Rabbit Osteochondral Defect Research Articles

Multifunctional polyphenol-based silk hydrogel alleviates oxidative stress and enhances endogenous regeneration of osteochondral defects

In osteochondral defects, oxidative stress caused by elevated levels of reactive oxygen species (ROS) can disrupt the normal endogenous repair process. In this study, a multifunctional hydrogel composed of silk fibroin (SF) and tannic acid (TA), the FDA-approved ingredients, was developed to alleviate oxidative stress and enhance osteochondral regeneration. In this proposed hydrogel, SF first interacts with TA to form a hydrogen-bonded supramolecular structure, which is subsequently enzymatically crosslinked to form a stable hydrogel. Furthermore, TA had multiple phenolic hydroxyl groups that formed interactions with the therapeutic molecule E7 peptide for controlled drug delivery. In vitro investigations showed that SF-TA and SF-TA-E7 hydrogels exhibited a multitude of biological effects including scavenging of ROS, maintaining cell viability, and promoting the proliferation of bone marrow mesenchymal stem cells (BMSCs) against oxidative stress. The proteomic analysis indicated that SF-TA and SF-TA-E7 hydrogels suppressed oxidative stress, which in turn improved cell proliferation in multiple proliferation and apoptosis-related pathways. In rabbit osteochondral defect model, SF-TA and SF-TA-E7 hydrogels promoted enhanced regeneration of both cartilage and subchondral bone as compared to hydrogel without TA incorporation. These findings indicated that the multifunctional SF-TA hydrogel provided a microenvironment suitable for the endogenous regeneration of osteochondral defects.

Platelet-Derived Growth Factor-Functionalized Scaffolds for the Recruitment of Synovial Mesenchymal Stem Cells for Osteochondral Repair.

Cartilage regeneration is still a challenge for clinicians because of avascularity, denervation, load-bearing, synovial movement, and the paucity of endogenous repair cells. We constructed a multilayered osteochondral bionic scaffold and examined its repair capacity using a rabbit osteochondral defect model. The cartilage phase and interface layer of the scaffold were prepared by freeze-drying, whereas the bone phase of the scaffold was prepared by high-temperature sintering. The three-phase osteochondral bionic scaffold was formed by joining the hydroxyapatite (HAp) and silk fibroin (SF) scaffolds using the repeated freeze-thaw method. Different groups of scaffolds were implanted into the rabbit osteochondral defect model, and their repair capacities were assessed using imaging and histological analyses. The cartilage phase and the interface layer of the scaffold had a pore size of 110.13 ± 29.38 and 96.53 ± 33.72 μm, respectively. All generated scaffolds exhibited a honeycomb porous structure. The polydopamine- (PDA-) modified scaffold released platelet-derived growth factor (PDGF) for 4 weeks continuously, reaching a cumulative release of 71.74 ± 5.38%. Synovial mesenchymal stem cells (SMSCs) adhered well to all scaffolds, but demonstrated the strongest proliferation ability in the HSPP (HAp-Silk-PDA-PDGF) group. Following scaffold-induced chondrogenic differentiation, SMSCs produced much chondrocyte extracellular matrix (ECM). In in vivo experiments, the HSPP group exhibited a significantly higher gross tissue morphology score and achieved cartilage regeneration at an earlier stage and a significantly better repair process compared with the other groups (P < 0.05). Histological analysis revealed that the new cartilage tissue in the experimental group had a better shape and almost filled the defect area, whereas the scaffold was nearly completely degraded. The new cartilage was effectively fused with the surrounding normal cartilage, and a substantial amount of chondrocyte ECM was formed. The SF/HAp three-layer osteochondral bionic scaffold exhibited favorable pore size, porosity, and drug sustained-release properties. It demonstrated good biocompatibility in vitro and encouraging repair effect at osteochondral defect site in vivo, thereby expected to enabling the repair and regeneration of osteochondral damage.

Type I collagen scaffold with WNT5A plasmid for in situ cartilage tissue engineering.

