CN114099775A - 负载SDF-1α/CGRP的HAp-SF人工骨膜及其制备方法 - Google Patents
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Abstract
本发明公开了负载SDF‑1α/CGRP的HAp‑SF人工骨膜及其制备方法,属于骨科植入材料领域,是由可降解高分子为主要原材料,并加入SDF‑1α和CGRP因子,通过静电纺丝的方法制备负载SDF‑1‑α和CGPR因子的nHA/SF可吸收人工骨膜,所制备的人工骨膜具有负载和缓释SDF‑1‑α和CGPR因子的性能,使其具有良好的成骨成血管活性和生物相容性。此外,本发明可在缺损局部发挥屏障作用,能够有效阻挡周围软组织长入以及异位骨化的发生,人工骨膜还具有较好的力学强度及生物可降解性,可作为骨组织缺损的修复材料使用。
Description
技术领域
本发明属于生物医学材料技术领域,具体涉及负载SDF-1α/CGRP的 HAp-SF人工骨膜及其制备方法。
背景技术
骨缺损是骨科临床的常见疾病,可由创伤、感染及肿瘤等多种因素引起, 大段骨缺损是骨科治疗临床中的挑战之一。当前,骨移植是临床最常使用的治 疗方法,即通过植入支架填充骨缺损,来诱导骨再生。但植入骨支架后,缺损 区域发生被动的连接和填充过程,缺少材料对骨缺损微环境的主动识别。虽然 有部分支架整合入外源性活性因子,具有促进血管新生及骨再生的作用,但仍 难以发生体内自然的骨重建过程,因此并未获得满意的治疗效果(T.G a rg,A.K.Goyal,Biomaterial-based scaffolds--current status andfuture directions,Expert Opin Drug Deliv,11(2014)767-789)。骨膜在骨再生中具有重要 的作用,据报道,骨膜移植能够成功修复骨组织缺损,如:文献 J.F.Funk,G.Matziolis,D.Krocker,C.Perka,Promotion of bone healing throug h clinical application of a utologousperiosteum derived stem cells in a case of atrophicnon-union,Z OrthopUnfall,145(2007)790-794中的报道。移植骨膜可在 缺损部位募集更多骨修复相关细胞,能够诱导形成血管网络和骨结构。因此, 通过移植骨膜和骨填充材料来提高骨再生能力是行之有效的方法。此外,骨膜 可在缺损局部充当屏障结构,避免周围软组织长入和异位骨化的发生,为缺损 修复提供良好的微环境。但是,自体骨膜或同种异体骨膜来源有限,且存在疾 病传染和免疫排斥的风险(X.Zhang,H.A.Awad,R.J.O 'Keefe,R.E.Guldberg,E.M.Schwarz,Aperspective:engineeringperiosteum for structuralbone graft healing,ClinOrthopRelat Res,466(2008)1777-1787.)。因此, 通过模拟天然骨膜的结构和功能来人工合成骨膜材料具有良好的发展前景。
静电纺丝技术可以生产出直径为纳米级聚合物纤维,所制备纤维形貌可控、 柔性好,具有高比表面积和孔隙率,且纤维形貌类似细胞外基质,因此在生物 医学工程领域得到广泛的关注J.X u e,J.X i e,W.L i u,Y.X i a,ElectrospunNanofibers:NewConcepts,Materials,and Applications,AccChem Res,50(2017)1976-1987。
