cryogel Enriched with Exosomes Regenerates and Maintains Cartilage Architecture and Phenotype in Goat Osteochondral Injuries.

Treatment of critical-size osteochondral (OC) injuries at load-bearing sites has remained a major clinical challenge in orthopedic surgery. This is due to the anisotropic characteristics of OC tissue and the stratified structure of the cartilage. Here, we developed a multilayered OC scaffold by employing cryogelation technology. Gelatin, chitosan, and chondroitin sulfate were utilized for designing three distinct, 2425 ± 120 μm thick layers of cartilage having different alignments, while nanohydroxyapatite and gelatin were used for the subchondral bone layer. Exosomes derived from articular chondrocytes in the range of 60-110 nm were used to promote chondrogenesis. The biocompatibility and cartilage formation potential of the scaffold and exosomes were initially evaluated in rat OC defects. The application of exosome-loaded scaffolds was then investigated in a critical-size OC injury (8 × 10 mm) created in the goat knee. Artificial synovial fluid was designed and utilized as a carrier for exosomes for a booster dose administered as an intra-articular injection. X-ray imaging and micro-CT analysis revealed that the treatment resulted in improved subchondral bone regeneration. The defect region exhibited healthy hyaline cartilage formation, as detected by MRI imaging. Moreover, histological examination revealed that the treatment group showed augmented cell proliferation, matrix deposition, secretion of proteoglycans, and the formation of stratified hyaline cartilage over a long-term (6 and 12 months), whereas the control group demonstrated the formation of fibrocartilage. Treatment-induced upregulation of collagen II, aggrecan, and SOX 9 genes (∼10 fold) further provided evidence that the cartilage phenotype was well preserved. Hence, the proposed treatment has significant translational potential for treating adverse OC clinical injuries.

Molybdenum nanodots act as antioxidants for photothermal therapy osteoarthritis

Osteoarthritis (OA) manifests as the degradation of cartilage and remodeling of subchondral bone. Restoring homeostasis within the joint is imperative for alleviating OA symptoms. Current interventions primarily target singular aspects, such as anti-aging, inflammation inhibition, free radical scavenging, and regeneration of cartilage and subchondral bone. Herein, we developed molybdenum nanodots (MNDs) as bionic photothermal nanomaterials to mimic the antioxidant synthase to concurrently protected cartilage and facilitate subchondral bone regeneration. With near-infrared (NIR) irradiation, MNDs effectively eliminate reactive oxygen and nitrogen species (ROS/RNS) from OA chondrocytes, thereby reversed mitochondrial dysfunction, mitigating chondrocyte senescence, and simultaneously suppresses inflammation, hence preserving the inherent homeostasis between cartilage matrix synthesis and degradation while circumventing safety concerns. RNA sequencing of OA chondrocytes treated with MNDs-NIR revealed the reinstatement of chondrocyte functionality, activation of antioxidant enzymes, anti-aging properties, and regulation of inflammation. NIR irradiation induces thermogenesis and synergistically promotes subchondral bone regeneration via MNDs, as validated through histological assessments and microcomputed tomography (Micro-CT) scans. MNDs-NIR effectively attenuate cellular senescence and inhibit inflammation in vivo, while also remodeling mitochondrial dynamics by upregulating fusion proteins and inhibiting fission proteins, thereby regulating the oxidative stress microenvironment. Additionally, MNDs-NIR exhibited remarkable therapeutic effects in alleviating articular cartilage degeneration in an OA mouse model, evidenced by a 1.67-fold reduction in subchondral bone plate thickness, an 88.57 % decrease in OARSI score, a 5.52-fold reduction in MMP13 expression, and a 6.80-fold increase in Col II expression. This novel disease-modifying approach for OA utilizing MNDs-NIR offers insight and a paradigm for improving mitochondrial dysfunction by regulating the accumulation of mitochondrial ROS and ultimately alleviating cellular senescence. Moreover, the dual-pronged therapeutic approach of MNDs-NIR, which addresses both cartilage erosion and subchondral bone lesions in OA, represents a highly promising strategy for managing OA.

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.

Enhancement of hyaline cartilage and subchondral bone regeneration in a rat osteochondral defect model through focused extracorporeal shockwave therapy.