Cartilage tissue lacks the ability to heal. Cartilage tissue engineering using cell-free scaffolds has been increasingly used in recent years. This study describes the use of a type I collagen scaffold combined with WNT5A plasmid to promote chondrocyte proliferation and differentiation in a rabbit osteochondral defect model. Type I collagen was extracted and fabricated into a collagen scaffold. To improve gene transfection efficiency, a cationic chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) vector was used. A solution of TMC/WNT5A complexes was adsorbed to the collagen scaffold to prepare a WNT5A scaffold. Osteochondral defects were created in the femoral condyles of rabbits. The rabbits were divided into defect, scaffold, and scaffold with WNT5A groups. At 6 and 12 weeks after creation of the osteochondral defects, samples were collected from all groups for macroscopic observation and gene expression analysis. Samples from the defect group exhibited incomplete cartilage repair, while those from the scaffold and scaffold with WNT5A groups exhibited "preliminary cartilage" covering the defect. Cartilage regeneration was superior in the scaffold with WNT5A group compared to the scaffold group. Safranin O staining revealed more proteoglycans in the scaffold and scaffold with WNT5A groups compared to the defect group. The expression levels of aggrecan, collagen type II, and SOX9 genes were significantly higher in the scaffold with WNT5A group compared to the other two groups. Type I collagen scaffold showed effective adsorption and guided the three-dimensional arrangement of stem cells. WNT5A plasmid promoted cartilage repair by stimulating the expression of aggrecan, type II collagen, and SOX9 genes and proteins, as well as inhibiting cartilage hypertrophy.

Hyaluronan size alters chondrogenesis of mesenchymal stem cells cultured on tricalcium phosphate-collagen-hyaluronan scaffolds.

Hyaluronan (HA) provides a favorable environment for chondrogenesis of bone marrow mesenchymal stem cells (BMSCs). A previous report from our group indicated that addition of HA increases the chondro-inductive capacity of scaffolds. Therefore, this study aimed to investigate whether the Mw of the HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. Human BMSCs (hBMSCs) and rabbit BMSCs (rBMSCs) were isolated and expanded. TCP-COL scaffolds and TCP-COL-HA scaffolds with two different HA Mws were assessed for their capacity to induce cartilage regeneration from hBMSCs in vitro and in vivo. The results showed that about 96.96% of hBMSCs expressed CD44. Moreover, Hyal-1 and chondrogenic marker genes expressions were increased in hMSCs seeded on TCP-COL-HA scaffolds, and blocking the HA-CD44 interaction with an anti-CD44 antibody reduced the expression levels of Hyal-1 and chondrogenic marker genes. Additionally, TCP-COL-HA scaffolds with 2000 kDa Mw showed greater induction of BMSC chondrogenesis induction compared with those with 80 kDa Mw. Similar results were observed in an ectopic implantation nude mouse model. In a rabbit osteochondral defect repair model, rBMSCs seeded on TCP-COL-HA scaffolds with 2000 kDa Mw showed greater cartilage regeneration than those seeded with 80 kDa Mw. In addition, hBMSC-seeded TCP-COL-HA scaffolds with 2000 kDa Mw showed a significantly higher mechanical strength than those with 80 kDa Mw. Collectively, these results indicate that the Mw of HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. The TCP-COL-HA scaffolds might be used as allogenic off the shelf products in cartilage tissue engineering in future.

Hydrogel-hydroxyapatite-monomeric collagen type-I scaffold with low-frequency electromagnetic field treatment enhances osteochondral repair in rabbits

BackgroundCartilage damage is a common medical issue in clinical practice. Complete cartilage repair remains a significant challenge owing to the inferior quality of regenerative tissue. Safe and non-invasive magnetic therapy combined with tissue engineering to repair cartilage may be a promising breakthrough.MethodsIn this study, a composite scaffold made of Hydroxyapatite-Collagen type-I (HAC) and PLGA-PEG-PLGA thermogel was produced to match the cartilage and subchondral layers in osteochondral defects, respectively. Bone marrow mesenchymal stem cells (BMSC) encapsulated in the thermogel were stimulated by an electromagnetic field (EMF). Effect of EMF on the proliferation and chondrogenic differentiation potential was evaluated in vitro. 4 mm femoral condyle defect was constructed in rabbits. The scaffolds loaded with BMSCs were implanted into the defects with or without EMF treatment. Effects of the combination treatment of the EMF and composite scaffold on rabbit osteochondral defect was detected in vivo.ResultsIn vitro experiments showed that EMF could promote proliferation and chondrogenic differentiation of BMSCs partly by activating the PI3K/AKT/mTOR and Wnt1/LRP6/β-catenin signaling pathway. In vivo results further confirmed that the scaffold with EMF enhances the repair of osteochondral defects in rabbits, and, in particular, cartilage repair.ConclusionHydrogel-Hydroxyapatite-Monomeric Collagen type-I scaffold with low-frequency EMF treatment has the potential to enhance osteochondral repair.

