| Objective: Periodontitis is recognized as a chronic inflammatory disease destroying periodontal support tissues(gingiva,the periodontal ligament and alveolar bone).Slowly over time,the teeth will become mobile and due to lack of the support from the surrounding bone,teeth may collapse and then tooth loss in severe periodontitis.Severe periodontitis is one of the main causes of tooth loss that occurs in later life,a condition that has significant impacts on the quality of life in individuals.Studies of modalities to treat periodontitis have been constantly performed by many investigators.Conventionally,efforts have been focused on periodontal scaling and root planing,periodontal flap surgery,and bone grafting.However,these therapies cannot truly achieve the regeneration of alveolar bone,with lacking ability of self-repair and re-construction in periodontal tissue.Thus,it is imperative to develop a method to accelerate alveolar bone regeneration in severe periodontitis with alveolar bone loss.With the rapid development of tissue engineering technology,tissue engineering regeneration technology provides a new strategy to the regeneration of human tissues and organs.In the field of dentistry,periodontal tissue engineering need three elements: seed cells,growth factors,and three-dimensional scaffolds to achieve periodontal bone regeneration.It provides a new possibility for the treatment of periodontitis with a periodontal bone defect.Human periodontal ligament is a heterogeneous group of cells composed of a variety of cells.Human periodontal ligament cells(hPDLCs)are the main cell component of the periodontal ligament with the potential to multi-differentiates into fibroblasts,cementoblasts,and osteoblasts.In periodontal tissue engineering,hPDLCs are potential “seed cells”.Besides,periodontal membrane cells play an essential role in periodontal tissue regeneration due to their ability to differentiate into osteoblasts.Bone regeneration is a complex process involving multiple growth factors with different functions.Bone morphogenetic proteins(BMPs)play an essential role in bone regeneration and have been extensively studied by many scholars.BMPs are a member of the transforming growth factor-?(TGF-?)superfamily,which are very important in embryonic development and bone formation.Bone morphogenetic protein 9(BMP9)has significantly stronger osteogenic differentiation ability than other BMPs,which has been proved to be one of the most influential factors in inducingosteogenic differentiation and an essential restorative osteogenic growth factor.As the main inorganic component of human bone tissue,hydroxyapatite(HA)can adsorb bone induction factors such as BMPs and vascular endothelial growth factor(VEGF)in the fabric.It has excellent biocompatibility,bone conductivity,bone induction,as well as mechanical strength.However,HA have a hydrophobic surface and a low degradation rate.Therefore,HA was combined with tricalcium phosphate(TCP)to form hydroxyaptite-tricalcium phosphate(HA-TCP),which can significantly improve the surface hydrophobicity of HA and increase the low degradation rate of HA,showing better biological performance.The structure of HA-TCP is porous and similar to human bone cancellous material,which is conducive to the adhesion,growth,and proliferation of cells.Gradually,HA-TCP also can be degraded and replaced by bone tissue in the body.Therefore,it is an ideal artificial bone scaffold material.The present study constructed new complex of recombinant adenoviral viruses BMP9(Ad-BMP9)transfected hPDLCs and HA-TCP scaffold materials for the first time.To explore the osteogenic differentiation ability of the new complex and implant the new compound into subcutaneous tissue of nude mice and the periodontal bone defect area of SD rats,to determine the function of periodontal bone repair,and provide a new idea for the regeneration of periodontal bone tissue.Methods: The experiment was divided into the following two parts:1.The effects of transfection of Ad-BMP9 on osteogenic differentiation of hPDLCs: Human derived periodontal ligament was digested with type I collegenase and primary h PDLCs were culture by tissue adhenrent method.Cell morphology of primary hPDLCs was observed under inverted phase contrast microscope on the 3rd,and 8th day.Cell morphology of the third generation hPDLCs was observed under an inverted phase contrast mincroscope.Ad-BMP9 was transfected into the third generation of hPDLCs.Fluorescence expression was observed under an inverted fluorescence microscope to screen suitable adenovirus transfection concentration.Quantitative real-time PCR(qRT-PCR)and Western blot(WB)were used to detect the transfection of Ad-BMP9.The early and late osteogenic differentiation of Ad-BMP9 was detected by ALP staining,ALP activity,alizarin red S staining,and alizarin red semi-quantitative analysis.2.The effects of the Ad-BMP9-transfected hPDLCs and HA-TCP scaffold complex on promoting periodontal bone regeneration: The Ad-BMP9 transfected hPDLCs and HA-TCP scaffold complex were constructed in vitro,and scanning electron microscope(SEM)was used to observe the adhesion,growth and proliferation of cells on the complex on the 1st,3rd,5th and 7th day respectively.qRT-PCR was used to detect the expression of ALP in the early stage of the complex and the manifestationof osteocalcin(OCN)and osteopontin(OPN)in the late stage.ALP activity was used to detect the early osteogenesis of the new complex.In vivo experiments,the complex was implanted subcutaneously in nude mice and rat periodontal bone defect areas.Results: 1.On the 3rd day,hPDLCs crawled out of the tissue mass,and on the 8th day,the cells crawled.The third generation of hPDLCs was mainly fusiform,with abundant cytoplasm and uniform morphology.After hPDLCs transfection with adenovirus at different concentrations,green fluorescence expression was observed under an inverted phase-contrast microscope,and the appropriate adenovirus transfection concentration was selected.qRT-PCR and WB results showed that after transfection of hPDLCs with Ad-BMP9,BMP9 expression was significantly increased at the gene and protein levels.ALP staining,ALP activity,alizarin red S staining,and alizarin red semi-quantitative analysis all indicated that Ad-BMP9 could promote the osteogenic differentiation of hPDLCs.2.After the construction of the Ad-BMP9-transfected hPDLCs and HA-TCP scaffold complex in vitro,the SEM observation results showed the Ad-BMP9-transfected hPDLCs could adhere,grow,and proliferate on the HA-TCP scaffold material.The complex is loose and porous,and a large number of pores can be seen on the surface.qRT-PCR results showed that the ALP expression of the compound reached its peak on the 5th day of osteogenesis induction,and the OCN and OPN expressions of lateosteogenic markers were significantly increased.The activity of ALP showed that the osteogenic differentiation ability of the complex was significantly enhanced.After 6 weeks of subcutaneous implantation of the complex in nude mice and periodontal defects in rat,histological analysis showed that the complex significantly increased new bone formation in both heterotopic and situ animal experiments.Conclusion: Ad-BMP9 promotes early and late osteogenic differentiation of hPDLCs.HA-TCP has good biocompatibility,and Ad-BMP9-transfected hPDLCs can stick,grow,and proliferate on HA-TCP.In this study,Ad-BMP9,hPDLCs,and HA-TCP were used as three elements of tissue engineering to construct the scaffold complex of Ad-BMP9-transfected hPDLCs and HA-TCP.In vitro experiments,the combination of Ad-BMP9-transfected hPDLCs and HA-TCP scaffolds enhanced early and late osteogenic differentiation.In vivo experiments,the Ad-BMP9-transfected hPDLCs and HA-TCP scaffold complex can effectively promote the formation of new bone in heterotopic location and promote the regeneration of periodontal bone tissue in situ,which provides a new idea for the development of periodontal tissue regeneration engineering. |
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