Evaluation Of Chitosan Thermo - Sensitive Hydrogel Containing BMP2 Plasmid DNA On Periodontal Fibroblast Compatibility

Purpose: The aim of this study was to prepared CS/CSn(p DNA-BMP2)-GP compound system. Chitosan nanoparticles was loaded with plasmid DNA of bone morphogenetic protein-2(p DNA-BMP2)(p DNA-BMP2). Chitosan/α,β-glycerophosphate(CS/α,β-GP) thermosensitive hydrogel was prepared by ionic crosslinking method. CS/CSn(p DNA-BMP2)-GP was constructed by mixing CSn(p DNA-BMP2) and the CS/α,β-GP thermosensitive hydrogel. The cytocompatibility of CS/CSn(p DNA-BMP2)-GP compound system was evaluated in vitro.Methods: 1. Preparation and characterization of CSn(p DNA-BMP2). Chitosan nanoparticles(CSn) were prepared with ionic cross-linking method. CSn were linked with plasmid DNA of bone morphogenetic protein-2(p DNA-BMP2)through electrostatic adsorption. The particle size, morphology and distribution were observed by scanning electron microscopy(SEM) and zeta-potential analysis.The binding ability and protective effect of plasmid DNA of bone morphogenetic protein-2(p DNA-BMP2) were evaluated by agarose gel electrophoresis analysis. 2.Preparation and the characterization of CS/CSn(p DNA-BMP2)-GP compound system:(1) Chitosan based thermosensitive hydrogel was prepared by chitosan andα, β-glycerophosphate(CS/α,β-GP) using ionic crosslinking method. Morphology of CS/α,β-GP thermosensitive hydrogel was observed by SEM. And temperature sensitivity of CS/α,β-GP thermosensitive hydrogel was identified using invert tube method.(2) CSn(p DNA-BMP2) were dispersed into CS/α,β-GP thermosensitive hydrogel. A novel CS/CSn(p DNA-BMP2)-GP compound system t was successfully constructed. Morphology and temperature sensitivity of CS/CSn(p DNA-BMP2)-GP compound system were identified.3. Evaluation the cytocompatibility of CS/CSn(p DNA-BMP2)-GP compound system to human periodontal ligament fibroblasts(HPDLCs) in vitro:(1) HPDLCs were cultured by tissue explant culture method.(2) HPDLCs was cultured in 3D culture system of CS/CSn(p DNA-BMP2)-GP compound system, and proliferation and morphology of HPDLCs were observed by crystal violet staining.(3) HPDLCs were cultured in the extraction of CS/CSn(p DNA-BMP2)-GP compound system.CCK8 assay showed the proliferation and morphology of HPDLCs under the microscope. Using SPSS18.0 statistical software to deal with the obtained data.Results:1.The particle dispersity of CSn and CSn(p DNA-BMP2) were uniform and in spherical shapes. The average diameter of CSn(p DNA-BMP2) is 270.1nm, PDI of0.486 and zeta potential of 27.0mv. The diameter of CSn(p DNA-BMP2) was larger than CSn. The average diameter of CSn is 170 nm, with PDI of 0.249, and the zeta potential of 45.5mv. The results of agar gel electrophoresis showed that relatively strong conjunction with different N/P ratio in 1:1、1.5:1、2:1、2.5:1 between CSn and p DNA-BMP2. DNAsel protection assay showed that CSn prevent p DNA-BMP2 from degradation at DNAsel concentrations with differernt N/P ratio in 1:1、1.5:1、2:1、2.5:1.2. The phase of CS/CSn(pDNA-BMP2)-GP compound system could change from the sol state to gel state at 37℃ in 3min, which indicated excellent thermo-sensitivity of it. SEM observation showed that CSn(p DNA-BMP2) were dispersed uniformly in the 3D network structure of CS/α,β-GP thermosensitive hydrogel.3.HPDLCs were successfully obtain in vitro using tissue explant culture method.Immunocytochemistry staining proved that HPDLCs is origined from mesoderm source with positive waveform silk protein and negative keratin. The results of crystal violet staining showed that the HPDLCs were in good condition and good proliferate activity after 48 h cultured. CCK8 results showed that different concentrations of extractions of CS / CSn(p DNA-BMP2)-GP composite system in1 d and 3d can promote the proliferation of HPDLCs. And the OD value in extraction of CS / CSn(p DNA-BMP2)-GP composite system is higher than the extraction of CS/α, β-GP thermosensitive hydrogel.Conclusion:1. CSn exhibited good shapes, proper diameter and zeta potential.2. CSn can bind and prevent p DNA-BMP2 from the degradation of DNase I. CSn( p DNA-BMP2) showed great shapes, proper diameter and zeta potential with N/Pratio at 2.5:1.3. CS/CSn(p DNA-BMP2)-GP compound system can promote the proliferation of HPDLCs and showed excellent cytocompatibility.