Effects Of TGF-β3,FGF18 Combined With Photosensitive Hyaluronic Acid Hydrogel On Chondrogenic Differentiation Of HUCMSCs

Objective1.Different combinations of TGF-β3 and FGF18 were used to 3D spheroid culture of human umbilical cord mesenchymal stem cells(HUCMSCs)in vitro to explore the optimal addition timing of FGF18 and observe the culture protocol that can most effectively promote the directional differentiation of HUCMSCs into chondrocytes and simultaneously inhibit the hypertrophic phenotype.2.Synthesize a photosensitive hyaluronic acid hydrogel based on hyaluronic acid,and explore the biocompatibility of this hydrogel to HUCMSCs,and compare whether the optimal culture scheme in the photosensitive hyaluronic acid hydrogel can promote chondrogenic differentiation of HUCMSCs more than the simple culture in vitro.Methods1.CCK8 was used to test the effect on proliferation ability after the addition of TGF-β3,FGF18 as well as TGF-β3 + FGF18,respectively.HUCMSCs of passage 3were cultured in a 15 ml centrifuge tube for three-dimensional spheroidization.The cartilage induction system was composed of different combinations of TGF-β3 and FGF18.Specifically,HUCMSCs were induced and cultured for 21 days according to blank group(i.e.no TGF-β3 and FGF18),TGF-β3 group,FGF18 group,TGF-β3 +FGF18 group and TGF-β3 + FGF18(added on the 14 th day),HE and Alcian blue staining were used to compare the synthesis of extracellular matrix and secretion of proteoglycan.Real time PCR was used to detect the expression of cartilage related genes COL2A1,COL10A1,SOX9 and ACAN.2.Add methacrylate anhydride to hyaluronic acid solution and adjust p H to 8.5,react in 4℃ in dark environment for 24 hours to make hyaluronic acid methacrylate anhydride,after lyophilized,observe whether the reaction is successful to form methacrylate esterification hyaluronic acid(HAMA)by Fourier transform infrared spectroscopy and hydrogen nuclear magnetic resonance spectroscopy.Different concentrations of HAMA solution were added to lithium phenyl-2-4-6trimethylbenzoylphosphate(LAP),the photosensitive hyaluronic acid hydrogel was synthesized with ultraviolet light irradiation for 1 min,the internal structure was observed by electron microscopy,the stress-strain curve was obtained by universal mechanical testing machine and the compressive modulus was calculated to detect the swelling ratio of different concentrations of hydrogels.CCK8 compared the cytotoxicity of different concentrations of hydrogels,chose the least toxic concentration and HUCMSCs to prepare the cell / hydrogel complex,and cultured them under the best induction scheme under the first chapter of the experiment.At the same time,the pellets were cultured under the same induction regimen.After 21 days,HE and Alcian blue staining were used to compare the extracellular matrix synthesis and proteoglycan secretion of each group,the expressions of COL2A1,COL10A1,SOX9 and ACAN were detected by real-time PCR.Results1.CCK8 results found that all three induction regimens promoted proliferation,and the TGF-β3 + FGF18 group showed the highest proliferation ability.HE and Alcian blue staining revealed that the extracellular matrix synthesis as well as proteoglycan secretion were increased in the TGF-β3,FGF18,TGF-β3 + FGF18,and TGF-β3 +FGF18(added on the 14 th day)groups compared with the blank group,whereas the TGF-β3 + FGF18(added on the 14 th day)group had the highest extracellular matrix density and the most abundant proteoglycan with blue staining.Real time PCR showed that the expression of chondrogenic differentiation marker genes COL2A1,SOX9,and ACAN was significantly increased in the TGF-β3 + FGF18(added on the 14 th day)group relative to the other groups,and the expression of hypertrophic marker gene COL10A1 was the lowest,and P < 0.05,indicating that this induction protocol had both the ability to promote the chondrogenic differentiation of HUCMSCs and the ability to inhibit the chondrogenic differentiation.2.HAMA has been successfully synthesized by anhydride of hyaluronic acid methacrylate as evidenced by Fourier transform infrared spectroscopy,nuclear magnetic resonance hydrogen spectroscopy.The electron microscope observation of photosensitive hyaluronic acid hydrogel with concentration of 1% and 3% showed that the 3% concentration group had a compact structure,a complete pore wall and a larger pore density.The calculated compression modulus of the 3% concentration group was higher and the swelling rate was slightly lower.CCK8 experiments showed that the absorbance of the 3% concentration group was higher and promoted the proliferation ability of cells.Therefore,3% concentration photosensitive hyaluronic acid hydrogel was used to prepare the cell / hydrogel complex.HE and Alcian blue staining found that the extracellular matrix was significantly more abundant,the blue stained sections were more numerous and many cells presented a chondrocyte like round morphology in the cell / photosensitive hyaluronic acid hydrogel group compared with the blank group and simple spheroidizing culture group.Real time PCR showed that the expression of chondrogenic differentiation marker genes,COL2A1,SOX9,and ACAN,was significantly increased in the cell / photosensitive hyaluronic acid hydrogel group.Conclusions1.In the in vitro 3D spheroid culture of HUCMSCs,TGF-β3 was added at the beginning,after which the induced differentiation protocol of combined culture with FGF18 added at 14 th day was the most suitable chondrogenic differentiation strategy,which could achieve both effects of promoting normal differentiation and inhibiting hypertrophic differentiation.2.The photosensitive hyaluronic acid hydrogel can be synthesized by UV irradiation under the irradiation of HAMA solution with LAP.It has good porosity,compression modulus,swelling rate and biocompatibility.Encapsulation of HUCMSCs through the photosensitive hyaluronic acid hydrogel under the same protocol of induced differentiation resulted in a better ability to promote differentiation to cartilage relative to simple cell spheroid culture.