ObjectiveTo study the preparation method of acellular dermal matrix (ADM) for cartilage tissue engineering and analyze its biocompatibility. MethodsThe dermal tissues of the calf back were harvested, and decelluarized with 0.5% SDS, and the ADM was reconstructed with 0.5% trypsin, cross-linked with formaldehyde, and modified with 0.5% chondroitin sulfate which can promote the proliferation of chondrocytes. And the porosity, cytotoxicity, and biocompatibility were determined. Co-cultured 2nd passage chondrocytes and bone marrow stromal cells in a proportion of 3 to 7 were used as seed cells. The cells were seeded on ADM (experimental group) for 48 hours to observe the cell adhesion. The expressions of mRNA and protein of collagen type Ⅱ were tested by RT-PCR and Western blot methods, respectively. And the expressions were compared between the cells seeded on the scaffold and cultured in monolayer (control group). ResultsAfter modification of 0.5% trypsin, the surface of ADM was smooth and had uniform pores; the porosity (85.4%±2.8%) was significantly higher than that without modification (72.8%±5.8%) (t=-4.384, P=0.005). The cell toxicity was grade 1, which accords to the requirements for cartilage tissue engineering scaffolds. With time passing, the number of inflammatory cells decreased after implanted in the back of the rats (P<0.05). The scanning electron microscope observation showed that lots of seed cells adhered to the scaffold, the cells were well stacked, displaying surface microvilli and secretion. The expressions of mRNA and protein of collagen type Ⅱ were not significantly different between experimental and control groups (t=1.265, P=0.235;t=0.935, P=0.372). ConclusionThe ADM prepared by acellular treatment, reconstruction, cross-linking, and modification shows perfect characters. And the seed cells maintain chondrogenic phenotype on the scaffold. So it is a proper choice for cartilage tissue engineering.
Bone marrow-derived mesenchymal stem cells (BMSCs) for repairing damaged heart tissue are a new kind of important treatment options because of their potential to differentiate into cardiomyocytes. We in this experiment investigated the effect of different electrical stimulation time on the expression of myocardial specificity gene and protein in rat bone marrow mesenchymal stem cells (rBMSCs) in vitro. The rBMSCs of second or third generation were randomly divided into three groups, i.e. electrical stimulation (ES) group, 5-Azacytidine (5-Aza) group and the control group. The rBMSCs in the ES groups with complete medium were exposed to 2 V, 2 Hz, 5 ms electrical stimulation for 0.5 h, 2 h, 4 h, and 6 h respectively every day for 10 days. Those in the 5-Aza group were induced by 5-Aza (10 μmol/L) for 24 h, and then cultured with complete medium for 10 days. Those in the control group were only cultured with complete medium, without any treatment, for 10 days. The rBMSCs' morphological feature in each group was observed with inverted phase microscope. The mRNA expression of myocyte-specific enhancer factor 2C (MEF-2C) and connexin 43 (Cx43) were examined with Real-Time quantitative PCR and the protein expression of MEF-2C, Cx43 were detected with Western Blot method. The results showed that the mRNA expression level of the MEF-2C, Cx43 and the protein expression level of MEF-2C, Cx43 were significantly higher in the ES group and 5-Aza group than those in the relative control group (P < 0.05). It suggests that electrical stimulation could play a part of role in the induction of the rBMSCs to differentiate into the cariomyocyte-like cells in vitro and the effectiveness of the electrical stimulation with 2 h/d had the best in our experiement. But the mechanism how electrical stimulation promotes the differentiation of rBMSC into cardiomyocyte is still unclear.