基质细胞衍生因子-1α(SDF-1α)由基质细胞分泌,具有细胞趋化作用, 可介导干细胞归巢,如趋化内皮细胞,造血干细胞和骨髓间充质干细胞等 【180,181】。大量研究报道SDF-1α促进破骨细胞生成和干细胞生长发育,调 节成骨分化【182-185】。
降钙素基因相关肽(CGRP)是由感觉神经元突触释放的生物活性小分子 神经肽。CGRP可以扩张血管,增加血流量,是一种有效的血管扩张剂,而且 还是一种促血管生成和促淋巴管生成因子[186-188]。CGRP通过上调血管内皮 细胞中的VEGF型来诱导血管生成[186,189,190]。在骨膜生理机制中,骨膜 中的感觉神经末端释放CGRP,骨膜内增多的CGRP可进一步促进骨膜内干细 胞的成骨分化[191]。
目前,还没有成熟的“人工骨膜”产品在临床中应用,现有研究中的人工 骨膜有:①经电喷技术制备的负载BMP、VEGF等生物活性因子的可降解高分 子多孔膜(王身国,王宏鹤,刘浩,任红文.生物可吸收人工骨膜及其制备方 法.2008-02-27.);②使用纳米钙磷盐与胶原纤维经压制、冷冻干燥等过程制备 的矿化胶原人工骨膜(陶春生,仇志烨,宋天喜,张自强,崔福斋.一种矿化胶 原人工骨膜及其制备方法.2014-10-15.)等,尚未见具有缓释SDF-1α和CGRP 功能的人工骨膜的研究及相关产品。因此,有必要开发一种能够缓释SDF-1α和CGRP因子的人工骨膜,以改善大段骨缺损修复临床治疗的难题。
发明内容
为了克服上述技术缺点,本发明的目的在于提供一种负载SDF-1α和 CGRP因子的人工骨膜及其制备方法,以改善大段骨缺损修复的临床治疗。
为了达到上述目的,本发明采用以下技术方案予以实现:
负载SDF-1α/CGRP的HAp-SF人工骨膜的制备方法,该负载SDF-1-α和CGPR因子的nHA/SF人工骨膜是在以高分子溶液为溶剂的高分子材料溶液中, 加入负载CGRP因子的GPTMS改性nHA悬液,然后通过静电纺丝法制得负 载CGRP因子的GPTMS-HAp-SF膜,再将膜在多巴胺中改性,后置于含有生 物活性因子SDF-1α的溶液中浸泡后,制得负载SDF-1α和CGRP因子的 HAp-SF人工骨膜;所述负载SDF-1-α和CGPR因子的nHA/SF人工骨膜的纤 维具有无规则排列或定向排列的纳米纤维结构。
进一步地,所述负载SDF-1α和CGRP因子的HAp-SF人工骨膜厚度为 500~1000μm,纤维直径为0.5-1.5μm。
进一步地,所述高分子材料为可降解天然高分子材料或可降解合成高分子 材料。
进一步地,所述可降解天然高分子材料为丝素蛋白。
进一步地,无机材料为纳米羟基磷灰石,羟基磷灰石HAp占人工骨膜质 量的2%。
进一步地,所述高分子溶液为六氟异丙醇。
进一步地,所述丝素蛋白高分子材料占人工骨膜质量的7-8%。
进一步地,所述多巴胺是为了增强SDF-1-α因子粘附和装载。
进一步地,所述HAp表面改性为GPTMS,增加其水溶液的稳定性。
进一步地,人工骨膜中SDF-1-α的负载量,质量百分比:1:2*105~1:4*105。
进一步地,人工骨膜中CGRP的负载量,质量百分比:1:100~1:200。
一种负载SDF-1α/CGRP的HAp-SF人工骨膜,包括以下步骤:
1)丝素提取,采用碱脱胶法对天然蚕丝进行脱胶
取去蛹蚕茧100-120g浸入0.02-0.04mol/L的Na2CO3溶液中,加热至95℃, 不断搅拌30-60分钟,捞出丝素,用去离子水冲洗3-5次,置于60-80℃烘箱中 烘干,得到丝素;
2)丝素蛋白溶液的配置
首先,配置丝素溶液,取出脱胶后干燥过的丝素蛋白,溶于摩尔比为1:2: 8的CaCl2,CH3CH2OH,H2O三元溶剂中,溶解过程中加热至70-80℃,不断 搅拌直到丝素完全溶解,用脱脂纱布过滤溶解后的丝素溶液除去不溶解的杂质, 得到蚕丝蛋白溶液;