Chitosan coatings with distinct innate immune bioactivities differentially stimulate angiogenesis, osteogenesis and chondrogenesis in poly-caprolactone scaffolds with controlled interconnecting pore size

This study tested whether osseous integration into poly (ε-caprolactone) (PCL) bioplastic scaffolds with fully-interconnecting 155 ± 8 μm pores is enhanced by an adhesive, non-inflammatory 99% degree of deacetylation (DDA) chitosan coating (99-PCL), or further incorporation of pro-inflammatory 83% DDA chitosan microparticles (83-99-PCL) to accelerate angiogenesis. New Zealand White rabbit osteochondral knee defects were press-fit with PCL, 99-PCL, 83-99-PCL, or allowed to bleed (drill-only). Between day 1 and 6 weeks of repair, drill-only defects repaired by endochondral ossification, with an 8-fold higher bone volume fraction (BVF) versus initial defects, compared to a 2-fold (99-PCL), 1.1-fold (PCL), or 0.4-fold (83-99-PCL) change in BVF. Hematoma innate immune cells swarmed to 83-99-PCL, elicited angiogenesis throughout the pores and induced slight bone resorption. PCL and 99-PCL pores were variably filled with cartilage or avascular mesenchyme near the bone plate, or angiogenic mesenchyme into which repairing trabecular bone infiltrated up to 1 mm deep. More repair cartilage covered the 99-PCL scaffold (65%) than PCL (18%) or 83-99-PCL (0%) (p < 0.005). We report the novel finding that non-inflammatory chitosan coatings promoted cartilage infiltration into and over a bioplastic scaffold, and were compatible with trabecular bone integration. This study also revealed that in vitro osteogenesis assays have limited ability to predict osseous integration into porous scaffolds, because (1) in vivo, woven bone integrates from the leading edge of regenerating trabecular bone and not from mesenchymal cells adhering to scaffold surfaces, and (2) bioactive coatings that attract inflammatory cells induce bone resorption.

Cartilage tissue engineering by co-transplantation of chondrocyte extracellular vesicles and mesenchymal stem cells, entrapped in chitosan–hyaluronic acid hydrogel

Mesenchymal stem cells (MSCs) on injectable hydrogels are mostly used to regenerate articular cartilage, which would have a variety of outcomes. Chondrocyte extracellular vesicles (EVs) have attracted many attentions for their chondrogenic differentiation capacity; however, the roles of EVs in both chondrogenic differentiation of MSCs and cartilage regeneration are poorly understood yet. In the current study, to investigate the differentiation effects of human articular chondrocyte EVs on adipose-derived MSCs, they were cultured in injectable chitosan–hyaluronic acid (CS–HA) hydrogel and then treated with chondrocyte EVs for 21 days. The continuous treatment of EVs performed on MSCs increased chondrogenic genes’ expressions of SOX9 and COL2A1 and induced expression of Col II protein. In addition, glycosaminoglycans secretion was detected in the EV-treated MSCs after about 14 days. The therapeutic efficiency of this hydrogel and EVs was studied in a rabbit osteochondral defect model. MRI results revealed that the cartilage regeneration capacity of EV-treated MSCs with CS–HA hydrogel was greater than the untreated MSCs or the EV-treated MSCs without hydrogel. Moreover, histological results showed hyaline-like cartilage in the CS–HA/MSC and CS–HA/EV/MSC groups in the cartilage defect sites. These findings suggested that the chondrocyte-EVs and CS–HA hydrogel could provide the preferable niche for chondrogenic differentiation of MSCs and cartilage regeneration in osteoarthritis cartilage injuries.

Intra-articular Injection of Bevacizumab Enhances Bone Marrow Stimulation–Mediated Cartilage Repair in a Rabbit Osteochondral Defect Model

