Study On Osteogenic Potential Of Cryopreserved Bone Marrow Stromal Cells In Vitro And In Vivo

Objective: The bone marrow stromal cells (BMSCs) contribute to the regeneration ofmesenchymal tissues such as bone, ligament, and stroma. Being regarded as amajor cell source of bone tissue engineering, therapeutic applications ofBMSCs have been intensively studied in the resent years. By cryopreservingthe BMSCs, a large number of BMSCs can be stored for extended periods oftime for immediate use when needed. Here we investigated the growth andosteogenic potential of the cryopreserved canine BMSCs under induction andnon-induction conditions in vitro. Furthermore, we explored the osteogenicpotentiality of combining collagenic membrane and cryopreserved canineBMSCs under induction and non-induction condition in vivo.Method: The study consisted of two parts.Part 1: Study on osteogenic potential of cryopreserved bone marrow stromalcells in vitro. The BMSCs of beagle dog were cryopreserved for 11 months,then cultured in the induction medium with 10^-8mol/L dexamethasone, 50mg/L ascorbic acid and 10mmol/L Na-β-Glycerophosphate. The cells wereobserved under microscope. The proliferation status and alkaline phosphatase(ALP) level of cells were investigated with MTT assay. Mineralizationpotential was showed by Von-Kossa staining.Part 2: Study on osteogensis of the construct combining collagenic membraneand cryopreserved bone marrow stromal cells in nude mice. The BMSCs ofbeagle dog were cryopreserved for 12 months, then thawed and cultured invitro. 3 groups of compound materials were implanted into the nude mice.Group 1 was collagenic membrane with induction medium. Group 2 wasBMSCs and collagenic membrane, cultured with normal medium. Group 3was BMSCs and collagenic membrane, cultured with induction medium. 4, 8and 12 weeks after implantation, the specimens were harvested for histological observation.Results: In part 1, after recovered and cultured, the cryopreserved BMSCs showed astrong capability of proliferation and displayed a fibroblast-like morphology.Compared to control group, the proliferation of BMSCs in induction mediumwas decreased, but ALP level increased continually. 5 days after induction,BMSCs in induction medium had formed mineralized nodules.In part 2, HE staining showed the compound materials of the BMSCs andcollagenic membrane, cultured with induction medium, had more cells thanthe compound of BMSCs and collagenic membrane, cultured with normalmedium. Also more collagen membrane was decomposed and more collagenwas synthesized in implantation areas.Conclusion:1. The cryopreserved BMSCs maintain a strong activity of proliferation anddifferentiation in vivo. After induced in mineralization condition medium,the cryopreserved BMSCs displayed a strong osteogenic potential andcould be differentiated into osteoblasts as shown by high ALP level.2. After recovered and cultured, the cryopreserved BMSCs could beintegrated into the scaffold of collagen membrane. Construction of thecryopreserved BMSCs with collagenic membrane has strong activities ofproliferation and osteogenic potential in vivo, especially whencryopreserved BMSCs were induced by mineralization condition medium.3. The findings of our study indicate that the cryopreserved BMSCs could beused as the seed cells in the bone tissue engineering, which would greatlypromote the reconstruction of bone structures and periodontal defects.