To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration. The OCD lesion was created on the trochlear groove of left articular cartilage of femur per rat (40 rats in total). The experimental groups were Sham, OCD, and ESWT (0.25 mJ/mm2, 800 impulses, 4 Hz). The animals were euthanized at 2, 4, 8, and 12 weeks post-treatment, and histopathological analysis, micro-CT scanning, and immunohistochemical staining were performed for the specimens. In the histopathological analysis, the macro-morphological grading scale showed a significant increase, while the histological score and cartilage repair scale of ESWT exhibited a significant decrease compared to OCD at the 8- and 12-week timepoints. At the 12-week follow-up, ESWT exhibited a significant improvement in the volume of damaged bone compared to OCD. Furthermore, immunohistochemistry analysis revealed a significant decrease in type I collagen and a significant increase in type II collagen within the newly formed hyaline cartilage following ESWT, compared to OCD. Finally, SRY-box transcription factor 9 (SOX9), aggrecan, and TGF-β, BMP-2, -3, -4, -5, and -7 were significantly higher in ESWT than in OCD at 12 weeks. ESWT promoted the effect of TGF-β/BMPs, thereby modulating the production of extracellular matrix proteins and transcription factor involved in the regeneration of articular cartilage and subchondral bone in an OCD rat model.

Nanoclay Reinforced Integrated Scaffold for Dual-Lineage Regeneration of Cartilage and Subchondral Bone.

Tissue engineering is theoretically considered a promising approach for repairing osteochondral defects. Nevertheless, the insufficient osseous support and integration of the cartilage layer and the subchondral bone frequently lead to the failure of osteochondral repair. Drawing from this, it was proposed that incorporating glycine-modified attapulgite (GATP) into poly(1,8-octanediol-co-citrate) (POC) scaffolds via the one-step chemical cross-linking is proposed to enhance cartilage and subchondral bone defect repair simultaneously. The effects of the GATP incorporation ratio on the physicochemical properties, chondrocyte and MC3T3-E1 behavior, and osteochondral defect repair of the POC scaffold were also evaluated. In vitro studies indicated that the POC/10% GATP scaffold improved cell proliferation and adhesion, maintained cell phenotype, and upregulated chondrogenesis and osteogenesis gene expression. Animal studies suggested that the POC/10% GATP scaffold has significant repair effects on both cartilage and subchondral bone defects. Therefore, the GATP-incorporated scaffold system with dual-lineage bioactivity showed potential application in osteochondral regeneration.

Three‐dimensional bioprinting biphasic multicellular living scaffold facilitates osteochondral defect regeneration

AbstractDue to tissue lineage variances and the anisotropic physiological characteristics, regenerating complex osteochondral tissues (cartilage and subchondral bone) remains a great challenge, which is primarily due to the distinct requirements for cartilage and subchondral bone regeneration. For cartilage regeneration, a significant amount of newly generated chondrocytes is required while maintaining their phenotype. Conversely, bone regeneration necessitates inducing stem cells to differentiate into osteoblasts. Additionally, the construction of the osteochondral interface is crucial. In this study, we fabricated a biphasic multicellular bioprinted scaffold mimicking natural osteochondral tissue employing three‐dimensional (3D) bioprinting technology. Briefly, gelatin‐methacryloyl (GelMA) loaded with articular chondrocytes and bone marrow mesenchymal stem cells (ACs/BMSCs), serving as the cartilage layer, preserved the phenotype of ACs and promoted the differentiation of BMSCs into chondrocytes through the interaction between ACs and BMSCs, thereby facilitating cartilage regeneration. GelMA/strontium‐substituted xonotlite (Sr‐CSH) loaded with BMSCs, serving as the subchondral bone layer, regulated the differentiation of BMSCs into osteoblasts and enhanced the secretion of cartilage matrix by ACs in the cartilage layer through the slow release of bioactive ions from Sr‐CSH. Additionally, GelMA, serving as the matrix material, contributed to the reconstruction of the osteochondral interface. Ultimately, this biphasic multicellular bioprinted scaffold demonstrated satisfactory simultaneous regeneration of osteochondral defects. In this study, a promising strategy for the application of 3D bioprinting technology in complex tissue regeneration was proposed.