Background: Bone marrow stimulation (BMS) via microfracture historically has been a first-line treatment for articular cartilage lesions. However, BMS has become less favorable because of resulting fibrocartilage formation. Previous studies have shown that angiogenesis blockade promotes cartilage repair. Bevacizumab is a Food and Drug Administration–approved medication used clinically to prevent angiogenesis. Hypothesis: The intra-articular injection of bevacizumab would prevent angiogenesis after BMS and lead to improved cartilage repair with more hyaline-like cartilage. Study Design: Controlled laboratory study. Methods: The dose of bevacizumab was first optimized in a rabbit osteochondral defect model with BMS. Then, 48 rabbits (n = 8/group/time point) were divided into 3 groups: osteochondral defect (defect), osteochondral defect + BMS (BMS group), and osteochondral defect + BMS + bevacizumab intra-articular injection (bevacizumab group). Rabbits were sacrificed at either 6 or 12 weeks after surgery. Three-dimensional (3D) micro–computed tomography (microCT), macroscope score, modified O’Driscoll histology scores, collagen type 2, Herovici staining, and hematoxylin and eosin staining were performed. Angiogenesis markers were also evaluated. Results: The intra-articular dose of 12.5 mg/0.5 mL bevacizumab was found to be effective without deleteriously affecting the subchondral bone. Intra-articular injection of bevacizumab resulted in significantly improved cartilage repair for the bevacizumab group compared with the BMS or the defect group based on 3D microCT, the macroscope score (both P < .05), the modified O’Driscoll histology score (P = .0034 and P = .019 vs defect and BMS groups, respectively), collagen type 2, Herovici staining, and hematoxylin and eosin staining at 6 weeks. Cartilage in the bevacizumab group had significantly more hyaline cartilage than did that in other groups. At 12 weeks, the cartilage layer regenerated in all groups; however, the bevacizumab group showed more hyaline-like morphology, as demonstrated by microCT, histology scores (P < .001 and .0225 vs defect and BMS groups, respectively), histology, and immunohistochemistry. The bevacizumab injection did not significantly change mRNA expressions of smooth muscle actin, vascular endothelial growth factor, or hypoxia-inducible factor-1 alpha. Conclusion: Intra-articular injection of bevacizumab significantly enhanced the quality and quantity of hyaline-like cartilage after BMS in a rabbit model. Future large-animal and human studies are necessary to evaluate the clinical effect of this therapy, which may lead to improved BMS outcomes and thus the durability of the regenerated cartilage. Clinical Relevance: The use of bevacizumab may be an important clinical adjunct to improve BMS-mediated cartilage repair.

Continued sustained insulin-releasing PLGA nanoparticles modified 3D-Printed PCL composite scaffolds for osteochondral repair

• We have successfully fabricated the insulin-PLGA/PDA/PCL composite scaffolds. • The scaffolds can provide a slow, sustained release of insulin. • The scaffolds stimulated chondrocytes proliferation and BMSCs differentiation. • The scaffolds enhanced bone and cartilage repair in vivo. Due to the poor ability of self-repair and regeneration, articular cartilage and subchondral bone defects and degradation are still a big problem nowadays. The aim of the present study is to fabricate and investigate a 3D printed polycaprolactone (PCL) scaffold modified by insulin-releasing PLGA nanoparticles for osteochondral repair. The double-emulsion solvent evaporation method is used to fabricate the insulin-coated PLGA nanoparticles, and the physicochemical characterization of the nanoparticles and scaffolds are tested. The cell studies in vitro and animal experiments in vivo are also investigated. The results show the hydrophilicity of the scaffolds is highly improved when coated with polydopamine (PDA), and the insulin release curve of the insulin-PLGA/PDA/PCL scaffolds exhibit a slowly and sustained profile after an initial burst release. The in vitro studies show that the insulin-PLGA/PDA/PCL scaffolds significantly improve the osteogenic differentiation of rabbit bone mesenchymal stem cells (rBMSCs) and the proliferation of chondrocytes. And the in vivo studies show that the insulin-PLGA/PDA/PCL scaffolds significantly improve repair of cartilage and subchondral bone after 8- and 12-week implantation in rabbit osteochondral defects. These findings exhibit that the insulin-PLGA/PDA/PCL scaffolds can provide a slowly, sustained release of insulin and may be a promising strategy for osteochondral repair.

Development of a decellularized porcine bone graft by supercritical carbon dioxide extraction technology for bone regeneration.