之后冷却溶液,将丝素蛋白溶液装入3500分子量的透析袋中,将透析袋 置于去离子水中,并于4℃冰箱中低温透析,4-6h更换一次透析液,直至完全 去除溶剂中的无机盐离子,透析结束后将蚕丝蛋白溶液倒出,冷冻干燥,得到 丝素蛋白;
最后,将冻干后的丝素蛋白按质量体积比为8:100-10:100的比例溶于六氟 异丙醇(HFIP)中,室温下用磁力搅拌子以1000-1200转/分的速度搅拌,搅 拌24-48h,得到丝素蛋白溶液;
3)羟基磷灰石(HAp)表面改性处理
将HAp与浓度为2-2.5%v/v的GPTMS(-γ(2,3-环氧丙氧基)丙基三甲氧基 硅烷)乙醇溶液混合,60-70℃下以100-120rpm搅拌2-4h;
在80-90℃下继续加热2-4h,使乙醇在对流炉中蒸发;然后将GPTMS-HAp 在120-130℃真空烘箱中干燥24-48h;将GPTMS改性的HAp悬浮于蒸馏水中, 得到浓度为20-30wt%的悬浮液;
4)羟基磷灰石(HAp)悬浮液负载CGRP因子
将所得20-30wt%的GPTMS-HAp悬液超声处理5-8min,加入CGRP因子, 继续搅拌6-8h,得到负载CGRP因子的GPTMS-HAp悬浮液;
5)静电纺丝溶液配置
将所得负载CGRP因子的GPTMS-Hap悬浮液与所得丝素蛋白溶液按质量 比1:10-1.5:10的比例混合,搅拌6-8h,得到静电纺丝GPTMS-HAp-SF溶液;
6)静电纺丝负载CGRP因子的GPTMS-HAp-SF膜
采用19号静电纺丝针头,调节接收距离为10cm,加正高压12kV,负高 压1kV;调节液体流速0.5mL/h的速度,5mL注射器,接收滚筒转速600r//min; 得到负载CGRP的GPTMS-HAp-SF膜;
7)将上述膜浸入多巴胺溶液中(0.2mg/mL,10mMTris-HCl缓冲液,pH 8.5) 中室温浸泡10min,取出负载CGRP的GPTMS-HAp-SF膜,用去离子水清洗, 去除残留多巴胺,然后置于含有生物活性因子SDF-1α的溶液中浸泡10min后 取出,冷冻干燥,即得到负载SDF-1α/CGRP的HAp-SF人工骨膜。
与现有技术相比,本发明的有益效果是:
本发明公开的负载SDF-1α/CGRP因子的人工骨膜的制备方法,使用可降 解天然高分子化合物为基体材料,既具有优异的生物相容性,又具有良好的力 学性能;添加SDF-1α/CGRP因子增加了人工骨膜对大鼠骨膜来源干细胞的趋 化作用促与进干细胞成骨分化功能。同时,添加SDF-1α/CGRP因子的人工骨 膜具有良好的促血管生成作用。
同时,通过使用静电纺丝法制备的纳米纤维膜作为人工骨膜,具有如下特 点:第一,能够最大程度模拟细胞外基质结构,利于细胞的迁移、粘附与生长; 第二,大的比表面积使人工骨膜易于负载SDF-1α/CGRP因子;第三,高孔隙 率利于细胞的粘附、营养和代谢产物交换以及生物信号交流。纳米孔还能够阻 止细菌及纤维组织的长入,减少感染与异位骨化的发生。
本发明利用高分子化合物与SDF-1α/CGRP因子的特性和静电纺丝技术 制备具有成骨功能的纳米纤维膜,有作为人工骨膜的潜在价值,其具体优势如 下:
1、大比表面积——有利于细胞粘附;
2、负载SDF-1α因子——对干细胞产生趋化作用;
3、负载CGRP因子——促血管再生;
4、高孔隙率——有利于营养和代谢产物交换、细胞信号交流;
5、小于组织细胞尺寸的孔——防止感染与异位骨化;
6、可降解性——植入后无需二次手术取出;
7、良好的生物相容性——不易发生免疫排斥;
8、药物缓释——延长药物作用时间。
附图说明:
图1为本发明制得脱胶并冻干后的丝素蛋白;
图2为本发明制得人工骨膜的实物照片;
图3为本发明制得人工骨膜的扫描电镜图像;
图4为本发明制得人工骨膜的SDF-1α/CGRP因子体外释放曲线图;
图5为本发明制得人工骨膜对细胞增殖的影响;
