Performance And Mechanism Of Oral Mucosa Defect Repair By Photocrosslinkable DHAMMA Composite Hydrogel Based On GelMA

[Objective]:Clinical collection of human amniotic membrane(HAM)and preparation of human decellularized amniotic membrane(dHAM)by acellular treatment.dHAM was grafted with methacrylic anhydride(MA)to fabricate photocrosslinkable dHAMMA(dHAM methacrylate).Based on GelMA hydrogel,photocrosslinkable acellular amniotic membrane composite hydrogel of GelMA-dHAMMA with bicomponent polymer network(BCN)structure was synthesized.The characterization of GelMA-dHAMMA composite hydrogel was investigated,the ability of the composite hydrogel to promote the proliferation and differentiation of human fibroblasts was explored in vitro,the angiogenic ability was evaluated,and the ability to promote the repair of oral mucosa defect were studied in vivo as well.Ultimately,the mechanism of GelMA-dHAMMA composite hydrogel in promoting the repair of oral mucosa defect was elucidated by experimental research.[Methods]:Using fresh human amniotic membrane as raw material,dHAM was obtained by acellular treatment with EDTA,NaOH and NH4Cl.dHAM was immersed in 4%(V/V)MA solution(MA dissolved in PBS)and aminated with methacrylic acid to synthesize photocrosslinkable dHAMMA(dHAM methacrylate),then the characterization of dHAM and dHAMMA were both investigated.The GelMA-dHAMMA composite hydrogel scaffold with thickness of 2mm(GelMA,dHAMMA mass ratio is 2:1)was prepared by photocrosslinking technique.Due to the lack of necessary groups for photocrosslinking of dHAM,the dHAM was ground into powder and embedded in GelMA,then the GelMA/dHAM hydrogel(GelMA/dHAM mass ratio was 2:1)was prepared by physical composite as a comparative study group.The characterization of GelMA,GelMA/dHAM and GelMA-dHAMMA were also investigated.GelMA,GelMA/dHAM and GelMA-dHAMMA were used as experimental group,while no material was added as blank control group,CCK8 and immunofluorescence were detected in vitro.Using GelMA,GelMA/dHAM and GelMA-dHAMMA as experimental group and microporous membrane group as control group,the angiogenic ability of GelMA-dHAMMA was studied by CAM model.New Zealand white rabbits were selected as animal model of mucosal defect,and the implant materials were divided into four groups:GelMA group,GelMA/dHAM group,GelMA-dHAMMA group and completely blank control group.The macroscopic observation,wound healing rate,HE staining,immunohistochemistry and Masson detection were used to evaluate the wound healing ability and clarify the healing mechanism.[Results]:By scanning electron microscope,it was observed that dHAM and dHAMMA showed porous fiber network structure,and the pore diameters were 6.1±1.77μm and 8.54±2.39μm,respectively.Through grafting modification,the pore size of dHAMMA is larger than that of dHAM.GelMA has a three-dimensional porous structure,uniform pore size distribution and good pore connectivity,the pore size is about 54.94±11.15μm.The three-dimensional pore diameter of GelMA/dHAM is nearly round,the wall is thin,and the size is uniform.The average pore size is about 10.16±2.77μm.dHAM powder is distributed in the pore wall and cavity.GelMA-dHAMMA also has a three-dimensional porous structure with nearly round pores,thin walls and uniform size distribution.the interface between GelMA and dHAMMA is interpenetrating and continuous,and the average pore diameter is about 27.87 ±9.28μm.The difference of pore diameter was statistically significant(p<0.05).FTIR showed the formation of new chemical bonds(double bonds forming methacrylamide groups)after dHAM grafting modification.Compared with the peak shape of GelMA,there is no obvious absorption peak of new group in GelMA/dHAM infrared spectrum.Compared with GelMA/dHAM,the vibration of the characteristic peak of acrylamide can still be seen in GelMA-dHAMMA,but the structure of the main chain does not change obviously.The swelling rates of dHAM,dHAMMA,GelMA,GelMA/dHAM and GelMA-dHAMMA were basically stable and reached saturation after water absorption at 24 h.At 36 h,the mass of