A series of novel decellularized porcine collagen bone graft (DPB) materials in a variety of shapes and sizes were developed by the supercritical carbon dioxide (SCCO2 ) extraction technique. The complete decellularization of DPB was confirmed by hematoxylin and eosin staining, 4,6-diamidino-2-phenylindole (DAPI) staining, and residual DNA analysis. The native intact collagen remained in the DPB after the SCCO2 process was confirmed by Masson trichrome staining. The physicochemical characteristics of DPB were investigated by scanning electron microscopy and x-ray diffraction. The cytotoxicity and biocompatibility tests according to ISO10993 and its efficacy for bone regeneration in osteochondral defects in rabbits were evaluated. The rabbit pyrogen test confirmed DPB was non-toxic. In vitro and in vivo biocompatibility tests of the DPB did not show any toxic or mutagenic effects. The bone regeneration potential of the DPB presented no significant histological differences compared to commercially available deproteinized bovine bone. In conclusion, DPB produced by SCCO2 exhibited similar chemical characteristics to human bone, no toxicity, good biocompatibility, and enhanced bone regeneration in rabbits comparable to that of deproteinized bovine bone. Results from this study could shed light on the potential application of the SCCO2 extraction technique to generate a native decellularized scaffold for bone tissue regeneration in human clinical trials.

Injectable Ultrasonication-Induced Silk Fibroin Hydrogel for Cartilage Repair and Regeneration.

Articular cartilage lacks both a nutrient supply and progenitor cells. Once damaged, it has limited self-repair capability. Cartilage tissue engineering provides a promising strategy for regeneration, and the use of injectable hydrogels as scaffolds has recently attracted much attention. Silk fibroin (SF) is an advanced natural material used to construct injectable hydrogels that are nontoxic and can be used efficiently in crosslinking applications. The objective of the present work was to develop an injectable hydrogel using SF in a novel one-step ultrasonication crosslinking method. Gelation kinetics and the characteristics of ultrasonication-induced SF (US-SF) hydrogels were systematically evaluated. The cytocompatibility of US-SF hydrogels was evaluated using rabbit chondrocytes, the Cell Counting Kit-8 testing, and immunofluorescence staining. Furthermore, the in vivo cartilage regenerative ability of US-SF hydrogels was confirmed following subcutaneous administration in nude mice and in situ injections in rabbit osteochondral defect models. These results suggest that US-SF hydrogels could be potential candidates for cartilage repair and regeneration. Impact statement Injectable silk fibroin hydrogel is a promising strategy for cartilage tissue engineering. The transition from solution state to gel state can be fabricated by both physical and chemical methods. However, the complexing protocol and toxicity of these methods remain hindrances to further application. In this study, a one-step ultrasonication method was developed. The novel ultrasonication-induced silk fibroin hydrogel showed satisfactory physicochemical and biomechanical properties. In vitro and in vivo experiments proved that it could promote cartilage regeneration, indicating that it may be a potential solution for cartilage repair and regeneration.

Decellularized xenogenic cartilage extracellular matrix (ECM) scaffolds for the reconstruction of osteochondral defects in rabbits.

The use of decellularized native allogenic or xenogenic cartilaginous extracellular matrix (ECM) biomaterials is widely expanding in the fields of tissue engineering and regenerative medicine. In this study, we aimed to develop an acellular, affordable, biodegradable, easily available goat conchal cartilaginous ECM derived scaffolding biomaterial for repair and regeneration of osteochondral defects in rabbits. Cartilages harvested from freshly collected goat ears were decellularized using chemical agents, namely, hypotonic-hypertonic (HH) buffer and Triton X-100 solution, separately. The morphologies and ultrastructure orientations of the decellularized cartilages remained unaltered in spite of complete cellular loss. Furthermore, when the acellular cartilaginous ECMs were cultured with murine mesenchymal stem cells (MSCs) (C3H10T1/2 cells), cellular infiltration and proliferation were thoroughly monitored using SEM, DAPI and FDA stained images, whereas the MTT assay proved the biocompatibility of the matrices. The increasing amounts of secreted ECM proteins (collagen and sGAG) indicated successful chondrogenic differentiation of the MSCs in the presence of the treated cartilage samples. In vivo biocompatibility studies showed no significant immune response or tissue rejection in the treated samples but tissue necrosis in control samples after 3 months. Upon implantation of the constructs in rabbits' osteochondral defects for 3 months, the histological and micro-CT evaluation revealed significant enhancement and regeneration of neocartilage and subchondral bony tissues. The IGF-1 loaded cartilaginous constructs showed comparatively better healing response after 3 months. Our results showed that decellularized xenogenic cartilaginous biomaterials preserved the bioactivity and integrity of the matrices that also favored in vitro stem cell proliferation and chondrogenic differentiation and enabled osteochondral regeneration, thus paving a new way for articular cartilage reconstruction.