Diatomite-incorporated hierarchical scaffolds for osteochondral regeneration

Osteochondral regeneration involves the highly challenging and complex reconstruction of cartilage and subchondral bone. Silicon (Si) ions play a crucial role in bone development. Current research on Si ions mainly focuses on bone repair, by using silicate bioceramics with complex ion compositions. However, it is unclear whether the Si ions have important effect on cartilage regeneration. Developing a scaffold that solely releases Si ions to simultaneously promote subchondral bone repair and stimulate cartilage regeneration is critically important. Diatomite (DE) is a natural diatomaceous sediment that can stably release Si ions, known for its abundant availability, low cost, and environmental friendliness. Herein, a hierarchical osteochondral repair scaffold is uniquely designed by incorporating gradient DE into GelMA hydrogel. The adding DE microparticles provides a specific Si source for controlled Si ions release, which not only promotes osteogenic differentiation of rBMSCs (rabbit bone marrow mesenchymal stem cells) but also enhances proliferation and maturation of chondrocytes. Moreover, DE-incorporated hierarchical scaffolds significantly promoted the regeneration of cartilage and subchondral bone. The study suggests the significant role of Si ions in promoting cartilage regeneration and solidifies their foundational role in enhancing bone repair. Furthermore, it offers an economic and eco-friendly strategy for developing high value-added osteochondral regenerative bioscaffolds from low-value ocean natural materials.

Architecture of β-lactoglobulin coating modulates bioinspired alginate dialdehyde-gelatine/polydopamine scaffolds for subchondral bone regeneration

In this study, we developed polydopamine (PDA)-functionalized alginate dialdehyde-gelatine (ADA-GEL) scaffolds for subchondral bone regeneration. These polymeric scaffolds were then coated with β-Lactoglobulin (β-LG) at concentrations of 1 mg/ml and 2 mg/ml. Morphological analysis indicated a homogeneous coating of the β-LG layer on the surface of network-like scaffolds. The β-LG-coated scaffolds exhibited improved swelling capacity as a function of the β-LG concentration. Compared to ADA-GEL/PDA scaffolds, the β-LG-coated scaffolds demonstrated delayed degradation and enhanced biomineralization. Here, a lower concentration of β-LG showed long-lasting stability and superior biomimetic hydroxyapatite mineralization. According to the theoretical findings, the single-state, representing the low concentration of β-LG, exhibited a homogeneous distribution on the surface of the PDA, while the dimer-state (high concentration) displayed a high likelihood of uncontrolled interactions. β-LG-coated ADA-GEL/PDA scaffolds with a lower concentration of β-LG provided a biocompatible substrate that supported adhesion, proliferation, and alkaline phosphatase (ALP) secretion of sheep bone marrow mesenchymal stem cells, as well as increased expression of osteopontin (SPP1) and collagen type 1 (COL1A1) in human osteoblasts. These findings indicate the potential of protein-coated scaffolds for subchondral bone tissue regeneration. Statement of SignificanceThis study addresses a crucial aspect of osteochondral defect repair, emphasizing the pivotal role of subchondral bone regeneration. The development of polydopamine-functionalized alginate dialdehyde-gelatine (ADA-GEL) scaffolds, coated with β-Lactoglobulin (β-LG), represents a novel approach to potentially enhance subchondral bone repair. β-LG, a milk protein rich in essential amino acids and bioactive peptides, is investigated for its potential to promote subchondral bone regeneration. This research explores computationally and experimentally the influence of protein concentration on the ordered or irregular deposition, unravelling the interplay between coating structure, scaffold properties, and in-vitro performance. This work contributes to advancing ordered protein coating strategies for subchondral bone regeneration, providing a biocompatible solution with potential implications for supporting subsequent cartilage repair.

Micro-current stimulation could inhibit IL-1β-induced inflammatory responses in chondrocytes and protect knee bone cartilage from osteoarthritis.

