The Effect Of Bone Marrow Mesenchymal Stem Cells (BMSCs) On Chondrocytes Was Investigated By Tissue Engineering Method

ObjectiveTo observe the repair results of filling the empty spaces remaining after autologous osteochondral mosaicplasty with tissue-engineered cartilage or BMNC-PLGA composites.Methods(1) Groups: Both knees of 18 miniature pigs which were 7–8 months of skeletal maturity were randomly assigned to the following four groups. ① the control group: single mosaicplasty; ② the scaffold group: mosaicplasty + filling the empty spaces with cell-free PLGA scaffolds; ③ the TE(tissue engineering) group: mosaicplasty + filling the empty spaces with tissue-engineered cartilage; ④ the composite group: mosaicplasty + filling the empty spaces with BMNC-PLGA composites.(2) Creation of the defects and formation of tissue-engineered grafts: A standardized full-thickness defect(diameter, 6 mm) was created in the weight-bearing area of each medial femoral condyle in both knees of 18 miniature pigs. In the TE group, the autologous chondrocytes were harvested at the same operation of creating the defect model, and the expanded chondrocytes of Passage 2 were then seeded onto PLGA scaffolds to construct tissue-engineered cartilage. In the composite group, before masaicplasty, BMNCs were harvest by selective cell retention to construct BMNC-PLGA composites.(3) Surgical repair: The defects were initially repaired by autologous osteochondral mosaicplasty. Simultaneously, any empty spaces between the multiple plugs were filled with cell-free PLGA scaffolds(in the scaffold group), tissue-engineered cartilage(in the TE group) or BMNC-PLGA composites(in thecomposite group). The empty spaces were left untreated as control(in the control group).(4) Evaluation of repair results: Six months after surgery, the repair results were assessed via macroscopic observation, histological evaluation, magnetic resonance imaging(MRI), biomechanical assessment and glycosaminoglycan(GAG) contents.ResultsAccording to the corresponding grading of macroscopic and histological evaluation, the MOCART scores and the T2 index, the repair results of the TE group and the composite group were much higher than those of the control group or the scaffold group with statistically significant differences(P < 0.05). The compressive modulus of regenerated cartilage was 26.9 MPa in the control group, 28.3 MPa in the scaffold group, 38.1 MPa in the TE group and 36.2 MPa in the composite group, indicating that the compressive moduli of both the TE group and the composite group were much higher than those of the control group and the scaffold group and the differences were statistically significant(P < 0.05). The amount of GAG was 10.81±0.79 mg/g in the TE group and 9.49±1.26 mg/g in the composite group, with statistically significant differences(P < 0.05).ConclusionFilling the empty spaces with tissue-engineered cartilage produced a good repair result, but is time-consuming, expensive and should be a two-step operation. Filling the empty spaces with BMNC-PLGA composites could achieve a similar repair result, which is a cost-effective, time-saving and convenient performance to repair cartilage defects.ObjectiveTo investigate the mechanisms of interactions between articular chondrocytes(ACs) and bone marrow mesenchymal stem cells(BMSCs) in co-culture system.MethodsCo-culture Experiment Ⅰ(1) Groups: ① ACs cultured alone; ② BMSCs cultured alone; ③ ACs co-cultured without direct cell-cell contact; ④BMSCs co-cultured without direct cell-cell contact; ⑤mixture of ACs and BMSCs co-cultured with direct cell-cell contact; ⑥mixture of ACs and BMSCs co-cultured without direct cell-cell contact.(2) Experimental process: Rat ACs and rat BMSCs were co-cultured in vitro with or without direct cell-cell contact at the ratio of 2:1, and the samples mentioned above were harvested on day 21.(3) Evaluation: The m RNA and protein expression of SOX9, COL2, Aggrecan, COL10 and COL1 were analyzed by quantitative real-time RT-PCR and western blot.Co-culture Experiment Ⅱ(1) Groups: ①ACs cultured alone; ②ACs co-cultured without direct cell-cell contact; ③ACs co-cultured with direct cell-cell contact; ④GFP-BMSCs cultured alone; ⑤GFP-BMSCs co-cultured without direct cell-cell contact; ⑥GFP-BMSCs co-cultured with direct cell-cell contact.(2) Experimental process: Rat ACs were also co-cultured with GFP-BMSCs from transgenic rats with or without direct cell-cell contact at the ratio of 2:1. On day 21, ACs and GFP-BMSCs co-cultured with direct cell-cell contact were separated by flow cytometry, and the samples mentioned above were harvested.(3) Evaluation: The m RNA and protein expression of SOX9, COL2, Aggrecan,COL10 and COL1 were analyzed by quantitative real-time RT-PCR and western blot.Resultsm RNA levels and protein expression of SOX9, COL2 and Aggrecan were significantly up-regulated in both BMSCs and ACs co-cultured with or without direct cell-cell contact, compared with ACs and BMSCs cultured alone. m RNA levels and protein expression of SOX9, COL2 and Aggrecan in mixed cells co-cultured with direct cell-cell contact were the highest among all the six samples, and significantly higher than in mixed ACs and BMSCs co-cultured without direct cell-cell contact. SOX9, COL2 and Aggrecan m RNA levels and protein expression in ACs co-cultured with direct cell-cell contact were significantly higher than in ACs co-cultured without direct cell-cell contact. Similarly, the m RNA and protein expression of SOX9, COL2 and Aggrecan in BMSCs co-cultured with direct cell-cell contact were also significantly higher than in BMSCs co-cultured without direct cell-cell contact. After co-culture either with or without direct cell-cell contact, m RNA levels and protein expression of SOX9, COL2 and Aggrecan in BMSCs were significantly lower than those in ACs in the equivalent co-culture systems, even lower than those in ACs cultured alone.Conclusion1. The stimulating and supporting effects of BMSCs on ACs were more important in enhancing cartilage-matrix formation than the reciprocal effect of ACs on BMSCs.2. Both soluble factors and direct physical contact occur in AC/BMSC co-cultures, with physical contact playing a predominant, or at least very important role in AC/BMSC co-culture systems.