Evaluation of osteochondral-like tissues using human freeze-dried cancellous bone and chondrocyte sheets to treat osteochondral defects in rabbits.

Human freeze-dried cancellous bone combined with human chondrocyte sheets have recently been used to construct an osteochondral-like tissue, which resembled a cartilage layer on a subchondral bone layer. Nevertheless, the efficacy of these human tissues in a xenogeneic model has been rarely reported. Therefore, this study aimed to evaluate the potential of human freeze-dried cancellous bones combined with human chondrocyte sheets for the treatment of osteochondral defects in rabbits. The key roles of the extracellular matrix (ECM) and released cytokines in these tissues in osteochondral repair were also assessed. Triple-layered chondrocyte sheets were constructed using a temperature-responsive culture surface. Then, they were placed onto cancellous bone to form chondrocyte sheet-cancellous bone tissues. The immunostaining of collagen type II (COL2) and the proteomic analysis of the human tissues were carried out before the transplantation. In our in vitro study, the triple-layered chondrocyte sheets adhered well on the cancellous bone, and the COL2 expression was apparent throughout the tissue structures. From the proteomic analysis results, it was found that the major function of the secreted proteins found in these tissues was protein binding. The distinct pathways were focal adhesion and the ECM-receptor interaction pathways. Among the highly expressed proteins, laminin-alpha 5 (LAMA5) and fibronectin (FN) not only played roles in the protein binding and ECM-receptor interaction, but also were involved in the cytokine-mediated signaling pathway. At 12 weeks after xenogeneic transplantation, compared to the control group, the defects treated with the chondrocyte sheets showed more hyaline-like cartilage tissue, as indicated by the abundance of safranin-O and COL2 with a partial collagen type I (COL1) expression. At 4, 8, and 12 weeks, compared to the defects treated with the cancellous bone, the staining of safranin-O and COL2 was more apparent in the defects treated with the chondrocyte sheet-cancellous bone tissues. Therefore, the human chondrocyte sheets and chondrocyte sheet-cancellous bone tissues provide a potential treatment for rabbit femoral condyle defect. LAMA5 and FN found in these human xenografts and their culture media might play key roles in the ECM-receptor interaction and might be involved in the cytokine-mediated signaling pathway during tissue repair.

Cell-Free Biomimetic Scaffold with Cartilage Extracellular Matrix-Like Architectures for In Situ Inductive Regeneration of Osteochondral Defects.

The development of a biomimetic scaffold designed to provide a native extracellular matrix (ECM)-like microenvironment is a potential strategy for cartilage repair. The ECM in native articular cartilage is structurally composed of three different architectural zones, i.e., horizontally aligned, randomly arranged, and vertically aligned collagen fibers. However, the effects of scaffolds with these three different ECM-like architectures on in vivo cartilage regeneration are not clear. In this study, we aim to systematically investigate and compare their in situ inductive regenerative efficacy on cartilage defects. ECM-mimetic silk fibroin scaffolds with horizontally aligned, vertically aligned, and random pore architectures are fabricated using the controlled directional freezing technique. All of these scaffolds exhibit similar pore area, swelling ratio, and in vitro degradation behavior. Nevertheless, the aligned scaffolds have a higher pore aspect ratio and hydrophilicity, and increase the proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. When implanted into rabbit osteochondral defects, the scaffold with vertically aligned pore architectures provides a more cell-favorable microenvironment conducive to endogenous BMSCs than other scaffolds and supports the simultaneous regeneration of cartilage and subchondral bone. These findings indicate that scaffolds with vertically aligned ECM-like architectures serve as an effective cell-free and growth factor-free scaffold for enhanced endogenous osteochondral regeneration.

Extracellular matrix derived from allogenic decellularized bone marrow mesenchymal stem cell sheets for the reconstruction of osteochondral defects in rabbits

