| Background: Periodontal disease is an important disease that threatens human oral health.Clinical treatment methods for periodontal disease include the basic therapy combined with drugs,guide periodontal tissue regeneration,bone grafting and growth factor treatment.Although these methods promote tissue regeneration to a certain extent,still unable to meet the oral clinical requirements of periodontal bone tissue repair.Stem cell transplantation brings promise to the treatment of periodontal disease.However,the survival rate and cell activity of transplanted stem cells is low,which results in the poor treatment results due to the change of the microenvironment of damaged tissue in vivo.Tissue engineering therapy can improve the in vivo microenvironment that stem cells live in,and further enhance the tissue regeneration function of stem cell transplantation.Due to their good biological compatibility and biodegradability,gelatin hydrogels have been extensively studied.It is reported that polyethylene glycol?PEG?as a plasticizer can significantly improve the mechanical properties of gelatin hydrogels,so gelatin-PEG composite hydrogels may have great potential in periodontal tissue repair.The degradation property is a very important characteristic of tissue engineering scaffolds,which should be matched with the specific tissue repair.However,the degradation of gelatin-PEG composite hydrogel is not clear at present.In addition,the effect of the composite hydrogel on the affinity and osteogenic differentiation of stem cells remain to be further investigated.Aim: In this study,we prepared gelatin-PEG composite hydrogels,demonstrated the effect of main preparation parameters of composite hydrogels on the hydrogel degradation,and investigated the role of composite hydrogels in the adhension,growth and osteogenic differentiation of human umbilical cord mesenchymal stem cells.Methods: Firstly,we developed the methods for the preparation and degradation evaluation of composite hydrogels.Gelatin and PEG were dissolved in water,respectively.And these two solutions were mixed together.Then GA was added into the mixed solution to generate the stable gelatin-PEG composite hydrogel films via the cross-linking reaction at 4 °C for 18 h.Then in vitro enzymatic degradation experiments were carried out using I type collagenase solution to immerse the composite hydrogel films.After the vacuum drying,the hydrogel films were weighed and the degradation rate of these hydrogel films was calculated.After that,the Plackett-Burman experimental design was used to screen the significant factors that affected the degradation rate of composite hydrogels from three factors,including gelatin concentration?X1?,glutaraldehyde concentration?X2?and PEG concentration?X3?.Then,response surface method?RSM??box Behnken design?was used to investigate the interaction between these three factors?X1,X2 and X3?and the effect of these factors on the composite hydrogel degradation rate?response value Y?,and obtain a prediction mathematical model for composite hydrogel degradation rate.Then three composite hydrogels were randomly selected to verify the fit between the predicted values and the actual degradation rates,which indicated that this model had enough accuracy.Next,human umbilical cord mesenchymal stem cells were seeded on the surface of hydrogels with different preparation conditions.The cell counting was used to determine the initial adhesion cell number,and live/dead staining was used to observe cell activity and distribution.F-actin fluorescent staining was performed to evaluate the adhesion and spreading of stem cells on the hydrogel surface.In addition,CCK-8 was used to investigate stem cell proliferation.Finally,osteogenic induction medium was used to induce stem cells on the gel surface.Alkaline phosphatase?ALP?quantitative detection and alizarin red staining were used to investigate the effect of hydrogels with different preparation conditions on the osteogenic differentiation of stem cells.Significant factor screening and response surface design experiments were performed using Minitab and Design-Expert,other data were analyzed using Origin 8.5 software.Results: 1.Glutaraldehyde concentration was the significant factor affecting the degradation of gelatin-PEG composite hydrogel.2.Mathematical model for predicting the degradation rate of composite hydrogels was obtained: Y=2.23625-0.13467*X1-14.52844*X2+0.061178*X3+0.043950*X1*X2-0.00379432*X1*X3+0.23396*X2*X3+0.00975590*X12+27.47820*X22-0.023016*X32.The R2 of this equation was 0.97,and the p value was less than 0.0001,which indicated that this equation had a reliable predictive ability.3.The degree of agreement between the actual degradation rate and the predictive value calculated by the model was greater than 80%,which showed that this model had a high reliability.4.The effect of PEG on the composite hydrogel degradation might be regulated by glutaraldehyde concentration.When the higher glutaraldehyde concentration was used,the addition of PEG could significantly speed up the hydrogel degradation,while PEG could slow down the hydrogel degradation relatively when low glutaraldehyde concentration was employed.5.Compared with the conventional hydrogel without PEG,the composite hydrogel had no significant differences in cell adhesion,activity and proliferation.However,the composite hydrogel had a more compact gel structure.6.Compared with the conventional two-dimensional culture surface,the hydrogel surface?with or without PEG?could significantly improve the activity of ALP and promote calcium deposition,which significantly promoted the osteogenic differentiation of stem cells.In addition,it was found that compared with the conventional hydrogel,PEG did not significantly influence the osteogenic differentiation of stem cells.Conclusions: This study identified the glutaraldehyde concentration was the significant factor influencing the degrading of gelatin-PEG composite hydrogel.And a mathematical model with a high reliability for predicting the degradation rate was built by RSM,which made it possible to prepare the desired composite hydrogels with specific degradation rates.In addition,the effect of PEG on the degradation of the composite hydrogel might be regulated by the glutaraldehyde or gelatin concentration,and the addition of PEG could make the gel structure more compact.And the growth and adhesion of stem cells on the surface of composite hydrogels were good,and it was proved that the composite hydrogel had a good affinity with stem cells,which could be used as a carrier for stem cell transplantation.In addition,the composite hydrogel could promote the osteogenic differentiation of stem cells.Therefore,this study might provide new methods and new ideas for the regeneration of periodontal bone tissue. |
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