This study aimed to evaluate the inhibitory effects of micro-current stimulation (MCS) on inflammatory responses in chondrocytes and degradation of extracellular matrix (ECM) in osteoarthritis (OA). To determine the efficacy of MCS, IL-1β-treated chondrocytes and monosodium iodoacetate (MIA)-induced OA rat model were used. To evaluate the cytotoxicity and nitric oxide (NO) production in SW1353 cells, the presence or absence of IL-1β treatment or various levels of MCS were applied. Immunoblot analysis was conducted to evaluate whether MCS can modulate IL-1R1/MyD88/NF-κB signaling pathway and various indicators involved in ECM degradation. Additionally, to determine whether MCS alleviates subchondral bone structure destruction caused by OA, micro-CT analysis, immunoblot analysis, and ELISA were conducted using OA rat model. 25 and 50 µA levels of MCS showed effects in cell proliferation and NO production. The MCS group with IL-1β treatment lead to significant inhibition of protein expression levels regarding IL-1R1/MyD88/NF-κB signaling and reduction of the nucleus translocation of NF-κB. In addition, the protein expression levels of MMP-1, MMP-3, MMP-13, and IL-1β decreased, whereas collagen II and aggrecan increased. In animal results, morphological analysis of subchondral bone using micro-CT showed that MCS induced subchondral bone regeneration and improvement, as evidenced by increased thickness and bone mineral density of the subchondral bone. Furthermore, MCS-applied groups showed decreases in the protein expression of MMP-1 and MMP-3, while increases in collagen-II and aggrecan expressions. These findings suggest that MCS has the potential to be used as a non-pharmaceutical method to alleviate OA.

Wet 3D printing of biodegradable porous scaffolds to enable room-temperature deposition modeling of polymeric solutions for regeneration of articular cartilage

Tissue engineering has emerged as an advanced strategy to regenerate various tissues using different raw materials, and thus it is desired to develop more approaches to fabricate tissue engineering scaffolds to fit specific yet very useful raw materials such as biodegradable aliphatic polyester like poly (lactide-co-glycolide) (PLGA). Herein, a technique of ‘wet 3D printing’ was developed based on a pneumatic extrusion three-dimensional (3D) printer after we introduced a solidification bath into a 3D printing system to fabricate porous scaffolds. The room-temperature deposition modeling of polymeric solutions enabled by our wet 3D printing method is particularly meaningful for aliphatic polyester, which otherwise degrades at high temperature in classic fuse deposition modeling. As demonstration, we fabricated a bilayered porous scaffold consisted of PLGA and its mixture with hydroxyapatite for regeneration of articular cartilage and subchondral bone. Long-term in vitro and in vivo degradation tests of the scaffolds were carried out up to 36 weeks, which support the three-stage degradation process of the polyester porous scaffold and suggest faster degradation in vivo than in vitro. Animal experiments in a rabbit model of articular cartilage injury were conducted. The efficacy of the scaffolds in cartilage regeneration was verified through histological analysis, micro-computed tomography (CT) and biomechanical tests, and the influence of scaffold structures (bilayer versus single layer) on in vivo tissue regeneration was examined. This study has illustrated that the wet 3D printing is an alternative approach to biofabricate tissue engineering porous scaffolds based on biodegradable polymers.

Enhanced osteochondral regeneration with a 3D-Printed biomimetic scaffold featuring a calcified interfacial layer

The integrative regeneration of both articular cartilage and subchondral bone remains an unmet clinical need due to the difficulties of mimicking spatial complexity in native osteochondral tissues for artificial implants. Layer-by-layer fabrication strategies, such as 3D printing, have emerged as a promising technology replicating the stratified zonal architecture and varying microstructures and mechanical properties. However, the dynamic and circulating physiological environments, such as mass transportation or cell migration, usually distort the pre-confined biological properties in the layered implants, leading to undistinguished spatial variations and subsequently inefficient regenerations. This study introduced a biomimetic calcified interfacial layer into the scaffold as a compact barrier between a cartilage layer and a subchondral bone layer to facilitate osteogenic–chondrogenic repair. The calcified interfacial layer consisting of compact polycaprolactone (PCL), nano-hydroxyapatite, and tasquinimod (TA) can physically and biologically separate the cartilage layer (TA-mixed, chondrocytes-load gelatin methacrylate) from the subchondral bond layer (porous PCL). This introduction preserved the as-designed independent biological environment in each layer for both cartilage and bone regeneration, successfully inhibiting vascular invasion into the cartilage layer and preventing hyaluronic cartilage calcification owing to devascularization of TA. The improved integrative regeneration of cartilage and subchondral bone was validated through gross examination, micro-computed tomography (micro-CT), and histological and immunohistochemical analyses based on an in vivo rat model. Moreover, gene and protein expression studies identified a key role of Caveolin (CAV-1) in promoting angiogenesis through the Wnt/β-catenin pathway and indicated that TA in the calcified layer blocked angiogenesis by inhibiting CAV-1.

High quality repair of osteochondral defects in rats using the extracellular matrix of antler stem cells.