Bioactive scaffolds from synthetical polymers or decellularized cartilage matrices have been widely used in osteochondral regeneration. However, the risks of potential immunological reactions and the inevitable donor morbidity of these scaffolds have limited their practical applications. To address these issues, a biological extracellular matrix (ECM) scaffold derived from allogenic decellularized bone marrow mesenchymal stem cell (BMSC) sheets was established for osteochondral reconstruction. BMSCs were induced to form cell sheets. Three different concentrations of sodium dodecyl sulfate (SDS), namely, 0.5%, 1%, and 3%, were used to decellularize these BMSC sheets to prepare the ECM. Histological and microstructural observations were performed in vitro and then the ECM scaffolds were implanted into osteochondral defects in rabbits to evaluate the repair effect in vivo. Treatment with 0.5% SDS not only efficiently removed BMSCs but also successfully preserved the original structure and bioactive components of the ECM When compared with the 1% and 3% SDS groups, histological observations substantiated the superior repair effect of osteochondral defects, including the simultaneous regeneration of well-vascularized subchondral bone and avascular articular cartilage integrated with native tissues in the 0.5% SDS group. Moreover, RT-PCR indicated that ECM scaffolds could promote the osteogenic differentiation potential of BMSCs under osteogenic conditions while increasing the chondrogenic differentiation potential of BMSCs under chondrogenic conditions. Allogenic BMSC sheets decellularized with 0.5% SDS treatment increased the recruitment of BMSCs and significantly improved the regeneration of osteochondral defects in rabbits, thus providing a prospective approach for both articular cartilage and subchondral bone reconstruction with cell-free transplantation.

An interleukin-4-loaded bi-layer 3D printed scaffold promotes osteochondral regeneration

Multilayer scaffolds fabricated by 3D printing or other techniques have been used to repair osteochondral defects. However, it remains a challenge to regenerate the articular cartilage and subchondral bone simultaneously with higher performance. In the present study, we enhanced the repair efficiency of osteochondral defects by developing a bi-layer scaffold: an interleukin-4 (IL-4)-loaded radially oriented gelatin methacrylate (GelMA) scaffold printed with digital light processing (DLP) in the upper layer and a porous polycaprolactone and hydroxyapatite (PCL-HA) scaffold printed with fused deposition modeling (FDM) in the lower layer. An in vitro test showed that both layers supported cell adhesion and proliferation, as the lower layer promoted osteogenic differentiation and the upper layer with IL-4 relieved the negative effects of inflammation on murine chondrocytes, which were induced by interleukin-1β (IL-1β) and M1 macrophages. In a rabbit osteochondral defect repair model, the IL-4-loaded bi-layer scaffold group obtained the highest histological score (24 ± 2) compared to the nontreated (11 ± 1) and pure bi-layer scaffold (16 ± 1) groups after 16 weeks of implantation, which showed that the IL-4-loaded bi-layer scaffold promoted regeneration of both cartilage and subchondral bone with increased formation of neocartilage and neobone tissues. Thus, the IL-4-loaded bi-layer scaffold is an attractive candidate for repair and regeneration of osteochondral defects.

Human early mesenchymal stromal cells delivered on porous lightweight biodegradable polycaprolactone-based microcarriers result in improved cartilage formation

Porous lightweight polycaprolactone (LPCL)-based microcarriers (MC) can be used for large-scale production of human mesenchymal stromal cells (MSC) in stirred bioreactors. These biodegradable MC can serve in delivering MSC, which exhibit improved bone healing in a rat calvarial defect model. Therefore, this study aims to explore their use as scaffolds to create chondrogenically differentiated, MSC-laden LPCL MC constructs for cartilage formation in vitro and in vivo in a rabbit osteochondral defect model. In-vitro studies, defining critical parameters that resulted in efficient chondrogenic differentiation of MSC expanded on the surface of LPCL MC were performed. The optimized MSC-LPCL MC constructs were transplanted in a rabbit osteochondral defect model and evaluated after 5 months, with histological staining and scoring. Our in vitro studies demonstrate that about 4.7 × 103 porous LPCL MC seeded with 50 × 103 cells at early exponential growth phase (21% cell confluency) resulted in the best MSC-PCL MC construct, with efficient cell growth and chondrogenic differentiation, after 21 days. Implantation of chondrogenically differentiated MSC-LPCL MC constructs resulted in the best cartilage healing outcomes: (i) 75% of samples (6 out 8 defects) displaying good healing outcomes, (ii) the greatest histological score at 22 ± 7 and 26 ± 7 (without and with subchondral bone evaluation respectively), and (iii) the highest mean score for 8 out of 12 categories, as compared to other transplant groups. This is the first study demonstrating that chondrogenically differentiated MSC-LPCL MC constructs can induce the most efficient cartilage healing in rabbits with an osteochondral defect.

Hydrogel Containing Anti-CD44-Labeled Microparticles, Guide Bone Tissue Formation in Osteochondral Defects in Rabbits.