dHAM,dHAMMA,GelMA,GelMA/dHAM and GelMA-dHAMMA expanded to(7.38±2.44)%,(7.19±2.3)%,(494.58±11.41)%,(440.54±16.95)%and(418.21±18.95)%,respectively.There was no significant difference between dHAM and dHAMMA(p>0.05).There were significant differences among GelMA,GelMA/dHAM and GelMA-dHAMMA(p<0.05).Extended over time(14 days),the degradation of dHAM and dHAMMA were persistent existence obviously and almost complete within 14 days,the difference was not statistically significant(p>0.05).The degradation of GelMA,GelMA/dHAM and GelMA-dHAMMA persisted within 16 days,but the degradation rate of GelMA-dHAMMA was significantly lower than that of GelMA/dHAM and GelMA,the difference was statistically significant(p<0.05).According to the stress-strain curves of dHAM,dHAMMA,GelMA,GelMA/dHAM andGelMA-dHAMMA,it was found that GelMA-dHAMMA tensile strength>GelMA>GelMA/dHAM>dHAM and dHAMMA,the difference was statistically significant(p<0.05).While there was no difference in tensile strength between dHAM and dHAMMA(p>0.05).Through the cell experiment in vitro,we found that GelMA-dHAMMA promoted the proliferation of fibroblasts,and the difference was statistically significant(p<0.05).The fluorescence intensity of α-SMA expression in GelMA-dHAMMA was the most obvious.In the angiogenesis experiment of CAM model,the vascular area of GelMA/dHAM group and GelMA-dHAMMA group was larger than that of control group and GelMA group(p<0.05).In addition,the vascular promotion in GelMA-dHAMMA group was better than that in GelMA/dHAM group,and the difference between the two groups was statistically significant(p<0.05).In vivo animal experiment,the wound healing rate of each group was as follows:GelMA-dHAMMA group>GelMA/dHAM group>GelMA group>control group.GelMA-dHAMMA group significantly promoted wound healing,and most of the wounds were almost completely healed(p<0.05).14 days after operation,CD34 immunohistochemistry showed that there were no obvious neovascularization endothelial cells in the control group,but the newly formed capillary endothelial cells could be observed in the other groups,and the capillary endothelial cells increased significantly in the GelMA-dHAMMA group.On the 14th day after operation,Masson staining showed that the wound tissue of GelMA-dHAMMA group was dark blue,and the collagen fibers of wound surface were neatly arranged,which was better than that of GelMA/dHAM group and GelMA group.The wound tissue of control group was light blue,collagen sparse and disordered.[Conclusion]:dHAM can be grafted and modified successfully by MA,and the photocrosslinkable dHAMMA can be obtained.Based on photosensitive GelMA hydrogel,photocrosslinkable dHAMMA can be successfully compounded by photosensitizer to synthesize GelMA-dHAMMA composite hydrogel with BCN structure.GelMA-dHAMMA composite hydrogel not only retains the good mechanical properties and water retention of GelMA hydrogel,but also inherits the biological activity of dHAM,avoids the deficiency of bioactive components when GelMA hydrogel is used alone,and overcomes the limitation of uncontrollable mechanics and easy degradation when dHAM is used solo.The mechanical properties of GelMA-dHAMMA composite hydrogel are significantly stronger than that of dHAM,meanwhile,in terms of cell biological behavior,GelMA-dHAMMA is significantly better than GelMA hydrogel,and can promote angiogenesis.GelMA-dHAMMA composite hydrogel has a natural extracellular matrix component of dHAM.The BCN structure of GelMA-dHAMMA can better simulate the ECM microenvironment and can provide a stable environment for the proliferation and deposition of fibroblasts in the defect area.GelMA-dHAMMA composite hydrogel is beneficial to the adhesion,proliferation and differentiation of fibroblasts,thus showing its versatility.The improvement of cell performance can promote the repair and regeneration of oral mucosa tissue.Therefore,GelMA-dHAMMA composite hydrogel scaffold can be used as a very attractive and broad application prospect of oral mucosa substitute material to accelerate the repair and healing of defect.