Rat Osteochondral Defect Research Articles

Acid ceramidase treatment enhances the outcome of autologous chondrocyte implantation in a rat osteochondral defect model

The overall aim of this study was to evaluate how supplementation of chondrocyte media with recombinant acid ceramidase (rhAC) influenced cartilage repair in a rat osteochondral defect model. Primary chondrocytes were grown as monolayers in polystyrene culture dishes with and without rhAC (added once at the time of cell plating) for 7 days, and then seeded onto Bio-Gide® collagen scaffolds and grown for an additional 3 days. The scaffolds were then introduced into osteochondral defects created in Sprague-Dawley rat trochlea by a microdrilling procedure. Analysis was performed 6 weeks post-surgery macroscopically, by micro-CT, histologically, and by immunohistochemistry. Treatment with rhAC led to increased cell numbers and glycosaminoglycan (GAG) production (∼2 and 3-fold, respectively) following 7 days of expansion in vitro. Gene expression of collagen 2, aggrecan and Sox-9 also was significantly elevated. After seeding onto Bio-Gide®, more rhAC treated cells were evident within 4 h. At 6 weeks post-surgery, defects containing rhAC-treated cells exhibited more soft tissue formation at the articular surface, as evidenced by microCT, as well as histological evidence of enhanced cartilage repair. Notably, collagen 2 immunostaining revealed greater surface expression in animals receiving rhAC treated cells as well. Collagen 10 staining was not enhanced. The results further demonstrate the positive effects of rhAC treatment on chondrocyte growth and phenotype in vitro, and reveal for the first time the in vivo effects of the treated cells on cartilage repair.

Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model

This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(ɛ-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.

Osteochondral tissue regeneration through polymeric delivery of DNA encoding for the SOX trio and RUNX2

Native osteochondral repair is often inadequate owing to the inherent properties of the tissue, and current clinical repair strategies can result in healing with a limited lifespan and donor site morbidity. This work investigates the use of polymeric gene therapy to address this problem by delivering DNA encoding for transcription factors complexed with the branched poly(ethylenimine)–hyaluronic acid (bPEI–HA) delivery vector via a porous oligo[poly(ethylene glycol) fumarate] hydrogel scaffold. To evaluate the potential of this approach, a bilayered scaffold mimicking native osteochondral tissue organization was loaded with DNA/bPEI–HA complexes. Next, bilayered implants either unloaded or loaded in a spatial fashion with bPEI–HA and DNA encoding for either Runt-related transcription factor 2 (RUNX2) or SRY (sex determining region Y)-box 5, 6, and 9 (the SOX trio), to generate bone and cartilage tissues respectively, were fabricated and implanted in a rat osteochondral defect. At 6weeks post-implantation, micro-computed tomography analysis and histological scoring were performed on the explants to evaluate the quality and quantity of tissue repair in each group. The incorporation of DNA encoding for RUNX2 in the bone layer of these scaffolds significantly increased bone growth. Additionally, a spatially loaded combination of RUNX2 and SOX trio DNA loading significantly improved healing relative to empty hydrogels or either factor alone. Finally, the results of this study suggest that subchondral bone formation is necessary for correct cartilage healing.

Systemic administration of granulocyte colony-stimulating factor (G-CSF) for cartilage repair in rat osteochondral defect

Systemic administration of granulocyte colony-stimulating factor (G-CSF) for cartilage repair in rat osteochondral defect

Effects of doxycycline on mesenchymal stem cell chondrogenesis and cartilage repair

Strategies to improve cartilage repair tissue quality after bone marrow cell-based procedures may reduce later development of osteoarthritis. Doxycycline is inexpensive, well-tolerated, and has been shown to reduce matrix-metalloproteinases (MMPs) and osteoarthritis progression. This study tests the hypotheses that doxycycline reduces MMP, enhances chondrogenesis of human bone marrow-derived mesenchymal stem cells (hMSC), and improves in vivo cartilage repair. Ninety hMSC pellets were cultured in chondrogenic media with either 0-, 1- or 2-μg/mL doxycycline. Pellets were evaluated with stereomicroscopy, proteoglycan assay, qRT-PCR, and histology. Osteochondral defects (OCDs) were created in the trochlear grooves of 24-Sprague-Dawley rats treated with/without oral doxycycline. Rats were sacrificed at 12-weeks and repair tissues were examined grossly and histologically. hMSC pellets with 1-μg/mL (P = 0.014) and 2-μg/mL (P = 0.002) doxycycline had larger areas than pellets without doxycycline. hMSC pellets with 2-μg/mL doxycycline showed reduced mmp-13 mRNA (P = 0.010) and protein at 21-days. Proteoglycan, DNA contents, and mRNA expressions of chondrogenic genes were similar (P > 0.05). For the in vivo study, while the histological scores were similar between the two groups (P = 0.116), the gross scores of the OCD repair tissues in doxycycline-treated rats were higher at 12-weeks (P = 0.017), reflective of improved repair quality. The doxycycline-treated repairs also showed lower MMP-13 protein (P = 0.029). This study shows that doxycycline improves hMSC chondrogenesis and decreases MMP-13 in pellet cultures and within rat OCDs. Doxycycline exerted no negative effect on multiple measures of chondrogenesis and cartilage repair. These data support potential use of doxycycline to improve cartilage repair to delay the onset of osteoarthritis.