图6为本发明制得人工骨膜对细胞趋化作用的影响;
图7为本发明制得人工骨膜对细胞成骨标志物之一碱性磷酸酶(ALP)活 性的影响;
图8为本发明制得人工骨膜对细胞其他成骨标志物表达活性的影响;
图9为本发明制得人工骨膜对细胞成血管活性的影响;
图10为本发明制得人工骨膜对新西兰兔股骨原位灭活骨再活化缺损修复 的影响。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施 例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所 描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发 明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所 有其他实施例,都应当属于本发明保护的范围。
此外,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排 他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备 不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于 这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述:
参见图1,为本发明公开的负载SDF-1α和CGRP因子的人工骨膜的脱胶 后丝素蛋白原料,由可降解天然高分子为主要原材料,并加入具有成骨、细胞 趋化、成血管功能的SDF-1α和CGRP因子,通过静电纺丝的方法制备负载 CGRP因子的羟基磷灰石丝素蛋白共混人工骨膜,通过表面吸附SDF-1α,得 到负载SDF-1α和CGRP因子的人工骨膜,具备缓释因子的效果。
一、具体实施例
实施例1制备CGRP-HAp-SF人工骨膜
步骤1:取去蛹蚕茧100g浸入0.02mol/L的Na2CO3溶液中,加热至95℃, 不断搅拌30分钟,捞出丝素,用去离子水冲洗3-5次,置于60℃烘箱中烘干, 得到丝素;
步骤2:配置摩尔比为1:2:8的CaCl2,CH3CH2OH,H2O三元溶剂
步骤3:将步骤1脱胶后干燥过的丝素蛋白溶于步骤2中,加热至70℃, 不断搅拌直到丝素完全溶解;
步骤4:用脱脂纱布过滤步骤3溶解后的丝素溶液,除去不溶解的杂质, 得到蚕丝蛋白溶液;
步骤5:将步骤4所得丝素蛋白溶液装入3500分子量的透析袋中,将透析 袋置于去离子水中,并于4℃冰箱中低温透析,4-6h更换一次透析液,直至完 全去除溶剂中的无机盐离子;
步骤6:将步骤5透析后的蚕丝蛋白溶液倒出,冷冻干燥,得到丝素蛋白;
步骤7:将步骤6所得丝素蛋白,按质量体积比为8:100的比例溶于六氟 异丙醇(HFIP)中,室温下用磁力搅拌子以1000转/分的速度搅拌24h,得到 丝素蛋白溶液;
步骤8:配置浓度为2%(v/v)的GPTMS(-γ(2,3-环氧丙氧基)丙基三甲氧基 硅烷)乙醇溶液;
步骤9:将羟基磷灰石颗粒(Hap)与步骤8所得溶液混合,60℃下以120rpm 搅拌2h;
步骤10:将步骤9所得混合液在80℃下继续加热2h,得到GPTMS-HAp 在120℃真空烘箱中干燥24h;
步骤11:将步骤10得到GPTMS-Hap悬浮于蒸馏水中,得到浓度为20wt% 的悬浮液;
步骤12:将步骤11所得20wt%的GPTMS-HAp悬液超声处理5-8min, 加入CGRP因子,继续搅拌6-8h,得到负载CGRP因子的GPTMS-HAp悬浮 液;
步骤13:将步骤12所得负载CGRP因子的GPTMS-Hap悬浮液与步骤7 所得丝素蛋白溶液按1:10的比例混合,搅拌6-8h,得到GPTMS-HAp-SF静电 纺丝溶液;
