The Preliminary Experimental Studies Of Chondrogenesis Of Adipose Tissue-Derived Stem Cells Induced By Misshapen Auricular Chondrocytes From Microtia

Part one Biological Characteristics of Adipose Tissue-Derived Stem Cells and Misshapen Auricular Chondrocytes from Microtia Cultured in VitroObjective: To investigate the separation, cultivation and identification of human adipose tissue-derived stem cells and misshapen auricular chondrocytes from microtia, and to observe biological characteristics of two kinds of cells.Methods: The human misshapen auricular chondrocytes from microtia were isolated and cultured to observe morphological changes, growth curves and the expressions of collagen type II and aggrecan in chondrocytes from first to fifth passages with RT-PCR method. The second passage chondrocytes were identified by immunohistochemistry with collagen type II monoclonal antibody. The ADSCs were digested with collagenase type II, cultured and subcultured to 15th passage in vitro. Growth curves and cumulative population doubling were determined to evaluate the proliferation potential of ADSCs. The phenotypes of ADSCs were identified by immunohistochemistry and flow cytometry. The multilineage potential of ADSCs was confirmed by inducing them into osteogenic, chondrogenic, endothelioid and adipogenic differentiation.Results: The human misshapen auricular chondrocytes from microtia were transformed from polygon or triangle into the shape of the fibroblast-like cell with the generation increasing. Most cells of the fifth passage turned into that shape. The growth curve showed that the proliferation of chondrocytes from the first to the third passage was powerful. Collagen type II was expressed in chondrocytes identified by immunohistochemistry. The expression of collagen type II was high before the third passage and decreased with the passage increasing in the mRNA level, and that of the fifth passage was negative. The expression of aggrecan was high before the fourth passage and decreased with the passage increasing and that of the fifth passage is weak. The results of flow cytometry and immunocytochemistry showed that The expreesions of stem cell-related antigens (CD29, CD105, CD44, MHC-I) were positive, and those of hematopoiesis-related antigens (CD14, CD34, CD31) and other antigens(CD3, CD19, CD33, CD45, CD117, CD184 ) were negative. The calcified nodules could be observed by Von Kossa staining in osteogenic ADSCs. The intracellular lipid droplets could be observed by Oil Red staining in adipogenic ADSCs. Cells aggregation and glycosaminoglycan could be detected by Alcian Blue staining in chondrogenic ADSCs. Cells interconnect into cancellated structure and endothelial markers CD34 expressed by immunofluorescence detection on the differentiation of ADSCs into endotheliocytes.Conclusion: The misshapen auricular chondrocytes from the first to the third passage seem suitable for human auricular cartilage engineering. ADSCs can be abundantly harvested and have stable proliferation in vitro. ADSCs have multi-lineage potential and can be the suitable seed cells for constructing tissue-engineered cartilage.Part two Chondrogenesis of Adipose Tissue-Derived Stem Cells Induced by Misshapen Auricular Chondrocytes from Microtia in vivo Objective: To test the hypothesis that misshapen auricular chondrocytes from microtia can promote chondrogenic differentiation and chondrogenesis of human adipose tissue-derived stem cells at non-chondrogensis site in vivo.Methods: Human ADSCs and misshapen auricular chondrocytes were mixed at ratio (7:3) and 1.0×10~7 mixed cells were suspended in 0.2ml 30% Pluronic F-127, and then the mixture was injected into Balb/c nude mice subcutaneously as experimental group (Exp). Misshapen auricular Chondrocytes or ADSCs at the same cell number were mixed with 0.2 ml Pluronic F-127 and injected respectively as positive and negative control groups (Ctrl.1 and Ctrl.2). 0.3×10~7 misshapen auricular chondrocytes were mixed and injected as low concentration chondrocyte control group (Ctrl.3). All samples were collected at 8th week post-injection. Eight weeks after injection, the neonatal tissues in nude mice were taken out for morphological examination, Haematoxylin and Eosin (HE) staining, Verhoeff staining of elastin, Toluidine Blue stainings of acid mucopolysaccharide, Safranin O stainings of glycosaminoglycan, Masson staining of collagen and immunohistochemical staining ofcollagen type II.Results: In exp and ctrl.1 groups, all specimens formed semitransparently white mature cartilage-like tissue, looked like normal cartilage in appearance and elasticity. In Ctrl.2 groups, the specimens formed pallide-flavens fiber-like tissue. In Ctrl.3 groups, most of specimens formed analogic cartilaginous tissue, and weak in intension and lustreless on surface gloss. Between exp and ctrl.1 groups, average wet weight and GAG content of specimens had no significant difference in statistics (P>0.05). However, average wet weight and GAG content of specimens in Ctrl.2 and Ctrl.3 groups were lower than those in exp and ctrl.1 groups (P<0.05). In exp, ctrl.1 and Ctrl.3 groups, mature cartilage lacunas could be observed by histology staining and collagen type II could be detected for expression by immunohistochemistry in different extent in specimens. In Ctrl.3 groups, cartilage lacunas were incompact and inhomogeneous. The number of cartilage lacunas in Ctrl.3 groups was less than that in exp and Ctrl.1 groups. In Ctrl.2 groups, mature cartilage lacunas could not be observed by histology staining and collagen type II could not be detected for expression by immunohistochemistry.Conclusion: Misshapen auricular chondrocytes from microtia can promote chondrogenic differentiation and chondrogenesis of ADSCs at non-chondrogenesis sites in vivo.Part three Chondrogenesis of Adipose Tissue-Derived Stem Cells Induced by Misshapen Auricular Chondrocytes from Microtia in vitroObjective: To test the hypothesis that misshapen auricular chondrocytes from microtia can promote chondrogenic differentiation and chondrogenesis of human adipose tissue-derived stem cells in vitro.Methods: Human ADSCs and misshapen auricular chondrocytes were mixed at ratio (7:3) and 1.0×10~6 mixed cells were harvested as pellets as experimental group (Exp). Misshapen auricular Chondrocytes or ADSCs at the same cell number were harvested as pellets respectively as positive and negative control groups (Ctrl.1 and Ctrl.2). All samples were incubated in the centrifuge tubes for 28 days. Four weeks after incubation, the neonatal tissues in centrifuge tubes were taken out for morphological examination, expressions of collagen type II and aggrecan in pellets with RT-PCR, Haematoxylin and Eosin (HE) staining, Toluidine Blue stainings of acid mucopolysaccharide, Safranin O stainings of glycosaminoglycan, and collagen type II immunohistochemical staining.Results: Cell pellets concentrated into spherical tissue mass after 3 days incubation in three groups. After 4 weeks incubation in vitro, in exp and ctrl.1 groups, all specimens formed semitransparently stretchable disc tissue, smooth and glossy on surface. In Ctrl.2 groups, the specimens shrunk into spherical tissue in yellow and were not stretchable. The average wet weight and GAG content of specimens in exp group were all over eighty percent of those of specimens in ctrl.1 group. However, average wet weight and GAG content of specimens in ctrl.2 group were lower than those in exp group (P<0.05). In exp and ctrl.1 groups, the expressions of collagen type II and aggrecan could be dectected by RT-PCR, mature cartilage lacunas could be observed by histology staining and collagen type II could be detected for expression by immunohistochemistry in specimens. In ctrl.2 groups, the expressions of collagen type II and aggrecan could not be dectected by RT-PCR, mature cartilage lacunas could not be observed by histology staining and collagen type II also could not be detected for expression by immunohistochemistry.Conclusion: Misshapen auricular chondrocytes from microtia can promote chondrogenic differentiation and chondrogenesis of ADSCs in vivo.