Evaluation Of Combined Application Of Dentin Noncollagenous Proteins And Odontogenic Biphasic Calcium Phosphate In Rabbit Maxillary Sinus Lifting

Objective To simulate biomimetic mineralization of bone tissue composition and structure is one of the hotspots in tissue engineering research.In this study,dentin noncollagenous proteins（DNCPs）were extracted as growth factors.At the same time,odontogenic biphasic calcium phosphate（BCP）was prepared by sintering teeth as scaffold material,and the protein was loaded on the scaffold.The biocompatibility of materials and proteins was verified in vitro.The effects of odontogenic BCP incorporating DNCPs on volume maintenance,new bone formation and new bone quality in bone reconstruction after maxillary sinus floor lifting were evaluated in the rabbit maxillary sinus lifting models.Methods The dentin powder was successively placed in guanidine hydrochloride（GdmHCl）,EDTA and Gdm-HCl,containing protease inhibitor to extract DNCPs and freezedried after dialysis.The constituents were identified by Western blot and Coomassie brilliant blue staining.In terms of scaffold materials,the extracted teeth were calcined in a sintering furnace to prepare BCP.X-ray diffraction（XRD）,elemental analysis and Brunauer–Emmett–Teller（BET）were used to detect the phase,composition,specific surface area,pore volume and pore width,and the material surface was observed by scanning electron microscopy（SEM）.Then the cells were seeded on the materials and DNCPs-loaded materials;and compared with deproteinized bovine bone mineral（DBBM,Bio-Oss）and β-TCP materials commonly used in clinic.The cell adhesion rate,proliferation activity,cell morphology,cytoskeleton and spreading status were detected.In the animal experiment,New Zealand white rabbits were selected for maxillary sinus lifting.BCP,DBBM and β-TCP were filled respectively.The osteogenic ability of the combined application of proteins and materials in the maxillary sinus environment was evaluated by Micro-CT and histological analyses.Results Coomassie blue staining results revealed that the molecular weights of DNCPs complexes were mainly distributed at about 55 k Da,70 k Da,130 k Da and 180 k Da.Western blot results showed that DNCP contained and expressed DSPP,DMP1,OPN and BMP2.The phases of BCP were hydroxyapatite（HA）and β-tricalcium phosphate（β-TCP）,（HA / β-TCP : 71 / 29）,and the calcium-phosphorus ratio was 1.627.Although the specific surface area of BCP is the smallest,its average pore size is the largest,and many 200 nm pores exist.In addition,the material has micropores of about 1 nm.The SEM showed that there were traffic pores like dentinal tubules openings on the BCP and granular structures about 1 μm.In vitro experiments showed that DNCPs had no significant effect on cell adhesion and proliferation.The cell adhesion rate and proliferation ability of β-TCP were higher than those of BCP and DBBM.On culture Day1,the cells on the material were fusiform or oval,with elongated filopodia extending,and some cells began to spread.On culture Day 3,the cell spreading area increased,the morphology was normal and the proliferation was obvious.Micro-CT results showed that the sinus lifting tissue volume（TV）of β-TCP group was significantly lower than the TV of DBBM group and BCP group（P < 0.05）.The ratio of bone volume to total volume（BV/TV）of BCP-DNCPS group was significantly higher than the BV/TV of BCPControl and β-TCP-DNCPS groups.There was no significant difference in bone mineral density（BMD）,bone trabecular number（Tb.N）and other indicators among groups（P > 0.05）,while the bone trabecular separation（Tb.Sp）of BCP material group and DNCPs subgroup was significantly smaller（P < 0.05）,suggesting that their bone quality was higher.Histological analysis also showed that β-TCP materials could not maintain the space of maxillary sinus elevation.The new bone area ratio of BCP-Control and DBBM-Control groups was greater than that of β-TCP-Control group,and the difference was significant（P < 0.05）.Moreover,significant differences in the average integral optical density（IOD）values of green area were found between the Control subgroup and DNCPs subgroup of the three materials（P < 0.05）,indicating that DNCPs may promote bone matrix mineralization.Conclusion The combined application of DNCPs and odontogenic BCP in maxillary sinus lifting can well maintain the bone reconstruction volume after sinus floor lifting,promote the formation of new bone and improve the quality of new bone.