Study Of Micro-Nano Structured Hydroxyapatite Bioceramics In Regeneration Of Alveolar Bone Defect

Objectives:The objective of this present study was to evaluate the capacity of a tissue-engineering bone complex of combinant micro-nano structured hydroxyapatite(mnHA)bioceramics and dental pulp stem cells(DPSCs)to reconstruct critical-size alveolar bone defect in rats.Firstly,The DPSCs were isolated and the biological characteristics and osteogenic differentiation capacity were investigated.Secondly,the attachment and differentiation capability of DPSCs were established when cultured on traditional HA bioceramics with dense and flat surface and mnHA bioceramics with micro-nano structured surface in vitro.Finally,the regeneration effect on alveolar bone defect was evaluated in vivo.Materials and methods:1.Stro-1 positive human DPSCs were purified from dental pulp cells(DPCs)by magnetic-activated cell sorting.The characteristics of the cells were analyzed by CCK8 for proliferation,immunofluorescence staining for expression of Vimentin,S-100,Stro-1 and cytokeratin(CK),flow cytometric analysis for expression of stem cell markers CD29,CD44,CD90,CD 105,CD34 and CD45.2.Human DPSCs were induced for multilineage differentiation including osteogenic and adipogenic lineages respectively.Adipogenic potential was established by oil red O staining,while osteogenic capability was evaluated by alkaline phosphatase(ALP)staining and alizarin red staining.Furthermore,the expression level of osteogenic genes ALP,Collagen I(Col I),runt-related transcription factor 2(Runx2),osteocalcin(OCN)mRNA was established by Real-time PCR.3.Rat DPSCs were isolated and cultured,seeded on HA bioceramics with micro-nano structured surface(mnHA)and traditional HA bioceramics with dense and flat surface(HA).The attachment,proliferation,osteogenic and angiogenic ability of DPSCs were investigated by MTT,confocal laser scanning microscopy,scanning electron microscopy,alkaline phosphatase activity and quantitative Real-time PCR.4.Rat alveolar bone defects were established and filled with(1)traditional HA bioceramics with smooth and dense surface(HA),(2)HA bioceramics with micro-nano structured surface(mnHA),(3)HA bioceramics combined with rat DPSCs(HA+DPSCs),(4)mnHA bioceramics combined with rat DPSCs(mnHA+DPSCs)and(5)remained empty(Control)during implantation.The harvested samples were evaluated by micro-computed tomography,sequential fluorescent labeling of TE,AL and CA and histological images at week 8 to observe the new bone formation.Results:1.Human DPSCs were successfully isolated and purified by modified tissue culture with magnetic cell sorting.Majority of cells were spindle and exhibited high proliferation ability.Phenotype analysis revealed that human DPSCs expressed mesenchymal stem cell markers including CD44,CD90,CD 105,CD29,Stro-1 and negative for hematopoietic stem cell markers including CD34,CD45.2.Human DPSCs can form mineralized nodules or lipid vacuoles when induced to osteogenic or adipogenic lineages.Moreover,human DPSCs showed high osteogenic potential in vitro.When compared with the control group,ALP staining of the osteogenic induced DPSCs was significantly higher.Furthermore,the expression levels of osteogenic genes including ALP,Col I,Runx2 and OCN were up-regulated in osteogenic induced DPSCs.3.When seeded on HA bioceramics with 3D micro-nano structured surface(mnHA),DPSCs possesses greater cell proliferation,cell adhesion,ALP activity,enhanced mRNA expression levels of osteogenic genes including Runx2,OCN,dentin sialophosphoprotein(DSPP),dentin matrix protein-1(DMP-1)and angiogenic genes including vascular endothelial growth factor(VEGF),angiopoietin-1(Ang-1)compared with the traditional samples with flat and dense surface.4.In rat alveolar bone defect model,bone volume relative to tissue volume(BV/TV),trabecular thickness(Tb.Th),trabecular number(Tb.N),sequence fluorescent labeling area and new bone formation for the repaired groups were higher than those for control group.Moreover,the mnHA group had higher BV/TV,Tb.Th,Tb.N,sequence fluorescent labeling area and new bone area than those for HA group,while combination with DPSCs could further enhance new bone formation.Conclusions:1.DPSCs can be isolated and purified by modified tissue culture with magnetic cell sorting.Phenotype analysis revealed that human DPSCs expressed mesenchymal stem cell markers and exhibited osteogenic and adipogenic differentiation potential when induced in vitro.Moreover,the osteogenic capability of DPSCs was outstanding.2.HA bioceramics with micro-nano structured surface significantly enhanced the cell attachment,spreading,proliferation,and ALP activity of DPSCs compared to traditional HA bioceramics with dense and flat surface.Moreover,the mnHA bioceramics could not only enhance osteogenic but also angiogenic gene expression of DPSCs.This effect may be due to that the micro-nano hybrid surface topography of mnHA bioceramics was similar to the structure of nature bone.3.In rat alveolar bone defect model,mnHA bioceramics with micro-nano structured surface combined with rat DPSCs can significantly enhance new bone formation and mineralization.It is suggested that the HA bioceramics with micro-nano structured surface could act as promising grafts for alveolar bone regeneration.