【作者】 隋欣；

【导师】 刘志辉；

【作者基本信息】 吉林大学 ， 口腔医学（专业学位）， 2022， 硕士

【摘要】 研究目的:大面积骨缺损缺乏自行愈合的能力,需利用骨替代材料为其提供支持以促进其成骨,理想的骨替代材料需要具有足够的机械强度,充足的孔隙率,良好的生物活性及生物相容性并且来源充足。3D打印能够构建出满足上述要求的骨支架材料并且可以根据患者需要制备成特定的形状。在众多的3D打印技术中,最常用的是挤出型3D打印,因为其设置相对简单,运行成本低,打印速度快。本研究欲通过碱/尿素溶液通过反复冻-融的方式溶解壳聚糖(Chitosan,CS),以期获得更高机械强度及良好生物相容性的基质材料,随后将纳米羟磷灰石(Hydroxyapatite,HAP)及冻干的富血小板纤维蛋白(Lyophilized platelet-rich fibrin,L-PRF)粉末复合到基质中共同打印,赋予支架良好的骨传导及释放生长因子的能力。通过对最终的复合支架进行相关的检测,以期筛选出性能优良的骨支架材料。研究方法:1、通过反复冻-融的方式使CS溶解在碱/尿素溶液中,将不同质量分数的HAP加入CS溶液中配制CS-HAP生物墨水,流变学检测出HAP质量分数为20%时复合油墨的粘度最大,并将其作为后续实验的配比。2、将质量分数分别为0.5%、1%、2.5%的L-PRF粉末加入筛选后CS-HAP墨水中,并用醋酸-明胶溶液对其流变性能进行优化。并对优化后的PRF-CSHAP(P-C-H)复合油墨进行流变性能检测以评估其打印适性。随后在4℃条件下对P-C-H复合油墨进行3D打印,并用37℃去离子水浸泡使其凝胶化,最终采用冷冻干燥技术去除三维支架中多余的溶剂及水分。3、通过傅里叶变换红外光谱(FTIR)及X射线衍射实验(XRD)分别对复合支架进行成分及晶体结构分析,通过扫描电镜(SEM)对复合支架的孔径及内部结构进行观察。4、对复合支架进行机械性能,亲水性,降解性能及缓释性能检测,随后将支架与MC3T3-E1细胞进行共培养,检测复合支架的生物相容性及PRF中生长因子的活性。研究结果:1、分别配制了C-H、0.5%P-C-H、1%P-C-H及2.5%P-C-H复合油墨,经流变学分析其均具有“剪切稀释”行为,并且具有温敏的特性,储能模量与温度呈正相关。2、成功将4种油墨打印出三维支架,经过SEM扫描观察到4组支架的孔径约为500-700μm,并且线条连续均匀;通过FTIR分析,在图谱中发现了CS及HAP的特征峰;通过XRD分析发现支架体系中的HAP晶体结构并没有发生改变。3、随着加入L-PRF的增加,支架的压缩性能逐渐降低,但是仍然满足骨松质的机械强度;4组支架均表现出了较好的吸水能力,吸水率均超过了300%;降解性能检测后发现复合支架均具有较稳定的降解速度,但是2.5%P-C-H组的支架在降解80天后出现了线条断裂的情况,随后进行的p H检测得知支架降解后的p H值大多保持在7.5-8.4之间。4、对复合支架进行持续35天的缓释性能测定,发现复合支架在35天内均能检测到生长因子的释放,释放水平随着时间的延长呈下降的趋势;MC3T3-E1细胞与4组支架共培养后发现4组支架均能促进细胞的增殖,其中2.5%P-CH组的促细胞增殖能力最佳,证明了复合支架具有良好的生物相容性并且PRF经过冻干及打印后仍然具有活性。研究结论:2.5%P-C-H支架具有良好的生物相容性及促进细胞增殖的能力,虽然其机械强度相比于其他组有所下降,但是仍然满足骨松质的强度要求,因此认为2.5%P-C-H组为最有潜力的骨支架复合材料。更多还原

