Tissue Engineered Articular Cartilage With Decalcified Bone Matrix And Homologous Chondrocyte And/or Bone Marrow Mesenchymal Stem Cell In Rabbits' Knee: An Experimental Research

Objective To identificate the feasibility of cultivating tissue engineered articular cartilage in knee cavity and demonstrate the role of bone marrow mesenchymal stem cell in the cultivating of tissue engineered cartilage.Material and method All 90 New Zealand white rabbits,age from 6m to 8m,weight from 2.5～4.0kg,male,were randomly divided into three groups,each group for 30 rabbits.Group A,grafted in the knee with bone decalcified matrix only;10 specimens were taken out at the time of 8w,10w and 12w respectively for pathological observation. Group B,grafted in the knee with bone decalcified matrix and homologous articular chondrocyte;10 specimens were taken out at the time of 8w,10w and 12w respectively for pathological observation.Group C,grafted in the knee with bone decalcified matrix and articular chondroeyte and bone marrow mesenchymal stem cell;also 10 specimens were taken out at the time of 8w,10w and 12w respectively for pathological observation. All the specimens were to wrap up with auto-periosteum at the beginning of grafting surgery using micro-surgery technology.All the specimens were carried out with H-E stain and toluidine blue stain,and the number of cells which is toluidine blue positive under the observation of inverted microscope(10x40) were recorded for statistic analyze using SPSS v14.0.Measurement data were demonstrated with mean±standard deviation mode,One-way ANOVA and Post Hoc Tests(Dunnett) were carried out for group comparison. Results Group C(decalcified bone matrix and chondrocyte and bone marrow mesenchymal stem cell):After cultivating in the knee cavity for 8 weeks,H-E stain show that the chondrocyte has began to proliferate,and in good metabolism stasis,the cell bodies grown bigger,karyotins are reinforced,and already has secretion of cell matrix.At the 8^th week of growth,the specimens became hyaline cartilage substance, has cartilage lacuna,and surrounded by a pond of cellular matrix,decalcified bone matrix structure disappeared.On toluidine blue stain samples,group C at 8^th week show the same as H-E stain,the majority of cellular karyotins are in deep hyacinthine (positive);at 12^th week,there are a lot more toluidine positive cells in group C than group A and B.The cell are aligned in a line or clump appearance,there are a bulk of cells in deep hyacinthine(positive);Cartilage lacuna is obvious.Group A(decalcified bone matrix) and B(decalcified bone matrix and chondrocyte):At 8^th week,H-E stain show only a few chondrocyte spread on the scaffold structure,most of the cell became to shrink,the scaffold is in integrity.At 12^th week,group B samples show a little proliferation of the chondrocyte,the chondrocytes are in triangle or fusiform shape, cellular matrix is fewer than group C,did not fuse a lamella and no cartilage lacuna. Toluidine blue stain show fewer chondrocyte,spread over the sample,the karyotins are in light blue,no cartilage lacuna.In group A,toluidine blue stain positive cells are rare at 12^th week.In all groups,toluidine blue positive chondrocyte cells are counted at 8^th week,10^th week,and 12^th week respectively.The numbers of cells are analyzed using SPSS v14.0 software,the result show group C is better than Group A and B,the difference is significant.Conelusion We can conclude from the experimental research that knee cavity is an ideal environment for tissue engineered articular cartilage growth,and the theory of "grow in the knee cavity,graft in the same knee" is quite feasible.