Spinal reconstruction and orthopedic trauma demand a great amount of bones, but the imbalance between supply and demand restricts the usage of implant bone, so to find a new way is urgent.OBJECTIVE:
To explore the best experimental conditions for isolation and cultivation of bone marrow mesenchymal cells (BMSCs) and to observe its biological features in vitro.DESIGN, TIME AND SETTING:
The experiment was performed in the orthopedics laboratory of The Children's Hospital of Soochow University from May 2007 to May 2008.MATERIALS:
Four-week-old big ear male New Zealand white rabbits were provided by the Animal Center of Soochow University.METHODS:
BMSCs were separated by density gradient centrifugation method from bone marrow of New Zealand white rabbits, then were cultivated and sub-cultivated in vitro. The cells were removed into new culture medium 48 hours after primary inoculation, and then the total medium exchange was made every two or three days. Mixture of trypsin and EDTA was used for digestion and passage when cells reached 90% confluence.MAIN OUTCOME MEASURES:
Using inverted microscope, cell morphology was observed, cell viability was measured with the MTT method, according to which cell growth curve was drawn. Following P3 generation of cells in osteogenesis conditioned medium were chosen to culture for 4 weeks, alkaline phosphatase positive cells, marking osteogenic differentiation, were identified using Gomori staining method, and calcium nodules were determined with the modified Von-Kossa staining.RESULTS:
Following isolation and culture, BMSCs grew with normal appearance and vigor. After inoculation, cells presented from short fusiform to long fusiform, triangular and even polygon. Each generation of cells had remarkable latency, increased logarithmic phase and plateau phase, and cells could be cultured to P9 generation continuously. The rate of alkaline phosphatase positive cells was 93%. Staining of mineralization nodules was positiveCONCLUSION:
In vitro isolation and cultivation of BMSCs is relatively simple and feasible, and BMSCs can be differentiated into osteoblasts directionally.