Placenta DerivedStem Cells Produced Osteoid In Vitro And In Vivo, And The Constructive Effect Of Tissue-engineered Bone Cultured In Bioreactor

Seed cells-MSCs appear to be an attractive tool in the context of tissue engineering, we can separate it from autologous or allogenic tissue or animals. Bone marrow represents the main source of MSCs for both experimental and clinical studies. However, the number of bone marrow MSCs significantly decreases with age, which makes the search for adequate alternative sources of these cells for autologous and allogenic use necessary. In this connection, most attention should be paid to tissues containing cells with higher proliferative potency, capability of differentiation, and lower risk of viral contamination.Objective: This study is aimed to search for alternative sources of postnatal human mesenchymal stem cells (HPMSCs): candidate seed cells from placenta for bone tissue engineering. To establish a systemic protocol for the isolation, culture and osteogenic induction of HPMSCs as well as in vivo bone tissue formation of the induced cells, thus paving the way for science research or possible clinical use of MSCs-based bone tissue engineering.Method: Mononucleated cells were isolated from the human placentatissue perfusate by density gradient fractionation with Percoll at a density of 1.073g/ml. The cells were cultured according to the method of BMSCs. When the cultures reached 90%of confluence, cells were followed by passages. Growth characteristics and cell cycle analysis were performed. Analysis of cell surface molecules was made by using flowcytometry. The differentiation ability adipogenic and osteogenic differentiaton of HPMSCs was assessed at the fifth passages .The induce condition were based on the BMSCs.Bioreactor was also used to observe whether it could be suitable in cultivating HPMSCs in large quantity.The ability of HPMSCs to form osteoid in vivo was determined by loaded the cells onto β-TCP matrix and then implanted into the muscle of SCID mice.To observe whether the cell-Coralline hydroxyapatite complexes which cultured in bioreactor more effective in defect repairing, the complexes were implanted into dogs cranial defects. Samples were extracted 3 and 6 months after operation for X-ray , histological analysis.Result: After 6 to 12h, the inoculation, the placenta drived cells attached the plastic surface. About 3 to 7days later, the cells formed individual colonies displaying fibroblast-like morphology. 2 weeks or later a homogeneous layer of fibroblastoid (MSC-like) cells occupied nearly the whole plastic surface, and these adherent cells could be readily expanded in vitro by successive cycles without morphologic alteration.Flow-cytometry analysis demonstrated a phenotypical identity in CD34^-, CD45^-, CD29⁺, CD44⁺, CD166⁺ and HLA-DR^- indicating that the MSCs obtained with this method displaying a cell's characteristics, suchas mesenchymal cells, which differ the cells from clusters of HSCs. Cell cycle studies revealed 93.97% of the cells in G₀/G₁, showing that while a small fraction of MSCs are actively engaged in proliferation, the vast majority of the cells are standing at G₀/G₁ phase of the cell cycle.After 2 weeks induction, adipogenic differentiation of the cells was identified by oil-red-0 staining. The cells also underwent progressive osteogenic differentiation indicated by alizarin red staining, alkaline phosphatase histochemical staining and Von Kossa staining.Nine days after cultivated in bioreactor the result showed that a maximum viable cell concentration of 1.75×10⁹cells/L whereas the cell density increased to a maximum of 3.68×10⁸ cells/L on 7th day of static culture system.Four and eight weeks post-implantation, the histological studies showed new bone formed significantly in the construction implanted into the muscle of nude miceSix moths after implantet the bioceramics in the dogs cranial defects, the complexes cultured in bioreactor degraded faster and produced more bone formation than those without culturing in bioreactor. Conclusion:1. There large amount of MSCs exit in human placenta, they could be separated and isolated with suitable methods. We called these cells human placental MSCs(HpMSCs).2. Like the BMSCs HPMSCs were CD29, CD44, CD 166, positive, but were negative for CD34, CD45, CDHLA-DR.3. PHMSCs could differentiate into adipocytes and osteocytes. 4. After osteogenic differentiation it has the ability of produce osteoid in...