| BackgroundThe repair of cartilage defects caused by various factors has always been a great challenge in clinic because cartilage has a low spontaneous repair and regeneration capacity.Tissue engineering,based on the principles of biology and engineering,is expected to provide an advanced procedure for the regeneration of damaged cartilage tissue.However,this technology has not been applied in large-scale clinical transformation,and the limited source of seed cells limits its clinical application.Mesenchymal stem cells(MSCs)are a kind of adult stem cells which exist in a variety of tissues and have multipotential differentiation.MSCs are favored by researchers because they do not involve ethical problems and have great differentiation potential.Among them,adipose-derived stem cells(ADSCs)have become an important seed cell source for autologous and allogeneic stem cell based therapies and tissue engineering due to their abundant sources,easy access and low immunogenicity.After digestion of adipose tissue with collagenase,the pelleted stromal vascular fraction(SVF)is obtained.SVF represents a cell population composed of ADSCs,endothelial cells,endothelial progenitor cells,hematopoietic cells(including hematopoietic stem cells,granulocytes,monocytes,lymphocytes)and pericytes,among which there is a certain proportion of stem cells.SVF can not achieve allogeneic stem cell transplantation due to its immunogenicity,however,freshly acquired SVF does not require in vitro culture and expansion,so it can achieve first-stage surgical autologous transplantation and save clinical treatment time.The comparison of chondrogenic characteristics under three-dimensional conditions in vitro and injectable elastic cartilage regeneration in vivo between ADSCs and SVF will provide a theoretical basis for cartilage tissue engineering technology based on cell therapy and provide a reference for optimal selection of seed cells in the construction of elastic cartilage.Objective1.Compare biological characteristics of ADSCs and SVF isolated from rabbit adipose tissue and identify ADSCs,so as to provide cell source basis for chondrogenic research in vitro and in vivo.2.Compare the chondrogenic characteristics of rabbit derived ADSCs and SVF by histology,gene expression and GAG quantitative analysis,and establish the application strategy of seed cells based on the above cells,so as to provide a reference for the optimal selection of seed cells in the construction of tissue engineered elastic cartilage.Methods1.ADSCs were isolated and purified by collagenase digestion method and adherence culture method.SVF was obtained by collagenase digestion method.The cell morphological characteristics of ADSCs and SVF were observed under microscope,and the cell viability of the two groups was compared by MuseTM cell analyzer.Surface marker profiles of ADSCs and SVF,including CD31,CD34,CD45,CD73,CD90 and CD 105,were compared by flow cytometry.The ADSCs subsets in SVF were identified as CD45-CD34+ CD31-to clarify the proportion of ADSCs in SVF.In addition,ADSCs were induced by adipogenesis,osteogenesis and chondrogenesis,and self-identification of ADSC stem cell characteristics was carried out in combination with the expression of ADSC cell surface markers.2.Rabbit auricular chondrocytes(ACs)were isolated by trypsin and collagenase digestion.In three-dimensional culture conditions in vitro,ADSCs,SVF,ACs,ADSCs+ACs and SVF+ACs(1:1 mixture in co-culture groups)were induced in chondrogenic differentiation medium for 4 weeks and we compared the chondrogenic potential of ADSCs and SVF in vitro by gross observation,histological staining,tunel staining,Real-time PCR analysis and GAG quantitative analysis.3.Rabbit-derived ACs,ADSCs+ACs and SVF+ACs(1:1 mixture in co-transplantation groups)loaded with Pluronic F-127 gel were subcutaneously implanted into nude mice for 8 weeks,and we compared injectable elastic cartilage regeneration in vivo after co-transplantation of ADSCs and SVF with ACs by gross observation,histological staining,Real-time PCR chondrogenesis related gene detection and GAG quantitative analysis.Results1.The cell morphology of ADSCs and SVF was observed under inverted phase contrast microscope.It was found that ADSCs had a spindle-shaped fibroblast-like appearance and the cell morphology was uniform.SVF cells were of different sizes and they were in suspension.MuseTM cell viability analysis showed that the viability of ADSCs was higher than that of SVF.Flow cytometry showed that there were differences in surface marker profiles between ADSCs and SVF from rabbits.Isolated and expanded ADSCs contained a more homogenous cell population,which were CD31,CD34,CD45 negative(CD31:1.822±1.480%,CD34:1.935 ±0.1334%,CD45:2.105±0.9136%),but positive for MSC marker like CD73,CD105(CD73:88.07±5.444%,CD105:88.47±4.113%),and the expression rate of CD90 in ADSCs was 23.43±6.647%.SVF represented a heterogeneous cell population containing not only cells with typical MSC markers(CD73:31.65±5.279%,CD90:41.98±7.238%,CD105:19.08±1.493%),but also CD31+ endothelial cells/endothelial progenitors,CD34+ stem cells and CD45+hematopoietic cells(CD31:33.97±4.246%,CD34:26.72±1.845%,CD45:20.97±3.260%),and the proportion of ADSCs in SVF was 9.947±1.072%.The isolated and expanded ADSCs were able to differentiate into adipocytes,osteoblasts and chondrocytes.2.In three-dimensional culture conditions in vitro,ADSCs,SVF,ACs,ADSCs+ACs and SVF+ACs could form pellet after chondrogenesis induced for 4 weeks.Compared with ADSCs group and SVF group,the results of histological staining showed the secretion of extracellular matrix in ADSCs group was better than that in S VF group,and the total GAG quantification and GAG/DNA in ADSCs group were higher than those in SVF group.In coculture with ACs,histological staining results showed that there was no significant difference in cartilage matrix secretion between ADSCs+ACs group and SVF+ACS group in pellet surface region,while the matrix secretion of ADSCs+ACs group was significantly better than that of SVF+ACs group in pellet core and middle region.The expression of Real-time PCR cartilage related genes and GAG contents in ADSCs+ACs group were higher than those in SVF+ACs group.TUNEL staining results showed that cell apoptosis rate of ADSCs+ACs group was significantly lower than that of SVF+ACs group in pellet surface region,middle region and core region.3.ACs,ADSCs+ACs and SVF+ACs loaded with Pluronic F-127 could form cartilage-like tissue in nude mice subcutaneously,and the specimens of each group were different in size,and the specimens in SVF+ACs group were smaller than those of the other two groups.Histological staining results showed that the matrix secretion of ADSCs+ACs group was better than that of SVF+ACs group at the edge and center of the specimen.Real time PCR results showed that the relative expressions of ACAN and COMP in ADSCs+ACs group were higher than those in SVF+ACs group,but there was no significant difference in the relative expression of COL2A1 between the two groups.GAG quantitative results showed that GAG content in ADSCs+ACs group was higher than that in SVF+ACs group,and the difference was statistically significant.Conclusions1.There are differences in cell viability and surface marker profiles expression between rabbit-derived ADSCs and SVF.The isolated and purified ADSCs are homogeneous cell population with higher cell viability.The isolated SVF represents a heterogeneous cell population.And,the isolated and purified ADSCs have potential of multi-lineage differentiation.2.In three-dimensional culture conditions in vitro,ADSCs,SVF,ACs,ADSCs+ACs and S VF+ACs can be successfully induced to differentiate into cartilage.The chondrogenic properties of the ADSCs group are better than those of the SVF group,and compared with SVF+ACs,ADSCs+ACs have stronger chondrogenic characteristics.3.The regeneration of elastic cartilage in vivo in Pluronic F-127-loaded ADSCs+ACs is better than that in SVF+ACs.It can be preliminarily considered that ADSCs may be a better source of seed cells than SVF in vivo co transplantation with ACs in nude mice(or in vitro coculture with ACs).However,in terms of application convenience,SVF still has some advantages in clinical application. |
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