【Abstract】 Purposes:Large-scale bone defects lack self-healing ability,so bone substitute materials are used to support and promote their osteogenesis.An ideal bone substitute material needs to have sufficient mechanical strength,sufficient porosity,good bioactivity and biocompatibility,and sufficient supply.3D printing can fabricate bone scaffolds that meet the above requirements and can be prepared into specific shapes according to the needs of patients.Among various 3D printing techniques,extrusion-based printing is most commonly adopted due to its relatively simple set-up,low operation cost,and high printing speed.In this study,chitosan(CS)was dissolved by a repeated freezethaw method in alkali/urea solution,to obtain a matrix material with higher mechanical strength and good biocompatibility.Subsequently,hydroxyapatite(HAP)and lyophilized platelet-rich fibrin(L-PRF)powder were combined into the CS solution for co-printing,which endows the scaffold with good osteoconductivity and the ability to release growth factors.The final composite scaffolds were tested to screen out bone scaffolds with excellent performance.Method:1.CS is dissolved in alkali/urea solution by repeated freeze-thaw method,and then different mass fractions of HAP are added into CS solution to prepare CS-HAP bioink.The viscosity of the composite ink was the largest when the mass fraction of HAP was 20% by rheology,and it was used as the ratio of the subsequent experiments.2.L-PRF powders with mass fractions of 0.5%,1%,and 2.5% were added to the screened CS-HAP bioink,and the rheological properties were optimized with acetic acid-gelatin solution.The rheological properties of the optimized PRF-CS-HAP(P-CH)composite ink was tested to evaluate its printability.Subsequently,the P-C-H composite ink was 3D printed at 4℃,and crosslinked with deionized water at 37℃.Then,the excess water and solvent in the 3D scaffold were removed by freeze-dried.3.The composition and crystal structure of the composite scaffolds were analyzed by Fourier transform infrared spectroscopy(FTIR)and X-ray diffraction(XRD),respectively,and the pore size and internal structure of the composite scaffolds were observed by scanning electron microscopy(SEM).4.The mechanical,hydrophilic,degradation,and sustained-release properties of the composite scaffolds were tested.Subsequently,the scaffolds were co-cultured with MC3T3-E1 cells to detect the biocompatibility of the composite scaffolds and the activity of growth factors in PRF.Results:1.C-H,0.5%P-C-H,1%P-C-H,and 2.5%P-C-H composite inks were prepared respectively.The rheological analysis showed that all of them had "shear-thinning" behavior and were thermo-sensitive,which were shown as storage modulus was positively correlated with temperature.2.The 3D scaffolds were successfully printed with four kinds of inks.After SEM scanning,it was observed that the pore diameter of the four scaffolds was about 500-700 μm,and the lines were continuous and uniform.According to FTIR analysis,the characteristic peaks of CS and HAP were found in the spectrum;through XRD analysis,it was found that the crystal structure of HAP in the scaffold did not change.3.With the addition of L-PRF,the compressive properties of the scaffolds gradually decreased,but they still met the mechanical strength of cancellous bone.All4 groups of scaffolds showed good water absorption capacity,and the water absorption rate exceeded 300%.After the degradation performance test,it was found that the composite scaffolds had a relatively stable degradation rate,but the 2.5%P-CH scaffolds had broken lines after 80 days of degradation.Subsequent p H detection showed that the p H value of the scaffolds after degradation was mostly maintained between 7.5-8.4.4.The sustained release performance of the composite scaffolds was measured for 35 days,and it was found that the release of growth factors could be detected in the composite scaffolds within 35 days,and the release level showed a decreasing trend with the extension of time.After MC3T3-E1 cells were co-cultured with 4groups of scaffolds,it was found that all 4 groups of scaffolds could promote cell proliferation,and the 2.5% P-C-H scaffolds had the best ability to promote cell proliferation,which proved that the composite scaffolds had good biocompatibility and PRF was still active after lyophilization and printing.Conclusion:The 2.5% P-C-H scaffold has good biocompatibility and the ability to promote cell proliferation.Although its mechanical strength is lower than that of other groups,it still meets the strength requirements of cancellous bone.Therefore,the 2.5% P-C-H group is considered to be the most potential composite material for bone scaffolds.更多还原