Construction Of Bifunctional Bio-scaffold Based On Cell-loaded 3D Printing For Periodontal Tissue Regeneration

BackgroundPeriodontal disease is a chronic disease with oral microbial infections characterized by progressive destruction of periodontal support tissue.Numerous studies have confirmed that periodontitis is closely related to physical health and systemic diseases.Periodontal disease is a potential risk factor for systemic disease,and systemic disease may also promote or exacerbate the occurrence and development of periodontal disease.It can be seen that periodontal disease is extremely harmful to health.Control inflammation is the first step to treat periodontal disease,and the further step is to guide regeneration of the damaged tissue,such as guided tissue regeneration(GTR).Periodontal disease always leads to the destruction of both soft and hard periodontal tissues.Therefore,the ideal treatment requires the recovery of both of them at the same time.At present,the conventional GTR is not very effective,because it mainly acts as a barrier,and does not show the biological function of guiding tissue regeneration,which is far from meeting the needs of clinical treatment.Therefore,it is urgent to develop bioactive material,which is capable of effectively promoting the regeneration and repair of soft and hard tissues at the same time.Seed cells,scaffolds,and growth factors are the elements of ideal tissue engineering.Generally,stem cells are usually used as the seed cells,which have the potential of self-renewal and multi-directional differentiation.The scaffold provides a suitable environment for cell adhesion and stereoscopic growth of cells.Different growth factors can promote seed cells to proliferate and differentiate into different tissues.This project constructs a bifunctional bio-scaffold based on cell-loaded 3D printing,which can accurately locate and transport cells according to the model designed previously,and successfully print the ideal 3D structure.It not only promotes the regeneration of both hard and soft tissues in periodontal defect,but also provides new ideas and breakthroughs for the future therapy.ObjectiveTo prepare dual-function bioscaffold based on cell-loaded 3D printing in order to achieve the regeneration of both hard and soft tissues in periodontal defect at the same time.Material and Methods1.In part one,BG was prepared by the sol-gel method,and then Gel MA/SA scaffolds and BG/Gel MA/SA scaffolds were prepared in combination with 3D printing technology.Physical and chemical properties of the scaffolds were tested by scanning electron microscope(SEM),mechanical test,simulated mineralization experiment,swelling and degradation experiment.Moreover,the biocompatibility of the scaffolds was verified by cell proliferation,Live-Dead staining and cell adhesion while the osteogenesis of the scaffolds was tested by ALP and alizarin red staining,the quantification of alizarin red and ALP,and Real-Time quantitative PCR(RT-q PCR).2.In part two,bifunctional biological scaffolds based on cell-loaded 3D printing were prepared.The experiments in vitro were divided into three groups,including m MSCs scaffold-group,m MSCs/PDGF scaffold-group and m MSCs/BMP scaffold-group.The viability and biocompatibility of scaffolds were tested by Live-Dead staining and cell proliferation experiments.At the same time,the ALP activity and expression of genes related to osteogenesis and soft tissue were detected by the quantification of ALP and RT-q PCR.In addition,this researh also constructed acute periodontal defect models in beagle dogs to further explore the function of the scaffolds in vivo,which were divided into four groups: Blank-group,Scaffold-group,BMP/PDGF scaffold-group,and m MSCs/BMP/PDGF scaffold-group.Through Mirco-CT and histological examination,we explored the effect of soft and hard tissue regeneration of the dual-function scaffold based on cell-loaded 3D printing.Results1.The result of SEM showed that both scaffolds had interconnected porous structures.Mechanical test and swelling experiment showed both scaffolds had suitable mechanical properties and water content.The results of the simulated mineralization and degradation experiments indicated that the BG/Gel MA/SA scaffolds had higher mineralization ability and stability.Moreover,cell proliferation and cell adhesion experiments showed that both scaffolds had good biocompatibility.The results of ALP and alizarin red staining,the quantification of alizarin red and ALP,and RT-q PCR indicated that both Gel MA/SA and BG/Gel MA/SA scaffolds promoted the expression of genes related to osteogenesis,and the BG/Gel MA/SA scaffolds showed a more significant effect.2.Cell viability and cell proliferation experiments showed that the three kinds of scaffolds had good cell viability and biocompatibility.The result of quantification of ALP activity showed that m MSCs/BMP scaffold group significantly promoted osteogenic differentiation of m MSCs.The result of RT-q PCR showed that m MSCs/BMP scaffold-group significantly increased the expression of osteogenesis-related genes(OPN,OCN,Runx2),while m MSCs/PDGF scaffold-group obviously increased the expression of fibroblast-related genes(FN).In vivo experiment,the results of Mirco-CT and histological examination indicated that all experimental groups showed gingival regeneration and new bone formation.Moreover,the m MSCs/BMP/PDGF scaffold-group had the most significant effect for hard and soft tissue regeneration.ConclusionBG/Gel MA/SA scaffolds have good biocompatibility,mineralization ability,stability and osteogenesis.Based on BG/Gel MA/SA printing ink,m MSCs scaffolds,m MSCs/PDGF scaffolds and m MSCs/BMP scaffolds loaded with cells(m MSCs)and growth factors(PDGF,BMP)can be constructed,and all of them have good biocompatibility.Compared with m MSCs Scaffold-group,m MSCs/PDGF scaffolds have better ability of soft tissue differentiation,while the m MSCs/BMP scaffolds show better osteogenic effect.In vivo experiments,m MSCs/BMP/PDGF scaffolds possess superior ability of soft tissue formation and osteogenesis in the periodontal defect models of beagle dog.Therefore,bifunctional scaffolds based on cell-loaded3 D printing have the potential to be applied to compound periodontal defects.