Hydrogels are suitable for osteochondral defect regeneration as they mimic the viscoelastic environment of cartilage. However, their biomechanical properties are not sufficient to withstand high mechanical forces. Therefore, we have prepared electrospun poly-ε-caprolactone-chitosan (PCL-chit) and poly(ethylene oxide)-chitosan (PEO-chit) nanofibers, and FTIR analysis confirmed successful blending of chitosan with other polymers. The biocompatibility of PCL-chit and PEO-chit scaffolds was tested; fibrochondrocytes and chondrocytes seeded on PCL-chit showed superior metabolic activity. The PCL-chit nanofibers were cryogenically grinded into microparticles (mean size of about 500 µm) and further modified by polyethylene glycol–biotin in order to bind the anti-CD44 antibody, a glycoprotein interacting with hyaluronic acid (PCL-chit-PEGb-antiCD44). The PCL-chit or PCL-chit-PEGb-antiCD44 microparticles were mixed with a composite gel (collagen/fibrin/platelet rich plasma) to improve its biomechanical properties. The storage modulus was higher in the composite gel with microparticles compared to fibrin. The Eloss of the composite gel and fibrin was higher than that of the composite gel with microparticles. The composite gel either with or without microparticles was further tested in vivo in a model of osteochondral defects in rabbits. PCL-chit-PEGb-antiCD44 significantly enhanced osteogenic regeneration, mainly by desmogenous ossification, but decreased chondrogenic differentiation in the defects. PCL-chit-PEGb showed a more homogeneous distribution of hyaline cartilage and enhanced hyaline cartilage differentiation.

Effect of Platlate Riched Fibrin and mononuclear cells on regeneration of osteochondral defect in rabbits

Objective: To assess the effectiveness of Platlate riched fibrin (PRF) and bone marrow derived mononuclear cells (BMNCs) in regeneration of osteochondral defects in rabbits. Design: A randomized-controlled experimental study. Animals: Forty-eight adult New Zealand rabbits were allocated randomly into four groups (n=12). Procedures: An osteochondoral defect of a 4 mm diameter and 5 mm depth was made in the trochlear groove of the left stifle joints. The defect was left for spontaneous healing in group A, filled with Platlate riched fibrin (PRF) in group B, filled with BMNCs in group C and combination of PRF and BMNCs in group D. Healing of the defects was assessed grossly and microscopically at 3, 6 and 12 weeks postoperative. Results: Grossly, the degree of defect repair, integration to border zone and appearance of defect area were significantly higher in group D than other groups (P≤0.05). Microscopically, surface architecture, tissue morphology, cell distribution and safranin O staining of the matrix were significantly higher in group D than other groups (P≤0.05). Conclusion and clinical relevance: The results of the present study indicated that, combination of PRF and BMNCs encourages quicker and better healing of osteochondral defects.

An all-silk-derived functional nanosphere matrix for sequential biomolecule delivery and in situ osteochondral regeneration

Endogenous repair of osteochondral defect is usually limited by the insufficient number of cells in the early stage and incomplete cell differentiation in the later stage. The development of drug delivery systems for sequential release of pro-migratory and pro-chondrogenic molecules to induce endogenous bone marrow-derived mesenchymal stem cells (BMSCs) recruitment and chondrogenic differentiation is highly desirable for in situ osteochondral regeneration. In this study, a novel, all-silk-derived sequential delivery system was fabricated by incorporating the tunable drug-loaded silk fibroin (SF) nanospheres into a SF porous matrix. The loading efficiency and release kinetics of biomolecules depended on the initial SF/polyvinyl alcohol (PVA) concentrations (0.2%, 1% and 5%) of the nanospheres, as well as the hydrophobicity of the loaded molecules, resulting in controllable and programmed delivery profiles. Our findings indicated that the 5% nanosphere-incorporated matrix showed a rapid release of E7 peptide during the first 120 h, whereas the 0.2% nanosphere-incorporated matrix provided a slow and sustained release of Kartogenin (KGN) longer than 30 days. During in vitro culture of BMSCs, this functional SF matrix incorporated with E7/KGN nanospheres showed good biocompatibility, as well as enhanced BMSCs migration and chondrogenic differentiation through the release of E7 and KGN. Furthermore, when implanted into rabbit osteochondral defect, the SF nanosphere matrix with sequential E7/KGN release promoted the regeneration of both cartilage and subchondral bone. This work not only provided a novel all-silk-derived drug delivery system for sequential release of molecules, but also a functional tissue-engineered scaffold for osteochondral regeneration.