步骤14:将步骤13所得静电纺丝溶液进行静电纺丝,采用19号静电纺丝 针头,调节接收距离为10cm,加正高压12kV,负高压1kV;调节液体流速 0.5mL/h的速度,5mL注射器,接收滚筒转速600r//min,得到厚度为500~1000μm 负载CGRP的GPTMS-HAp-SF膜;
步骤15:配置多巴胺溶液(0.2mg/mL,10mM Tris-HCl缓冲液,pH 8.5);
步骤16:配置含有生物活性因子SDF-1α的溶液;
步骤17:将步骤14所得负载CGRP的GPTMS-HAp-SF的静电纺丝膜浸 入步骤15所得多巴胺溶液,室温浸泡10min,用去离子水清洗,然后置于步 骤16所得含有生物活性因子SDF-1α的溶液中浸泡10min后取出,冷冻干燥, 即得到负载SDF-1α/CGRP的HAp-SF人工骨膜。
实施例2
步骤1:取去蛹蚕茧110g浸入0.03mol/L的Na2CO3溶液中,加热至95℃, 不断搅拌45分钟,捞出丝素,用去离子水冲洗3-5次,置于70℃烘箱中烘干, 得到丝素;
步骤2:配置摩尔比为1:2:8的CaCl2,CH3CH2OH,H2O三元溶剂
步骤3:将步骤1脱胶后干燥过的丝素蛋白溶于步骤2中,加热至75℃, 不断搅拌直到丝素完全溶解;
步骤4:用脱脂纱布过滤步骤3溶解后的丝素溶液,除去不溶解的杂质, 得到蚕丝蛋白溶液;
步骤5:将步骤4所得丝素蛋白溶液装入3500分子量的透析袋中,将透析 袋置于去离子水中,并于4℃冰箱中低温透析,4-6h更换一次透析液,直至完 全去除溶剂中的无机盐离子;
步骤6:将步骤5透析后的蚕丝蛋白溶液倒出,冷冻干燥,得到丝素蛋白;
步骤7:将步骤6所得丝素蛋白,按质量体积比为9:100的比例溶于六氟 异丙醇(HFIP)中,室温下用磁力搅拌子以1100转/分的速度搅拌36h,得到 丝素蛋白溶液;
步骤8:配置浓度2.25%(v/v)的GPTMS(-γ(2,3-环氧丙氧基)丙基三甲氧基 硅烷)乙醇溶液;
步骤9:将羟基磷灰石颗粒(Hap)与步骤8所得溶液混合,65℃下以120rpm 搅拌3h;
步骤10:将步骤9所得混合液在80℃下继续加热2h,得到GPTMS-HAp 在120℃真空烘箱中干燥24h;
步骤11:将步骤10得到GPTMS-Hap悬浮于蒸馏水中,得到浓度为25wt% 的悬浮液;
步骤12:将步骤11所得25wt%的GPTMS-HAp悬液超声处理5-8min, 加入CGRP因子,继续搅拌6-8h,得到负载CGRP因子的GPTMS-HAp悬浮 液;
步骤13:将步骤12所得负载CGRP因子的GPTMS-Hap悬浮液与步骤7 所得丝素蛋白溶液按1.25:10的比例混合,搅拌6-8h,得到GPTMS-HAp-SF 静电纺丝溶液;
步骤14:将步骤13所得静电纺丝溶液进行静电纺丝,采用19号静电纺丝 针头,调节接收距离为10cm,加正高压12kV,负高压1kV;调节液体流速 0.5mL/h的速度,5mL注射器,接收滚筒转速600r//min,得到厚度为500~1000μm 负载CGRP的GPTMS-HAp-SF膜;
步骤15:配置多巴胺溶液(0.2mg/mL,10mM Tris-HCl缓冲液,pH 8.5);
步骤16:配置含有生物活性因子SDF-1α的溶液;
步骤17:将步骤14所得负载CGRP的GPTMS-HAp-SF的静电纺丝膜浸 入步骤15所得多巴胺溶液,室温浸泡10min,用去离子水清洗,然后置于步 骤16所得含有生物活性因子SDF-1α的溶液中浸泡15min后取出,冷冻干燥, 即得到负载SDF-1α/CGRP的HAp-SF人工骨膜。
实施例3
步骤1:取去蛹蚕茧120g浸入0.04mol/L的Na2CO3溶液中,加热至95℃, 不断搅拌60分钟,捞出丝素,用去离子水冲洗3-5次,置于80℃烘箱中烘干, 得到丝素;