Cartilage defects are some of the most common causes of arthritis. Cartilage lesions caused by inflammation, trauma or degenerative disease normally result in osteochondral defects. Previous studies have shown that decellularized extracellular matrix (ECM) derived from autologous, allogenic, or xenogeneic mesenchymal stromal cells (MSCs) can effectively restore osteochondral integrity. To determine whether the decellularized ECM of antler reserve mesenchymal cells (RMCs), a xenogeneic material from antler stem cells, is superior to the currently available treatments for osteochondral defects. We isolated the RMCs from a 60-d-old sika deer antler and cultured them in vitro to 70% confluence; 50 mg/mL L-ascorbic acid was then added to the medium to stimulate ECM deposition. Decellularized sheets of adipocyte-derived MSCs (aMSCs) and antlerogenic periosteal cells (another type of antler stem cells) were used as the controls. Three weeks after ascorbic acid stimulation, the ECM sheets were harvested and applied to the osteochondral defects in rat knee joints. The defects were successfully repaired by applying the ECM-sheets. The highest quality of repair was achieved in the RMC-ECM group both in vitro (including cell attachment and proliferation), and in vivo (including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular hyaline cartilage integrated with surrounding native tissues). Notably, the antler-stem-cell-derived ECM (xenogeneic) performed better than the aMSC-ECM (allogenic), while the ECM of the active antler stem cells was superior to that of the quiescent antler stem cells. Decellularized xenogeneic ECM derived from the antler stem cell, particularly the active form (RMC-ECM), can achieve high quality repair/reconstruction of osteochondral defects, suggesting that selection of decellularized ECM for such repair should be focused more on bioactivity rather than kinship.

3D Printing of Cobalt-Incorporated Chloroapatite Bioceramic Composite Scaffolds with Antioxidative Activity for Enhanced Osteochondral Regeneration.

Osteochondral defects are often accompanied by excessive reactive oxygen species (ROS) caused by osteoarthritis or acute surgical inflammation. An inflammatory environment containing excess ROS will not only hinder tissue regeneration but also impact the quality of newly formed tissues. Therefore, there is an urgent need to develop scaffolds with both ROS scavenging and osteochondral repair functions to promote and protect osteochondral tissue regeneration. In this work, by using 3D printing technology, a composite scaffold based on cobalt-incorporated chloroapatite (Co-ClAP) bioceramics, which possesses ROS-scavenging activity and can support cell proliferation, adhesion, and differentiation, is developed. Benefiting from the catalytic activity of Co-ClAP bioceramics, the composite scaffold can protect cells from oxidative damage under ROS-excessive conditions, support their directional differentiation, and simultaneously mediate an anti-inflammatory microenvironment. In addition, it is also confirmed by using rabbit osteochondral defect model that the Co-ClAP/poly(lactic-co-glycolic acid) scaffold can effectively promote the integrated regeneration of cartilage and subchondral bone, exhibiting an ideal repair effect in vivo. This study provides a promising strategy for the treatment of defects with excess ROS and inflammatory microenvironments.

PUD@HA/PEEK Scaffold Induces Subchondral Bone Regeneration to Repair Osteochondral Defect in Rabbits.

Osteochondral defects (OCDs) pose a significant challenge in clinical practice, and recent advancements in their repair indicate that satisfying subchondral bone repair may be critical for this. Herein, a series of hydroxyapatite/poly(ether ether ketone) (HA/PEEK) scaffolds were fabricated with varying mass percentages (0, 20, 30, and 40%) to induce subchondral bone regeneration. Subsequently, an optimal scaffold with 40% HA/PEEK was selected to establish osteochondral scaffolds with poly(ether urethane) urea-Danshensu (PUD) for repairing the OCD. The material characteristics of HA/PEEK and PUD were investigated using scanning electron microscopy, tensile, swelling, and fatigue tests, and cytological experiments. The effects of serial HA/PEEK scaffolds on subchondral bone repair were then assessed by using microcomputed tomography, hard tissue slicing, and histological staining. Furthermore, the optimal 40% HA/PEEK scaffold was used to develop osteochondral scaffolds with PUD to observe the effect on the OCD repair. HA/PEEK materials exhibited an even HA distribution in PEEK. However, when composited with HA, PEEK exhibited inferior mechanical strength. 40%HA/PEEK scaffolds showed an optimum effect on in vivo subchondral bone repair. Cartilage regeneration on 40%HA/PEEK scaffolds was pronounced. After PUD was introduced onto the HA/PEEK, the PUD@40%HA/PEEK scaffold produced the expected effect on the repair of the OCD in rabbits. Therefore, achieving satisfactory subchondral bone repair can benefit surficial cartilage repair. The PUD@40%HA/PEEK scaffold could induce subchondral bone regeneration to repair the OCD in rabbits and could provide a novel approach for the repair of the OCD in clinical practice.

