The Rabbit Living Bone Mediate Enhanced Homologous Osteoblasts From MSCs Adhesion On Scaffolds In Vitro

The bone defects due to trauma, inflammation and tumor are very common in clinical practice, which bring dysfunction and inferior life quality to patients. It is still an difficult problem to repair and reconstruct bone defects in orthopaedics, it's also one of the research focus in recent years. Until now, there have been a few methods to repair bone defects including bone autograft, allograft and external fixation. Although these methods have their advantages and disadvantages respectively, they can not meet the needs of perfect reconstruction in the end. Tissue engineering that aims to "replicate tissue and organ" provides another innovative choice for repair and regeneration of bone defects. The construction of the tissue engineered bone requires the interaction of three basic biologic elements: seeding cells, extracellular matrix scaffolds, adhesion and growth factors. Now, the study of seeding cells and scaffolds have been made a great progress, but why is not the quality of tissue engineered bone satisfying? Seeding cells adhesion on scaffolds play an important role in this process. So, the settlement of adhesion is one of most crucial problem in bone tissue engineering. Aimed at this problem, we cocultured the rabbit living bone and homologous MSCs or the osteoblasts from MSC to improve the adhesion characteristics of these cells; we seeded the osteoblasts from MSCs on the scaffolds which have been cocultured with homologous living bone to improve the quality of tissue engineered bone. Meanwhile, we studied the biological characteristics of seeded cells.Firstly, the rabbit marrow stromal cells (MSCs) were harvested from bone marrow by puncture, and then induced into osteoblasts. After the rabbit MSCs and osteoblasts from MSCs were cecultured with homologous living bone, the efficiency of attached cells was obtained. Results showed: 1. It took 10～14 d for MSCs to be confluent with plating concentration of 3×105cells/cm2. 2. The cells formed calcified nodule and expressed alkaline phosphatase, typeⅠcollagen and osteocalcin after osteoinduction. 3. Ultramicrostructure revealed naive condition in MSCs and mature state after osteoinduction with active secretion. 4. MSCs had the capacity to differentiate into osteoblasts. 5. the efficiency of attached cells of homologous MSCs or osteoblasts from MSCs, which have been cocultured with rabbit livingbone in vitro, were significantly increased.Secondly, the CPPf/PLLA scaffolds were fabricated with solvent-casting particulate-leaching method, following cocultured with rabbit living bone in vitro, and then we seeded the osteoblasts from MSCs in them. The efficiency of attached cells on the scaffolds was obtained by MTT method. The compounds of cells-scaffolds were implanted in the rabbit subcutaneous for 4 weeks, the samples were processed by histological and immunohistochemistrical method. Results suggested: 1. The CPPf/PLLA scaffolds had good cell biocompatibility. 2.There is a good linear correlation between directly counted rabbit osteoblasts from MSCs and MTT absorbency(492nm). 3. The efficiency of attached cells on the scaffolds, which have been cocultured with rabbit living bone, was significantly increased. 4. The quality of tissue engineered bone was improved by the method of accelerating cells adhesion. Conclusions can be drawn as follows from this experiment: 1. MSCs have great capability of proliferation and can differentiate into osteoblasts with expression of alkaline phosphatase, type Ⅰcollagen and osteocalcin, which is suitable for bone tissue engineering; 2. the efficiency of attached cells of homologous MSCs or osteoblasts from MSCs, which have been cocultured with rabbit living bone, were significantly increased. 3. CPPf/PLLA scaffolds have the characteristics of excellent biocompatibility and osteogenous ability. 4. There is a good linear correlation between directly counted rabbit osteoblasts from MSCs and MTT absorbency(492nm). 5. The efficiency of attached cells on the scaffolds, which have been cocultured with rabbit liv...