| Objectives The autologous cell based joint cartilage therapy has become the most potential spot in cartilage tissue engineering, and the non-cartilage derived MSCs were recently regarded as the alternative cells to overcome the shortcomings that are inherent to the conventional autologous chondrocytes. How to fully apply these MSCs has been an intensively competitive topic.This research focued on these updates and clinical demands, including four new ideas, such as the in vivo and in vitro experiments of autologous bMSC, 3D culture and induction, periodic stress incentives, and the injectable tissue engineering in mini-injury way, and provided a practical reference for clinical repairing of joint cartilage defects with cartilage tissue engineering methods.Methods By cell culture, animal model, histology, biochemistry measurements as baisic methods, we subsequently proceeded closely related three parts of experiments as following:1. Autologous bMSC 2D culture and chondrocyte induction:1.1 Comparison on the isolation methods of autologuos bMSCs of adult rabbit: Bone marrow aspirates were obtained from 3 months old New Zealand rabbits from bone marks' sites, such as femoral tuberosity tibial tuberosity and ischial tuberosity. Three isolation methods, including the whole blood culture, hemolysis, and density gradient centrifugation protocols were compared. The cloning formation rate, primary passagetime and success rate of each method were observed to confirm the most effective and convenient primary culture for bone marrow derived mesenchymal stem cell, and explored some useful aspiration tricks;1.2 Manufacture of multiporous microcarrier and rapid amplication of autologous rabbit bMSC: Multiporous microcarrier was made as toluene as oil phase medium by the method of oil-in-water-in-oil emulsion, surface modifying, elution, dehydration and Irradiation. Adult rabbit MSC was separated and inoculated on the microcarreirs. Compare effects of different culture parameters of rotating wall vessel on the growth of bMSC, of which includes the inoculation concentration, rotating frequency; and the curve of the adhesive, cell vitality, sugar consumption, protein production were also measured. The basic fibroblastic growth factor's (bFGF) influence on the suppression of the aging of the passage bMSC was also investigated;1.3 Effects of different growth factor on proliferation and choadrocyte inducing of bMSC: Several growth factor, including basic fibroblastic growth factor (bFGF) transforming growth factor P 1 (TGF P 1) and hepatocyte growth factor (HGF), were adopted to identified their concentration range and effects on the proliferatin of bMSC on plates, then we designed several chondrocyte inducing culture with low mediate and high concentration of above factors with or without serum;2. Three-dimensional chondrocyte induction with dynamic stress:2.1 In vitro effects of periodic stress on bMSC in 3D environment: bMSC from adult rabbits were separated by gradient-density centrifugation and amplified, and were mixed in fibrin sealant gel at final concentration of 2 l07/ml and then incubated in rotating wall vessel bioreactor at 37 C,5%CO2. The parallel static culture was performed as control group. Gross appearance, histological section, I and II typecollagen immunohistochemistry, cell vitality and PG and collagen contents were investigated after 2 weeks;2.2 Scan culture model in 3D dynamic system: Three dynamic stress enviroment for alginate drop mediated chondrogenesis form bMSC , including simple rotating wall or rotating wall plus rolling compression and stiring culture, were compared to identified the most suitable model for dynamic stress culure, on which different induction method were subsequently scanned to identify the best one;2.3 Establishment and modification of transitional chondrocyte inducing from autologous bMSC in 3D dynamic system: bMSC were expanded. Several in vitro inducing group such as (A) high density bMSC in alginate-drop, (B) cell pellet in alginat... |
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