Effectiveness of Bilayer Scaffold Containing Chitosan/Gelatin/Diclofenac and Bovine Hydroxyapatite on Cartilage/Subchondral Regeneration in Rabbit Joint Defect Models.

Subchondral defects are often caused by trauma involving cartilage damage, leading to subsequent damage to the underlying bone, specifically the subchondral region. Bilayer scaffolds made from biomaterials, such as bovine hydroxyapatite, possess biocompatible and biodegradable properties that mimic the natural environmental conditions of target tissues so that they can support the formation of new tissues. On the other side, diclofenac as an anti-inflammatory drug potentiates to inhibit the inflammatory excess regarding the damage. This study aims to study the effectiveness of diclofenac scaffold to rabbit joint defect model. The scaffold was implanted in the rabbit femoral trochlear bone hole, which had a diameter of 5 mm and a depth of 4 mm. After 28 days of intervention, the animals were examined using macroscopic evaluation, hematoxylin-eosin (HE) staining, and immunohistochemistry (IHC) for type I collagen and type II collagen. Subsequently, the cartilage was evaluated using the International Cartilage Repair Society (ICRS) scoring system. The macroscopic ICRS scores were significantly higher (p < 0.05) in the bilayer scaffold implantation group compared to the monolayer scaffold and control groups. Histological ICRS scores were also significantly higher (p < 0.05) in the bilayer scaffold group compared to the control group. Type II collagen expression was higher (p < 0.05) in the bilayer scaffold group compared to the monolayer scaffold and control groups, although type I collagen expression was lower in comparison. In conclusion, this research suggests that the diclofenac-loaded bilayer scaffold effectively enhances cartilage and subchondral bone regeneration.

Biomimetic injectable and bilayered hydrogel scaffold based on collagen and chondroitin sulfate for the repair of osteochondral defects

The simultaneous regeneration of articular cartilage and subchondral bone is a major challenge. Bioinspired scaffolds with distinct regions resembling stratified anatomical architecture provide a potential strategy for osteochondral defect repair. Here, we report the development of an injectable and bilayered hydrogel scaffold with a strong interface binding force. In this bilayer hydrogel, composed of carbonyl hydrazide grafted collagen (COL-CDH) and oxidized chondroitin sulfate (OCS), which are derivatives of osteochondral tissue components, in combination with poly (ethylene glycol) diacrylate (PEGDA), functions as a cartilage layer; while zinc-doped hydroxyapatite acts as a subchondral bone layer that is based on the cartilage layer. The strong interface between the two layers involves dynamic amide bonds formed between COL-CDH and OCS, and permanent CC bonds formed by PEGDA radical reactions. This bilayer hydrogel can be used to inoculate adipose mesenchymal stem cells which can then differentiate into chondrocytes and osteoblasts, secreting glycosaminoglycan, and promoting calcium deposition. This accelerates the regeneration of cartilage and subchondral bone. Micro-CT and tissue staining revealed an increase in the amount of bone present in new subchondral bone, and new tissues with a structure similar to normal cartilage. This study therefore demonstrates that injectable bilayer hydrogels are a promising scaffold for repairing osteochondral defects.

Effect of autologous lyophilized platelet‑rich fibrin on the reconstruction of osteochondral defects in rabbits.

