Electrospun Fibrous Scaffolds Combined With Nanoscale Hydroxyapatite Induce Osteogenic Differentiation Of Human Periodontal Ligament Cells

Periodontitis is a common chronic inflammatory disease. Periodontal defects resulting from periodontitis exhibit significant destruction of periodontal supported apparatus, eventually leading to loss of teeth if untreated. The ultimate goal of the treatment is the regeneration of the destroyed tissues. Periodontal repair is a complex process in which regeneration of osseous tissue is a vital component.It may be hypothesized that periodontal regeneration, based on bone repair, can be achieved through the use of biomaterials capable of inducing tissue regeneration combined with appropriate scaffolds. With the development of nanomaterials, it is discovered that hydroxyapatite, presents in the form of nanoscale monocrystal in the bone, is distributed in collagen stroma uniformly. Therefore, the direct introduction or in situ synthesis of nHA inserted inside or outside collagen fibers can obtain a biomimic scaffold. In this study, we embedded nHA in collagen to produce fibers with a structure conducive to the proliferation, metabolism, and osteogenic differentiation of human PDLCs. This fibrous scaffold might be innovatively applied in regeneration of alveolar bone regeneration.Part I The fabrication and characterization of COL/PCL/nHA-SBFObjective:To develop the COL/PCL/nHA-SBF scaffold and characterize its physical and chemical propertiesMethods:The COL/PCL/nHA-SBF was produced by electrospinning which was followed by being immersed in a 1.5 times simulated blood fluid (SBF) for 7 days. The characteristics of the scaffolds were evaluated by scanning electronic microscope (SEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction analysis (XRD), and water contact angle analysis (WCA).Results:All the scaffolds had a highly porous network with interconnected pores. The diameters of the ruleless fibers are between 300-400nm. Compared with COL/PCL, COL/PCL/nHA had a rough surface because of the nHA, which was demonstrated by EDX and XRD, packed inside the fibers. Precipitation of calcium phosphate was visible on COL/PCL/nHA-SBF after incubation for 7 days in SBF. The results of EDX and XRD showed that the crystal structure of the calcium phosphate is similar to hydroxyapatite. In addition, the introduction of nHA in COL/PCL/nHA-SBF leads to an increase in hydrophilicity.Conclusion:The COL/PCL/nHA-SBF scaffold produced by electrospinning followed by biomimetic mineralization was composed of electrospun fibers containing nHA.. The scaffold, with good hydrophilicity, had a rough surface because of the nHA packaged inside and deposited outside the fibers.Part Ⅱ The isolation and identification of periodontal ligament cellObjective:To isolate, culture and identify the origin of human periodontal ligament cells (PDLC).Methods:PDLC, isolated by explant method, was measured by immunohistochemical staining with anti-vimentin and anti-keratin antibody.Results:PDLC gradually climbed out from the organizations and proliferated stably on the tenth day of primary culture. With active proliferation, PDLC, which could cover the bottom of cell culture flasks until no less than 90% ten days later, were passaged using 0.25% trypsin and further expanded until passage 4. The immunohistochemistry of the PDLC obtained positive staining of vimentin and negative staining of kerati.Conclusion:PDLC was isolated successfully by explant method. The result of immunohistochemistry indicated that the PDLC did derive from mesodermic cells.Part III The biocompatibility and osteoinductive ability of COL/PCL/nHA-SBFObjective:To evaluate the biocompatibility and osteoinductive ability of COL/PCL/nHA-SBF.Methods:The morphology, cytoskeletal system, proliferation and differentiation of human periodontal ligament cells (PDLCs) seeded on the scaffold were examined by SEM, CLSM, cell counting kit and real-time quantitative polymerase chain reaction (PCR) analysis which focused on alkaline phosphatase, runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and osteopontin (OPN).Results:PDLC adhered to the COL/PCL/nHA-SBF scaffolds with obvious cilium, filopodia and actin filament had good growth activity. CCK8 assay showed that both scaffolds supported cell proliferation without any significant difference. However, real-time quantitative PCR analysis showed that expressions of the bone-related markers, including Runx2, OCN and OPN, was upregulated only on the COL/PCL/nHA-SBF scaffold, which might initially start mineralizaiton process.Conclusion:COL/PCL/nHA-SBF, which could promote the adhesion and proliferation of cells, had good biocompatibility and osteoinductive ability.In this study, we adopted the method of electrospinning followed by biomimetic mineralization to produce a COL/PCL/nHA-SBF scaffold composed of electrospun fiers containing nHA. The scaffold obtained had good biocompatiblity and osteoinductive ability, and may be innovatively applied to periodontal tissue engineering as a potential scaffold.