The Study Of Engineered Cartilage And Bone Tissue Constructed By Different Technologies

Objective:To explore the effective technologies to improve seeding efficiency in theconstruction of engineered cartilage and bone tissue in vitro which might layfoundations for constructing highly-qualified engineered osteochondral tissue.Methods:Experiment is divided into two parts. Part I: Chondrocytes were isolated fromrabbit (1 month old) articular cartilages and expanded in vitro. The second generationof rabbit chondrocytes (50μl, 4×107/ml ) and PLGA scaffold (5mm×5mm×2mm)were employed to construct engineered cartilage tissue in vitro using fibrin gel andthe static seeding techniques respectively. After 3 weeks of culture, the engineeredcartilage was harvested and examined by gross observation, histological staining,immunohistochemisty of collagenⅡ, the content of GAG, DNA and the percentageof collagenⅡdyeing area. Part II: Osteoblasts were isolated from rabbit (1 monthold) articular and expanded in vitro. The second generation of rabbit osteoblasts (50μl,4×107/ml ) andβTri-calcium phosphate scaffold (5mm×5mm×6mm)were used to construct tissue engineered bone in vitro with underpressure cell- seeding and thestatic seeding techniques respectively.The scaffolds were observed by SEM to see thenumber of the cells and its distribution. The adhersive rates were calculated afterseeding.At different time points,the number of cells was counted and alkalinephosphatase activity was detected. After 3 weeks of culture, immunohistochemisty ofcollagenⅠof the engineered bone tissue was examined.ResultResults:Part I: Egineered cartilage with fibrin gel techniques grew bigger than thecontrol groups, and their surfaces were smooth and glossy. The contents of GAG,DNA and the percentages of collagenⅡdyeing area were higher than control groups.The differences were statistically significant (P<0.05). Part II: Every scaffold couldbe observed cells by SEM, but cells on the experiment group were more than controlgroup and its distribution was more consistent and uniform.The mean adhersive rateof the engineered bone with underpressure cell-seeding technique was 68.57%,whichis higher than the control group.The osteoblasts in the scaffolds increased with culturetime for the two groups,at the same time point the difference of the cell number intwo groups had statistical significance(P<0.05).The expression of the ALP inexperiment group was higher than control group at every time point. (P<0.05).collagen I dyeing area in the experiment group was more homogenous.Conclusions:The fibrin gel and the underpressure cell- seeding are effective ways forseeding cells onto scaffolds ,it can help to regenerate more highly-qualifiedengineered cartilage and bone in vitro .