Expression of microRNA-892B in human mesenchymal stem cells promotes cartilage regeneration in athymic nude rat osteochondral defect model

Abstract

Purpose: Articular cartilage (AC) is poorly supplied by blood vessels, nerves, and the lymphatic system. Therefore, AC has a very limited capacity that can be repaired after an injury. Although several therapeutic measures have been developed to promote the healing of AC, no widely accepted treatment has been established. But transfection of a cocktail of genes into cells has recently been attractive to stem cell differentiation. Here, based on our previous in vitro studies, we confirm in vivo effectiveness of miR-892b for cartilage regeneration in the rat osteochondral defect model in vivo. Our strategy modulating miR-892b gene can suggest a new strategy for effective cartilage regeneration in MSC mediated tissue engineering fields. Methods: The knee joints of the male arthymic nude rat were exposed by a medial parapatellar incision, and the trochlear groove was severed due to the lateral obstruction of the patella. A 1.5 mm outer diameter trephine drill was employed to create osteochondral defects (1.5 × 3.0 mm) in the trochlear groove of the femur. The osteochondral defects was managed using one of the following methods : filling with HCF only (Group 1, n = 3), filling with TGF-â1-loaded HCF (Group 2, n = 3); and filling with lenti-control/hMSC-loaded HCF (Group 3, n = 3); and filling with lenti-control/hMSC and TGF-â1-loaded HCF (Group 4, n = 3); and filling with lenti-miR-892b/hMSC and TGF-â1-loaded HCF (Group 5, n = 3). An equal volume of thrombin solution was simultaneously injected with HCF in all groups. Each group consisted of 3 rats. After 8 weeks, the rats were sacrificed. Results: After genomic DNA PCR, a sequence correspond to pri-miR-892b was cloned into NheI-EcoRI sites of pCDH-copGFP lentiviral plasmid (Fig. 1a). As shown in Fig. 1b, lentiviral control and lenti-miR-892b transduced hMSCs were dually labeled with a far-red fluorescent dye (CellVeu) for in vivo visualization. After 8 weeks of post-surgery, this far-red fluorescent signals were only detected osteochondral defects of CellVeu labeled hMSCs transplanted groups (Fig. 1c). The ICRS macroscopic score was highest in group 5 (lenti-miR-892b-transduced hMSCs and TGF-β1: 11.0), Group 5 had significantly higher score than the other groups (Fig. 1d). Histological assessment showed that group 5 defects had good healing and covered with hyaline cartilage-like tissue. Although group 4 defects had complete healing with hyaline cartilage-like tissue, remodeling status in subchondral bone of the defects was irregular and incomplete. Defects of the rest group were mostly filled with fibrous tissue (Fig. 1f). According to the Wakitani Histological Grading Scale, group 5 was significantly lower than other group in surface, cell distribution, and subchondral bone (Fig. 1e). Conclusions: In conclusion, we previously found that miR-892b can enhance in vitro chondrogenesis of hMSCs. Here, we additionally evaluated its in vivo efficacy using rat osteochondral defect model. The macroscopic assessment of the regenerated cartilages demonstrated that the defect had become firm and smooth after 8 weeks in Groups 4, 5 while a visible defect was present in Group 1, 2 and 3. However, the reconstitution of subchondral bone was better in Group 5 than in Groups 4. The histological score was significantly better in Group 5 than in the other groups. These in vivo results confirmed that the expression of miR-892b in hMSC under TGF-β1 stinuli might promote the healing of osteochondral defects.