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The aim of the present study was to construct functional tissue-engineered bone with cell sheet technology and compare the efficacy of this method with that of traditional bone tissue engineering techniques. Canine bone mesenchymal stem cells (BMSCs) were isolated using density gradient centrifugation and then cultured. The BMSCs were induced to differentiate into osteoblasts and cultured in temperature-responsive culture dishes. The BMSCs detached automatically from the temperature-responsive culture dishes when the temperature was reduced to 20°C, forming an intact cell sheet. Demineralized bone matrix (DBM) and platelet-rich plasma (PRP) were prepared and used to construct a DBM/PRP/BMSC cell sheet/BMSC complex, which was implanted under the left latissimus dorsi muscle in a dog model. A DBM/PRP/BMSC complex was used as a control and implanted under the right latissimus dorsi muscle in the dog model. Immunoblot assays were performed to detect the levels of growth factors. Osteogenesis was observed to be induced significantly more effectively in the DBM/PRP/BMSC cell sheet/BMSC implants than in the DBM/PRP/BMSC implants. Immunoblot assay results indicated that the levels of the growth factors platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) in the experimental group were 3.2- and 2.5-fold higher compared with those in the control group, respectively. These results indicated that the BMSC cell sheets were functional and more effective than the control cell complex. Therefore, cell sheet technology may be used for the effective construction of functional tissue-engineered bone with ideal properties.

Construction of functional tissue-engineered bone using cell sheet technology in a canine model