步骤2:配置摩尔比为1:2:8的CaCl2,CH3CH2OH,H2O三元溶剂
步骤3:将步骤1脱胶后干燥过的丝素蛋白溶于步骤2中,加热至80℃, 不断搅拌直到丝素完全溶解;
步骤4:用脱脂纱布过滤步骤3溶解后的丝素溶液,除去不溶解的杂质, 得到蚕丝蛋白溶液;
步骤5:将步骤4所得丝素蛋白溶液装入3500分子量的透析袋中,将透析 袋置于去离子水中,并于4℃冰箱中低温透析,4-6h更换一次透析液,直至完 全去除溶剂中的无机盐离子;
步骤6:将步骤5透析后的蚕丝蛋白溶液倒出,冷冻干燥,得到丝素蛋白;
步骤7:将步骤6所得丝素蛋白,按质量体积比为10:100的比例溶于六氟 异丙醇(HFIP)中,室温下用磁力搅拌子以1200转/分的速度搅拌48h,得到 丝素蛋白溶液;
步骤8:配置浓度为2.5%(v/v)的GPTMS(-γ(2,3-环氧丙氧基)丙基三甲氧 基硅烷)乙醇溶液;
步骤9:将羟基磷灰石颗粒(Hap)与步骤8所得溶液混合,70℃下以120rpm 搅拌4h;
步骤10:将步骤9所得混合液在80℃下继续加热2h,得到GPTMS-HAp 在120℃真空烘箱中干燥24h;
步骤11:将步骤10得到GPTMS-Hap悬浮于蒸馏水中,得到浓度为30wt% 的悬浮液;
步骤12:将步骤11所得30wt%的GPTMS-HAp悬液超声处理5-8min, 加入CGRP因子,继续搅拌6-8h,得到负载CGRP因子的GPTMS-HAp悬浮 液;
步骤13:将步骤12所得负载CGRP因子的GPTMS-Hap悬浮液与步骤7 所得丝素蛋白溶液按1.5:10的比例混合,搅拌6-8h,得到GPTMS-HAp-SF静 电纺丝溶液;
步骤14:将步骤13所得静电纺丝溶液进行静电纺丝,采用19号静电纺丝 针头,调节接收距离为10cm,加正高压12kV,负高压1kV;调节液体流速 0.5mL/h的速度,5mL注射器,接收滚筒转速600r//min,得到厚度为500~1000μm 负载CGRP的GPTMS-HAp-SF膜;
步骤15:配置多巴胺溶液(0.2mg/mL,10mM Tris-HCl缓冲液,pH 8.5);
步骤16:配置含有生物活性因子SDF-1α的溶液;
步骤17:将步骤14所得负载CGRP的GPTMS-HAp-SF的静电纺丝膜浸 入步骤15所得多巴胺溶液,室温浸泡10min,用去离子水清洗,然后置于步 骤16所得含有生物活性因子SDF-1α的溶液中浸泡20min后取出,冷冻干燥, 即得到负载SDF-1α/CGRP的HAp-SF人工骨膜。
二、本发明制得的负载SDF-1α/CGRP的HAp-SF人工骨膜的性能检测
1.人工骨膜的体外SDF-1α/CGRP因子释放曲线图
将直径约1.5cm大小的SDF-1α/CGRP人工骨膜圆片浸于1mLPBS缓冲液 中,于37℃下连续搅拌,并于6、12h、1、3、7、10、14d时,取出培养板吸 取上清液备用。采用SDF-1α因子ELISA检测试剂盒(MM-21265R2,江苏 酶免和CGRP因子ELISA检测试剂盒(MM-0817H2,江苏酶免)对上清液中 的SDF-1α、CGRP因子浓度进行检测。
从图4的释放曲线可以看到,本发明制得的人工骨膜中的SDF-1α和 CGRP因子能够在14天内实现稳定释放,由此满足骨缺损修复过程所需的释 放性能要求。
2.人工骨膜对细胞增殖的影响
将SDF-1α/CGRP人工骨膜圆片置于96孔板底部,按照5×103/孔的密度 接种大鼠股骨的骨膜来源干细胞(rPDSC)于37℃,5%CO2培养箱中培养, 隔天换培养基。于种板的第1、3、5、7天采用CCK-8检测试剂盒对孔板中细 胞活力进行检测。
结果如图5所示,nHA/SF、CGRP-nHA/SF、SDF-1α-nHA/SF、SDF-1α /CGRP-nHA/SF各组人工骨膜各组对rPDSC细胞的增殖没有明显的影响,细胞 增殖能力在各组无明显差异。每次至少3个样本,实验重复3次。
