| Objective: To compare and study the expression changes of wound repair related factors of Human amniotic mesenchymal stem cells(hAMSCs)under the ordinary two-dimensional and three-dimensional cultivation conditions of hydrogel,so as to provide preliminary research data for the further transformation of hAMSCs using hydrogel as carrier into clinical application to promote wound healing.Methods: The hAMSCs were isolated by mechanical method combined with enzymatic digestion and cultured under ordinary two-dimensional conditions.The third-generation well-grown cells were harvested,and the isolated cells were identified by flow cytometry(FCM)technique,three lines(adipogenic,osteogenic,chondrogenic)induced differentiation,vimentin immunofluorescence staining.The third generation of hAMSCs with good cell vitality was mixed with ShakeGelTM3 D hydrogel(volume ratio: 1:1)and cultured together.After 7 days of culture,the cells were detected as follows and compared with the corresponding detection indexes of hAMSCs cultured under ordinary two-dimensional conditions:(1)Inverted phase contrast microscope observation of two kinds of culture under the condition of cell morphology and growth.(2)Vimentin immunofluorescence staining.(3)Confocal laser microscope observation of Live/Dead cells staining to identify cell vitality.(4)The hAMSCs cultured under three-dimensional in the hydrogel were observed the immunofluorescence staining of PPAR-?(peroxisome proliferator-activated receptor gamma),osteocalcin(OCN)secreted by osteoblast,the key of Chondrocytes transcription factor SOX9 markers and Phalloidin by laser scanning confocal microscope to identify the state of differentiation.(5)The hAMSCs under two culture conditions were collected,RNA was extracted and the relative expression of mRNA related to wound repair related factors was determined by RT-qPCR.(6)The supernatant of cell culture was collected under the two culture conditions,and the protein expression levels of wound repair related factors were determined by ELISA.Results:(1)The second-generation hAMSCs cultured in two dimensions showed a long-swirl vortex-like typical mesenchymal-like cell morphology,which was placed in aflat spindle-like cell culture bottle wall.The hAMSCs showed high expression of CD44,CD73,CD90 and CD105,positive expression of vimentin,no or low expression of CD34,CD19,CD45,CD11 b and HLA-DR.Alizarin red staining showed the presence of mineralized nodules in the cells after osteogenic induction.Oil red O staining showed that lipid droplets were observed in the cytoplasm after adipogenic induction.Toluidine blue staining showed that a large amount of glycosamine was secreted in the cell matrix after cartilage induction.(2)The three-dimensional cultured hAMSCs in the hydrogel showed that the cells were uniform and dispersed in the hydrogel with a circular translucent and three-dimension shape.Under the high power microscope,the cells could be seen extending pseudopodia slightly to the periphery.The vimentin fluorescence staining was positive and distributed uniformly in the cells.Live/dead cell staining showed good cell viability in hydrogels,suggesting that hydrogels are not toxic to cells.Immunofluorescence staining of PPAR?,OCN and SOX9 markers after adipogenic,osteogenic and chondrogenic induction were positive results.It is suggested that it still has the potential to induce differentiation,but the cytoskeleton staining of phalloidin revealed that the morphology of the cells did not change significantly,suggesting that its differentiation potential may be decreased.(3)The relative expression of IL-6,IL-8,HGF,EGF,bFGF,VEGF,HA and Vimintin in hAMSCs after three-dimensional culture in hydrogel was higher than that in two-dimensional cultured hAMSCs,and the difference was statistically significant(P<0.05).The relative expressions of IL-4,IL-10,TIMP,MMP,TGF-?1 and KGF were higher than those of hAMSCs cultured in two-dimensional culture.There was no significant difference between the two groups(P>0.05).(4)The supernatant of three-dimensional cultured hAMSCs in hydrogel was higher than two-dimensional in the presence of IL-6,IL-8,IL-10,HGF,EGF,bFGF,EGF,KGF and VEGF.The expression increased and the difference was statistically significant(P<0.05).The protein content of IL-4,TIMP,MMP and TGF-?1 was higher than that of the supernatant of hAMSCs cultured in two-dimensional culture.There was no significant difference between the two groups(P>0.05).Conclusion: As a three-dimensional culture scaffold for hAMSCs and a microenvironment for cell growth,hydrogel has good biocompatibility,which may better present the cell morphology and the paracrine biological effects of wound repair related indicators.It is a good hAMSCs carrier,providing partial preliminary research data forfurther clinical transformation research. |
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