| PartⅠPreparation and characterization of mesoporous bioglass/small intestinal mucosal acellular matrix biomaterialsObjective To investigate the preparation method of mesoporous bioglass and the characterization of its material system after surface coating of small intestine submucosal acellular matrix.Methods（1）4.0g P123,6.7g tetraethyl orthosilicate,1.4g Ca（NO3）2?4H2O,0.73g triethyl phosphate and 1.0g 0.5M hydrochloric acid dissolved in 60g ethanol（Si/Ca/P=80:15:5,molar ratio）,and stirred at room temperature for 1 d to obtain a mesoporous bioglass precursor fluid;（2）Then 40ppi polyurethane sponge was completely immersed in this solution for 10 minutes,transferred to a petri dish,the excess solution was removed,and the solution was evaporated at room temperature for 12 hours.This step was repeated 5 times;（3）After drying at 60℃ for 48 hours,the sample was calcined in a muffle furnace at 700℃（with a temperature increase rate of 1℃/min）for 5 hours to obtain MBG scaffolds.The MBG scaffold was immersed in a dopamine solution and stirred at room temperature for 12 hours;（4）The SIS extracted through a series of steps was ball-milled in a ball mill to acquire SIS solution（0.5%）,and the SIS solution and the MBG scaffold were coated by a freeze-drying method to obtain a three-dimensional porous scaffold MBG/SIS;（5）Observation of morphology and structure by transmission electron microscope（TEM）and electron scanning microscope（SEM）,qualitative analysis of chemical structure of material surface by Fourier infrared spectrometer（FTIR）,and detection of porosity and mechanical strength of MBG/SIS scaffold.Results（1）TEM results indicate that the MBG scaffold has an ordered mesoporous structure with a pore size of about 5 nm.The SEM results showed that the MBG scaffold and the MBG/SIS scaffold had good interconnected structures;（2）SEM analysis showed that the surface of pure MBG scaffolds was relatively smooth,while a thin film was deposited on the surface of composite MBG/SIS scaffolds;（3）FTIR results showed that SIS successfully combined with MBG,and MBG/SIS had better compressive strength than MBG scaffolds.Conclusion MBG scaffolds with excellent compressive strength and interpenetrating structure can be successfully prepared by polyurethane foam template.SIS can successfully coat the surface of MBG scaffolds,change the surface microstructure of MBG scaffolds and strengthen its compressive strength.PartⅡ Experimental study on the in vitro osteogenesis of small molecule peptides delivered by bioglass / natural extracellular matrix compositesObjective To investigate the effects of small molecule peptide combined with mesoporous bioglass/small intestinal submucosal decellularized（MBG/SIS）biomaterials on the biological behaviors of MC3T3-E1 cells.Methods（1）MC3T3-E1 cells were cultured on MBG / SIS scaffolds,MBG / SIS-P28 scaffolds,and MBG / SIS-H-P28 scaffolds at appropriate densities,placed in 6-well plates and incubated in basic culture medium;（2）MTT cell proliferation experiments were performed after 1/3/5 / 7d of culture;（3）Calcein-AM / PI cell live/dead test was performed at 3 / 7d of culture;（4）Alkaline phosphatase（ALP）qualitative and quantitative were performed at 7 / 14 d of culture;（5）Qualitative and quantitative experiments of Alizarin Red（ARS）were performed at 14/21 days of culture;（6）After 7/14 days of culture,PCR experiments were performed to detect and analyze osteogenic related gene expression of Runx2,ALP,Ocn,and Opn.Results（1）MTT cell proliferation results showed that the scaffolds of the three different groups can promote cell proliferation.Compared with the MBG / SIS and MBG / SIS-P28 groups,the MBG / SIS-H-P28 group has the highest OD value and has statistics significance（P <0.05）;（2）Calcein-AM / PI results showed that no obvious dead cells were seen on different scaffolds,and the trend was consistent with the results of MTT;（3）The qualitative and quantitative results of ALP staining showed that MBG / SIS-H-P28 significantly promoted ALP activity compared with the MBG / SIS group;（4）ARS staining results showed that the MBG / SIS-H-P28 group had a darker color than the simple control group,indicating that more minerals were formed in this group;（5）The results of PCR analysis showed that the expression levels of bone related genes Runx2,ALP,Ocn,and Opn in the MBG / SIS-H-P28 scaffolds were significantly higher than those in the MBG / SIS group,and there was a statistical difference（P <0.05）.Conclusion The MBG/SIS-H-P28 scaffold has excellent biocompatibility.Compared with the MBG/SIS scaffold,the MBG/SIS-H-P28 scaffold offering a cell-friendly environment was onducive to cell proliferation and osteogenic differentiation.Part Ⅲ Experimental study on the repair of cranial bone defects in rats by delivery of small molecule peptides using bioglass / natural extracellular matrix compositesObjective To evaluate the effect of MBG/SIS-H-P28 porous composite scaffolds in repairing bone defects in vivo by rat calvarial critical-sized defect.Methods（1）According to different groups of biological materials,30 SD rats（male）were randomly divided into three groups: MBG / SIS group;MBG / SIS-P28 group;MBG / SIS-H-P28 group;（2）The scaffolds were sterilized at 37 ° C with ethylene oxide to implant into the right skull defect;（3）After 6/12 weeks of surgery,SD rats were sacrificed by intraperitoneal injection of high-dose chloral hydrate.Skull specimens were collected and soft tissues were removed to perform Micro-CT scans.The software was used to calculate the new bone area percentage（BV / TV%）;（4）The free specimen was decalcified by 4% paraformaldehyde,embedded in paraffin embedding method,and then sectioned for HE staining;（5）The Paranomic viewer system was used to take pictures of the defect area.The results of the new bone area can be further analyzed by Image Pro Plus software.Results（1）Micro-CT results showed that compared with MBG / SIS group,MBG / SIS-P28 group and MBG / SIS-H-P28 group had significant new bone formation.BV / TV% quantitative analysis showed that at 6/12 weeks,the BV / TV% of MBG / SIS-H-P28 group and MBG / SIS-P28 group was significantly higher than that of MBG / SIS group（p <0.05）;（2）After panoramic scanning of HE sections,it was observed that the MBG / SIS group was mainly filled with fibrous connective tissue 6 weeks after the operation,and the new bone began to grow from the defect margin.Compared with the simple control group,more new bone formation was observed in the MBG / SIS-P28 and MBG / SIS-H-P28 groups;（3）At 12 weeks after operation,a large amount of new bone was observed in the MBG / SIS-H-P28 group under high-power HE staining,while the pure MBG / SIS group was filled in a small amount of new bone.At the same time,quantitative analysis of the new bone area in the defect area showed that the new bone area of the MBG / SIS-P28 and MBG / SIS-H-P28 groups was significantly higher than that of the MBG / SIS control group at weeks 6 and 12.Conclusion The MBG/SIS scaffold has good compatibility and osteoinductivity.MBG/SIS-P28 significantly enhances bone regeneration compared with MBG/SIS group,indicating that BMP-2 related small peptides can significantly induce new bone formation.The MBG/SIS-H-P28 scaffolds provides a friendly bone regeneration microenvironment and has the best bone regeneration effect. |
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