3.人工骨膜对细胞趋化作用的影响
将人工骨膜裁剪成24孔板孔大小的圆片,放置在transwell小室的下室内, 将生长状态良好的rPDSC消化离心后,重悬,以浓度为106/mL接种在transwell 小室的上室,下室加入常规的DMEM培养基500μL,放于5%的二氧化碳的 培养箱中培养24h后,取出培养板,去除培养基,用棉签轻轻将上室底面滤膜 上的细胞抹去,然后将滤膜放在甲醇中固定10min取出滤膜并用结晶紫染液染 色15min,显微镜下观察并拍照。随机选取5个不同的视野,统计穿过滤膜的 细胞数并分析,P<0.05和P<0.01定义为具有统计学差异。
结果如图6所示,负载了SDF-1α的SDF-1α-nHA/SF人工骨膜组与SDF-1 α/CGRP-nHA/SF人工骨膜组对rPDSC细胞具有明显的趋化作用,其穿过 transwell小室底面膜的细胞明显多于未负载SDF-1α的nHA/SF与 CGRP-nHA/SF人工骨膜组。
4.人工骨膜对细胞碱性磷酸酶(ALP)活性的影响
将人工骨膜裁剪成24孔板孔大小的圆片,放置在24孔板中。每孔加入成 骨诱导培养基2mL,放于培养箱中24h后取出培养板,收集培养板上的细胞培 养基,备用。
ALP测定,将rPDSC细胞以1x106/mL接种到24孔板中,400μL/孔,置 于5%二氧化碳培养箱过夜,次日,更换培养基为上述成骨诱导培养基,隔日 更换,于成骨诱导第7、14d取出细胞培养板,吸弃培养基,PBS清洗2遍, 然后每孔加入0.2%triton X-100裂解液4℃过夜。利用碱性磷酸酶检测试剂盒 (P0321M,碧云天)和BCA试剂盒(P0009,碧云天)测定ALP的相对含量。 P<0.05和P<0.01定义为具有统计学差异。
ALP染色:细胞培养同ALP测定。于成骨诱导第7、14d取出细胞培养板 去除培养基,PBS清洗2遍,后每孔加入00μL的4%多聚甲醛固定液(P0099, 碧云天)常温固定10min,PBS冲洗3遍,每次5min。然后利用BCIP/NBT碱 性磷酸酯酶显色试剂盒C3206,碧云天),对细胞内ALP进行染色。于显微镜 下观察,并拍照。
结果如图7所示,CGRP-nHA/SF、SDF-1α/CGRP-nHA/SF人工骨膜组表 现出明显地促rPDSC细胞成骨分化的作用。
5.人工骨膜对细胞其他成骨标志物表达活性的影响;
将人工骨膜裁剪成6孔板孔大小的圆片,放置在6孔板中。将rPDSC以 106/mL的浓度,每孔加入2mL细胞悬液,在培养箱中培养,并隔日更换细胞 培养基。于7、14天取出细胞培养板,采用E.Z.N.A.Total RNA Kit I(R6834-01 Omega Bio-Tek,美国)试剂盒提取细胞内的总RNA,利用qPCR(One-Step gDNA Removal)试剂盒(AE341-02北京全式金生物技术有限公司,中国北京) 进行逆转录,利用All-in-OneTMqPCR Mix试剂盒(P003,广州易锦生物技术有 限公司,中国广州)对cDNA实时定量扩增检测。PCR所使用的引物序列列 于表中。P<0.05和P<0.01定义为具有统计学差异。
结果如图8所示,ALP、BMP-2基因在SDF-1α/CGRP-nHA/SF和 CGRP-nHA/SF人工骨膜组在7、14天d都明显的上调。Col-Ⅰ基因在7d各组 间无显著性差异,但是随着时间的延长,于第14天在SDF-1α/CGRP-nHA/SF 和CGRP-nHA/SF人工骨膜组的表达都有显著的上调,分别达到nHA/SF人工 骨膜组的3.2倍和2.4倍。OPN基因的相对表达量在SDF-1α/CGRP-nHA/SF和CGRP-nHA/SF人工骨膜组的第14天有上升的趋势,但是在各组间的表达 没有表现出显著性差异。
6.人工骨膜对细胞成血管活性的影响;