Osteochondral defects caused by degenerative diseases of joints, traumas and inflammation are important issues in clinical practice. Different types of autologous platelet concentrate (PCs) are used in bone and cartilage regeneration. The present study aimed to investigate the effect of lyophilized platelet-rich fibrin (L-PRF) on the repair of osteochondral defects in rabbits. L-PRF was first prepared from fresh PRF (F-PRF) through freeze-drying, and histological and microstructural observations were performed to compare the characteristics of L-PRF and F-PRF. Thereafter, these bioactive scaffolds were implanted into osteochondral defects surgically created in rabbits to assess their effects on tissue repair using micro-CT scanning, histological observations and the evaluation scoring method for cartilage repair established by the International Cartilage Repair Society (ICRS). L-PRF had a histological structure similar to F-PRF. At 16 weeks after implantation surgery, full-thickness osteochondral defects with a diameter of 5 mm and a depth of 4 mm were well-filled with newly regenerated tissues, exhibiting the simultaneous regeneration of avascular articular cartilage and well-vascularized subchondral bone, as proven through macroscopic and microscopic observations in PRF-treated groups compared with that in the untreated group. The application of L-PRF and F-PRF for osteochondral defects in rabbits contributed to massive host remodeling and reconstruction of osteochondral tissues, thus offering a prospective bioactive scaffold for the simultaneous reconstruction of articular cartilage and subchondral bone tissue.

Gelatin methacryloyl hydrogel with and without dental pulp stem cells for TMJ regeneration: An invivo study in rabbits.

In the last decade, tissue-engineering strategies for regenerating the temporomandibular joint (TMJ) have been investigated. This may be a promising strategy for the minimally invasive restoration of joint integrity. To evaluate whether dental pulp stem cells (DPSCs) loaded in a light-occured hydrogel made of gelatin methacryloyl (GelMA) enhance the regeneration of osteochondral defects in the rabbit TMJ. Defects were filled with GelMA alone (control group; n = 4) or filled with GelMA loaded with rabbit DPSCs (experimental group; n = 4), In one group, the TMJ capsule was opened without creating a defect (sham group; n = 2). The following micro-CT parameters were analysed: bone volume to total volume ratio (BV/TV%) and bone mineral density (BMD). Histological evaluation was performed to assess cartilage regeneration features. A semi-quantitative scoring system was also used to evaluate the defects. All groups had no statistical difference regarding the micro-CT parameters. The highest mean healing score was found for the experimental group. After 4 weeks, there were no signs of hydrogel in either group or no signs of inflammation in the adjacent tissues. The tissue formed in the defect was dense fibrous connective tissue. Adding DPSCs to GelMA did not provide a regenerative enhancement in TMJ osteochondral defects. This resulted in similar micro-CT parameters after 4 weeks of healing, with improved signs of subchondral bone regeneration but no cartilage regeneration.

Surface-Modified Nano-Hydroxyapatite Uniformly Dispersed on High-Porous GelMA Scaffold Surfaces for Enhanced Osteochondral Regeneration.

This study aims to investigate the impact of enhancing subchondral bone repair on the efficacy of articular cartilage restoration, thereby achieving improved osteochondral regeneration outcomes. In this study, we modified the surface of nano-hydroxyapatite (n-HAp) through alkylation reactions to prepare n-HApMA. Characterization techniques, including X-ray diffraction, infrared spectroscopy scanning, thermogravimetric analysis, particle size analysis, and electron microscopy, were employed to analyze n-HApMA. Bioinks were prepared using n-HApMA, high porosity GelMA hydrogel, and adipose tissue derived stromal cells (ADSCs). The rheological properties of the bioinks during photocuring were investigated using a rheometer. Based on these bioinks, a biphasic scaffold was constructed. The viability of cells within the scaffold was observed using live-dead cell staining, while the internal morphology was examined using scanning electron microscopy. The stiffness of the scaffold was evaluated through compression testing. Scaffolds were implanted into the osteochondral defects of New Zealand rabbit knees, and microCT was utilized to observe the subchondral bone repair. Hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and Safranin O/Fast Green staining were performed to assess the regeneration of subchondral bone and cartilage. Furthermore, immunohistochemical staining was employed to detect the expression of osteogenic and chondrogenic-related molecules. Scaffold characterization revealed that surface modification enables the uniform distribution of n-HApMA within the GelMA matrix. The incorporation of 5% n-HApMA notably enhanced the elastic modulus and stiffness of the 6% high-porosity GelMA in comparison to n-HAp. Moreover, in-vivo study showed that the homogeneous dispersion of n-HApMA on the GelMA matrix facilitated the osteogenic differentiation of adipose-derived stem cells (ADSCs) and promoted osteochondral tissue regeneration. These findings suggest potential applications of the n-HApMA/GelMA composite in the field of tissue engineering and regenerative medicine.

Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H2O2 to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.