The Research Of The Repair Of Cartilage Defects With Bone Marrow Mesenchymal Stem Cells Teansfected With C-type Natriuretic Peptide Gene And Chitosan/silk Fibroin Scaffolds

Objective: To study the effect of the composite CNP-modified bone marrow mesenchymal stem cells (BMSCs) and chitosan/silk porous scaffolds on the repair of the articular cartilage defects in the rats so as to further explore the ways to repair cartilage defects.Methods: 1.BMSCs were isolated from Sprague-Dawley rats by whole bone marrow adherence method and were subcultured, and the specific identification and ability of multidirectional differentiation were determined. 2. The BMSCs were then transfected by recombinant adenovirus vector containing C-type natriuretic peptide gene and enhanced green fluorescent protein (Ad-NPPC-eGFP) with the specific multiplicity of infection (MOI). Flow cytometry (FCM) was used to detect the transfection efficacy,Real-time PCR was conducted to detect CNP mRNA expression, MTT assay was performed to examine the effect of transfection on cell proliferation, and alcian blue staining was performed to detect the extracellular matrix accumulation of the cells after chondrogenic differentiation in vitro for 21 days. And the expression of cartilage-related genes such as collagen II, Aggrecan and Sox9 were further detected by real-time PCR.3. Silk and chitosan were used as raw materials to develop 2% silk fibroin and 2%chitosan solution. Then the two solutions were mixed with 50/50 blending ratio, and by freeze-drying technology to obtain chitosan/silk fibrous scaffold material. The characters and porosity of the scaffold was observed by gross morphology and scanning electron microscopy (SEM). The porosity of the scaffold was calculated by volume method. The proliferation of BMSCs transfected with Ad-NPPC-eGFP was observed by fluorescence microscopy. 4. Forty eight Sprague Dawley rats were grouped into fourgroups: control group(C), Scaffolds group(S), MSC/Scaffolds group (MS), and CNP-modified MSCs/Scaffolds group (CMS). Osteochondral defects (1 mm diameter and 1 mm depth) were created and the effect of cartilage repair were observed. Group C was a natural healing group and group S was treated with chitosan/silk fibroin scaffold,group MS was treated with the composite of chitosan/silk fibroin scaffold and BMSCs.The CMS group was treated with the composite of the CNP-modified BMSCs and chitosan/silk porous scaffolds. At 4, 8 and 12 weeks post-surgery, the animals were killed. The cartilage repair results were examined by gross morphology, HE staining,and toluidine blue stain. And eventually made the histological scores.Results: 1. The rat BMSCs were successfully obtained,and their morphology was mainly spindle type, oval and polygonal and with radially, vortex-like growth. The FCM results showed high expression of mesenchymal stem cell surface markers, and without expression of hematopoietic stem cells surface markers. Osteogenic and adipogenic differentiation results also revealed a good multi-differentiation capacity. 2. Adenovirus transfected cells were in good condition, high transfection efficiency, and compared with the control group, although the proliferation decreased slightly, but the difference was not statistically significant (P>0.05). The expression of CNP mRNA in BMSCs after transfection was significant in Ad-NPPC-eGFP-transfected group (P<0.05). Alcian blue staining indicated significantly improved extracelluar matrix secretion after chondrogenic differentiation in vitro for 21 days, which was better than the groups without transfected with CNP gene (P<0.05). And the real-time PCR results futher showed that the cartilage-related genes such as collagen ?, Aggrecan, and Sox9 mRNA were in high expression (P<0.05). 3. The chitosan/silk scaffolds were milky porous materials with rough surface. The front side pores were larger and the the bottom side pores were smaller. Besides, the channels were interconnected in the interior of the scaffolds. The porosity of this chitosan/silk scaffolds was between 83% and 90%. SEM photographs reveal that the front side of scaffold was of relative uniform pores and the sizes of the apertures were 150-200 ?m. While the sizes of the apertures on the bottom side were 10-20?m. Under the green fluorescence microscope, it can be seen that the cells proliferated significantly after implantation of scaffold material for 72 hours than 12 hours. 4. The animal models of articular cartilage defects were successfully constructed. The gross morphology examination, histological staining, and histological score of repaired tissue showed that the CMS group had a better repair effect of cartilage defects than the C group, S group, and MS group (P<0.05).Conclusion: The composite of the CNP-modified BMSCs and chitosan/silk porous scaffolds has a good effect on the repair of the articular cartilage defects in the rats,which further enrich the ways of repairing of articular cartilage defects.