Evaluation Of PVA/CS Electrospun Nanofiber Scaffold Delivered With HAMSCs Induced By TGFβ1-bFGF For Tendon Repair In Rabbit Models)

Objective:(1)The construction of polyvinyl alcohol(PVA)/ chitosan(CS)electrospun nanofiber scaffold delivered with hAMSCs.(2)To investigate the therapeutic effect of hAMSCs /PVA/CS nanofilms on rabbit models of patellar ligament injury.Methods:(1)hAMSCs were obtained from isolated placenta by enzymatic digestion,cultured and passed to the third generation,and then the third generation cells(P3)were cultured in medium supplemented with TGFβ1-bFGF(10ng/ml)for 7 days.Cell morphology was observed and collagen secretion was evaluated by Sirius Red staining.(2)Preparation of PVA/CS nano-scaffold: 4 kinds of mixture solutions of PVA and CS(proportions of 9:1,8:2,7:3,6:4)were electrospun into PVA/CS nanofiber scaffolds.After drying,the scaffolds was heat treated at 100℃ or 160℃.Scanning electron microscopy(SEM)was used to observe the structure of the scaffolds,contact angle measuring instrument was used to test the hydrophilicity,Fourier transform infrared absorption spectroscopy(FTIR)and X-ray diffraction(XRD)were used to test the composition and crystallinity,and mechanical testing machine was used to test the tensile strength.Determine the best concentration ratio and suitable heat treatment temperature according to the result.(3)Construction and biological evaluation of hAMSCs /PVA/CS biomaterials: PVA/CS nanofiber scaffolds of 9:1 and 8:2 were cut into a 2cm circle and spread on a 24-well plate,and then disinfected by ultraviolet irradiation.The cells were then planted on the biomaterials and cultured for another seven days.Real-time fluorescent quantitative PCR(q RT-PCR)was ued to detect m RNA expression level of Col-I,fibronectin and TNC,immunofluorescence was uesd to asses protein secretion,living dead cell staining kits and cell count-8(CCK 8)were used to evaluate the biocompatibility of biomaterials,SEM was used to observe the growth of cells on the scaffolds.(4)To observe the therapeutic effect of HAMSCs /PVA/CS nanofilms on animal patellar ligament injury: Rabbit patellar ligament defect models were made,and 9:1 hAMSCs /PVA/CS nanofilms were implanted into the defect area of rabbit patellar ligament for the following treatments: Group 1: no special treatment was done(the control group);Group 2: PVA/CS nanoscaffolds were implanted only(PVA/CS group);The third group: implantation of hAMSCs/PVA/CS nanofilms(hAMSCs /PVA/CS group).The animals were sacrificed 2,4 and 8 weeks after surgery,and the patellar ligaments were taken for observation,histological examination(HE staining,Masson trichrome staining),immunohistochemical examination(Col-I,Col-III),and Western blot(Col-I,Col-III).Results:(1)After 7 days of culture and passage,the third generation of amniotic mesenchymal stem cells were obtained.The cells were fusiform and polygonal,with a few being elliptic and in good condition.After 7 days of TGFβ1-bFGF culture,the cell gradually became longer,with fusiform growth,protruding cytoplasm,and radial growth.Sirius Red stains show that cells were making large amounts of collagen.((2)Among the 4 kinds of PVA/CS nanofiber scaffolds,the fiber diameter of 9:1 is the largest,while that of 6:4 is the smallest,and 9:1 has the best mechanical properties.Compared with the nanofiber scaffolds after heat treatment at 100℃,the nanofiber scaffolds at 160℃ has higher crystallinity and insoluble in water,and the crystallinity of the nanofiber scaffolds at 9:1 is the highest,and the nanofiber scaffolds at 6:4 is the lowest.(3)Cells grew well on both 9:1 and 7:3 nanofiber scaffolds,and more cells were found in 9:1.With the TGF beta 1-bFGF culture,m RNA expression level of Col-I,fibronectin and TNC increased over time,and the expression level of Col-I in 9:1 is better than 8:2.(4)8 weeks after biomaterials implantation in the patellar ligament defect model,the local inflammatory response in the HAMSCs /PVA/CS group basically disappeared,and the biomaterials was almost completely degraded.Compared with the blank group and the PVA/CS group,the damaged site was replaced by new tissue,and the repair effect was the best.Conclusion:(1)hAMSCs were successfully induced by TGFβ1-bFGF,and PVA/CS electrospun nanofiber scaffold delivered with hAMSCs were successfully prepared.(2)hAMSCs/PVA/CS biomaterials have good potential in repairing injuries of animal patellar ligaments.