Bioinspired nanofibers support chondrogenesis for articular cartilage repair

Articular cartilage repair remains a significant and growing clinical challenge with the aging population. The native extracellular matrix (ECM) of articular cartilage is a 3D structure composed of proteinaceous fibers and a hydrogel ground substance that together provide the physical and biological cues to instruct cell behavior. Here we present fibrous scaffolds composed of poly(vinyl alcohol) and the biological cue chondroitin sulfate with fiber dimensions on the nanoscale for application to articular cartilage repair. The unique, low-density nature of the described nanofiber scaffolds allows for immediate cell infiltration for optimal tissue repair. The capacity for the scaffolds to facilitate cartilage-like tissue formation was evaluated in vitro. Compared with pellet cultures, the nanofiber scaffolds enhance chondrogenic differentiation of mesenchymal stems cells as indicated by increased ECM production and cartilage specific gene expression while also permitting cell proliferation. When implanted into rat osteochondral defects, acellular nanofiber scaffolds supported enhanced chondrogenesis marked by proteoglycan production minimally apparent in defects left empty. Furthermore, inclusion of chondroitin sulfate into the fibers enhanced cartilage-specific type II collagen synthesis in vitro and in vivo. By mimicking physical and biological cues of native ECM, the nanofiber scaffolds enhanced cartilaginous tissue formation, suggesting their potential utility for articular cartilage repair.

Zinc‐finger protein 145, acting as an upstream regulator of SOX9, improves the differentiation potential of human mesenchymal stem cells for cartilage regeneration and repair

Human mesenchymal stem cells (hMSCs) represent one of the most promising stem cell therapies for traumatic injury and age-related degenerative diseases involving cartilage. However, few genetic factors regulating chondrogenesis of MSCs have been identified. One study showed that zinc-finger protein 145 (ZNF145), a transcription factor, was up-regulated during 3-lineage differentiation of hMSCs. The present study was undertaken to validate whether this novel transcription factor is useful for the repair and regeneration of cartilage. Human MSCs were transfected with lentiviral short hairpin RNA (for small interfering RNA knockdown of ZNF145) and a lentiviral vector for overexpression of ZNF145, and the effects of ZNF145 on chondrogenesis were studied using quantitative polymerase chain reaction and immunostaining. Microarray and transient expression analyses were used to determine whether ZNF145 is a factor operating upstream of SOX9. Allogeneic transplantation of hMSCs into osteochondral defects in rats was performed to determine the effects of ZNF145 on repair of cartilage in vivo. Small interfering RNA-mediated gene silencing of ZNF145 slowed down chondrogenesis, whereas overexpression of ZNF145 enhanced chondrogenesis. Global gene expression profiling showed up-regulated gene expression in ZNF145-overexpressing MSCs, and transient overexpression of ZNF145 enhanced the expression of SOX9, suggesting that ZNF145 acts as a factor upstream of SOX9, the master regulator of chondrogenesis. Moreover, allogeneic transplantation of hMSCs into osteochondral defects of rat knees showed that ZNF145-overexpressing MSCs repaired cartilage defects better and earlier than empty control MSCs. These findings suggest that ZNF145 gene therapy may be a very useful strategy for improving the quality of cartilage regeneration and repair.

In vivo commitment and functional tissue regeneration using human embryonic stem cell-derived mesenchymal cells

Development of clinically relevant regenerative medicine therapies using human embryonic stem cells (hESCs) requires production of a simple and readily expandable cell population that can be directed to form functional 3D tissue in an in vivo environment. We describe an efficient derivation method and characterization of mesenchymal stem cells (MSCs) from hESCs (hESCd-MSCs) that have multilineage differentiation potential and are capable of producing fat, cartilage, and bone in vitro. Furthermore, we highlight their in vivo survival and commitment to the chondrogenic lineage in a microenvironment comprising chondrocyte-secreted morphogenetic factors and hydrogels. Normal cartilage architecture was established in rat osteochondral defects after treatment with chondrogenically-committed hESCd-MSCs. In view of the limited available cell sources for tissue engineering applications, these embryonic-derived cells show significant potential in musculoskeletal tissue regeneration applications.

Blocking vascular endothelial growth factor with soluble Flt‐1 improves the chondrogenic potential of mouse skeletal muscle–derived stem cells

To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt-1 (sFlt-1), on chondrogenesis, using muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle. The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real-time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP-4) were coimplanted with MDSCs transduced to express either VEGF or sFlt-1 (a VEGF antagonist) to provide a gain- and loss-of-function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups. Hyaline-like cartilage matrix production was observed in both VEGF-treated and VEGF-blocked (sFlt-1-treated) pellet cultures, but quantitative PCR revealed that sFlt-1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP-4 and transforming growth factor beta3 (TGFbeta3). In vivo testing of articular cartilage repair showed that VEGF-transduced MDSCs caused an arthritic change in the knee joint, and sFlt-1 improved the MDSC-mediated repair of articular cartilage, compared with BMP-4 alone. Soluble Flt-1 gene therapy improved the BMP-4- and TGFbeta3-induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage.