Amplification And Chondrogenic Differentiation Of Adipose-derived Mesenchymal Stem Cells By Cultured On Silk Fibroin/Poly-L-lactic Acid Microcarriers

BackgroundCartilage defect repair and cartilage scaffold transplantation have always been a serious challenge faced by plastic surgeons and orthopaedic surgeons.Since the late 1990s,the engineered cartilage in the back of nude mice were reported,the technology of cartilage tissue engineering has made great progress in the past 20 years.Adipose-derived mesenchymal stem cells(ADSC)as a sort of mesenchymal stem cell(MSC)have been more and more important in the cartilage tissue engineering researching because of its advantages,such as the source of which is sufficient,caused less trauma,low immunogenicity,and the ability to regulate inflammation.However,due to the limited number of ADSCs extracted from bodies,it is difficult to meet the demand of a large number of seed cells for tissue engineering,and in vitro amplification must be carried out.At present,CultiSpher microcarriers was the most widely used of commercial absorbable microcarriers.But,due to the fact that such microcarriers are made of gelatin,their physical and chemical properties cannot meet the needs of chondrogenic induction of ADSC.Silk fibroin(SF),extracted from natural silk,is a kind of natural material with great application potential in cartilage tissue engineering in recent,but its mechanical strength is insufficient.We combined it with poly-L-lactic acid(PLLA)to prepare SF-PLLA microcarriers.We used these for the culture and chondrogenesis induction of ADSCs,and the chondrogenesis induction products were transplanted into nude mice in vivo,in order to find a more suitable microcarrier for the cartilage tissue engineering.Objectives1.ADSCs were inoculated into CultiSpher G microcarriers and SF-PLLA microcarriers at a lower density(concentration:2.5×106 cells/150mg),respectively.And cultured in serum-containing medium.Effects of different microcarriers on the amplification efficiency,differentiation potential and surface markers of ADSC were compared.2.ADSCs were inoculated into CultiSpher G microcarriers and SF-PLLA microcarriers(concentration:2×107 cells/150mg)at high density,respectively.And chondrogenic induction was performed using chondrogenic induction medium which containing TGF-?3.Effects of different microcarriers on ADSC cell survival,differentiation ability,and secretion ability of GAG and COL2 were compared.3.Tissue engineered cartilage constructed in vitro combined with rabbit ear chondrocytes was transplanted subcutaneously into nude mice to construct tissue engineered cartilage in vivo,and the differences of the cartilage constructed by the two microcarriers were analyzed.Methods1.Human ADSCs were extracted from fat extracted from liposuction operation,and cultured to passage 3.Flow cytometry and three-line differentiation identification were carried out to confirm that the expanded cells were ADSCs.2.ADSCs were inoculated on CultiSpher G microcarriers and SF-PLLA microcarriers,respectively.These was cultured for 18 days under three-dimensional conditions in vitro.The proliferation,cell distribution and survival of the two groups were compared,and the related gene expressions of chondrogenic,osteogenic and adipogenic were detected by qPCR after culture.Flow cytometry was used to detect the surface markers after amplification of ADSCs,and the differentiation ability of ADSCs after amplification was detected by three-line induction differentiation.3.ADSCs were inoculated on CultiSpher G microcarriers and SF-PLLA microcarriers,and cultured for chondrogenic induction under three-dimensional conditions in vitro for 21d.Cell survival rate,cell distribution and viability of the two groups were compared,and the expression of chondrogenesis related genes in ADSCs after culture was detected by qPCR.The secretion capacity of GAG and COL2 was detected by histological staining.4.In vitro chondrogenic induced microcarrier-ADSC+chondrocyte complexes of the two microcarriers were transplanted into the lateral back of nude mice.The appearance and histological staining of the two groups were observed 8 weeks later.Results1.There was no statistically significant difference in the total amplification efficiency of ADSC for 18 days between the two groups(p<0.05).After 18 days of culture under three-dimensional conditions in vitro,the genes expression related to chondrogenic differentiation of ADSCs on the SF-PLLA microcarriers were significantly upregulated,and the genes expression related to adipogenic differentiation of ADSCs on the CultiSpher G microcarriers were significantly upregulated.The ADSCs amplified on the two microcarriers still maintained the three-line differentiation ability and stem cell surface markers.2.There was no significant difference in total cell survival between the two groups after 21 days of chondrogenic induction in vitro(p<;0.05),the genes expression of SOX9,AC AN and COL2 in ADSCs after chondrogenic induction in SF-PLLA group were more than those in CultiSpher G group,and the difference was statistically significant.Histological staining results showed that ADSCs in both group successfully induced chondrogenesis in vitro.3.In vivo experimental results showed that the morphology and texture of tissue engineered cartilage were better in the SF-PLLA microcarriers group.Histological staining results showed obvious cartilage lacunae in the induction products of both groups,and there were a large amount of GAG and COL2 accumulation.Conclusions1.SF-PLLA microcarriers can be used for the amplification of ADSCs,and the cells still maintain their stem cell characteristic after amplification,and the chondrogenic potential is up-regulated.2.ADSCs on SF-PLLA microcarriers can be successfully inducted chondrogenic differentiation,and the effect was better than that of CultiSpher G microcarriers.3.Both kinds of microcarriers with chondrogenic differentiation in vitro ADSC combined with chondrocytes as implants can realize the construction of mature tissue engineered cartilage in nude mice,among which the tissue engineered cartilage constructed based on SF-PLLA microcarriers has better morphology and texture,and can be used as a better choice of cartilage tissue engineered scaffold materials.