将人工骨膜裁剪成6孔板孔大小的圆片,放置在6孔板中。将HUVEC细 胞以106/mL的浓度,每孔加入2mL细胞悬液,在培养箱中培养,并隔日更换 细胞培养基。1、3天时,E.Z.N.A.Total RNA Kit I(R6834-01 Omega Bio-Tek, 美国)试剂盒提取细胞内的总RNA,利用全式金的All-in-One First-Strand cDNA Synthesis SuperMix for qPCR(One-StepgDNA Removal)试剂盒 (AE341-02,北京全式金生物技术有限公司,中国北京)进行逆转录利用 All-in-OneTMqPCR Mix试剂盒(QP003,广州易锦生物技术有限公司,中国广 州对cDNA实时定量扩增检测。PCR所使用的引物序列列于表中。P<0.05和 P<0.01定义为具有统计学差异。
结果如图9所示,VEGF基因在nHA/SF、CGRP-nHA/SF、SDF-1α-nHA/SF、SDF-1α/CGRP-nHA/SF组中的表达表现出逐渐上升的趋势,且各组间的差异 具有显著性差异。
7.人工骨膜对新西兰兔股骨灭活骨再活化修复的影响。
实验动物:30只雄性新西兰大白兔,体重2-2.5kg/只。新西兰大白兔来源 于南方医科大学动物实验中心,饲养在洁净级环境中,常规进食进水。
1)术前给予3%的戊巴比妥钠30mg/kg耳缘静脉注射麻醉。
2)手术处理过程:麻醉功后,碘伏酒精消毒双侧后肢股骨远端皮肤,沿 股骨长轴切开约5cm切口,向两侧分离筋膜及肌肉,在肌肉组织与股骨之间垫 上湿盐水纱布隔离开,充分显露股骨。保留骨膜及部分肌肉组织覆盖在股骨上, 股骨中下段位置处,于股骨外侧钻取一直径约2mm大小微波消融孔,穿破一 侧皮质骨,直达髓腔。然后以此孔为中心,间隔0.5cm向两侧分别钻取两个测 温孔,测温孔直径约1.0mm大小,同样也只穿透一侧皮质骨。然后在消融孔 插入直径约2mm的微波消融针,设定功率20w模拟临床骨肿瘤的消融治疗过程,红外测温仪监测局部消融温度,维持温度在75-85持续时间5min,充分灭 活局部骨段,灭活后,除去股骨表面坏死的骨膜组组,生理盐水冲洗干净,然 后将植入人工骨膜材料包裹在消融灭活后的股骨周围,缝合肌肉、皮下组织及 皮肤。
3)所有新西兰大白兔手术后予以肌肉注射青霉素钠40万单位/只,连续 三天抗感染。并注意监测新西兰大白兔的活动及生活状况。局部伤口愈合情况。
Micro-CT检测不同骨膜材料的成骨修复作用。对微波热消融骨段的皮质 骨代谢重构的动态分析,对消融部位上下各5mm区域进行扫面。并对扫描区 域骨代谢相关指标:骨矿物密度(BMD)骨体积分数(BV/TV)进行测量分 析。
结果如图10所示,无论4周后还是12周后,未添加因子的nHA/SF人工骨膜组的灭活骨未见明显的骨吸收和骨痂形成。CGRP-nHA/SF人工骨膜组的灭活骨的骨吸收和骨痂形成主要在灭活骨与正常骨的交界处。SDF-1α-nHA/SF人工骨膜组早期即4周时骨痂形成与CGRP-nHA/SF组类似,而在12周时,在整个灭活骨段都有明显的骨吸收和骨痂形成。而相对于其他三组,SDF-1α/CGRP-nHA/SF组的整个灭活骨段在4周)和12周都有明显的骨吸收及大量骨痂的形成。通过对骨密度的定量检测,发现骨密度在早期各组都有降低,然后随着骨痂的形成和重塑,骨密度逐渐升高,且在 SDF-1α/CGRP-nHA/SF人工骨膜组骨密度升高速度最明显(图 4-图10)。通过对骨体积比及骨表面积进行定量分析,BV/TV与 BS在SDF-1α/CGRP-nHA/SF人工骨膜组较其他三组高。说明在同样的体积中,SDF-1α/CGRP-nHA/SF人工骨膜组所含的骨组织成分最多,成骨转化最活跃。
Claims (10)
1.负载SDF-1α/CGRP的HAp-SF人工骨膜的制备方法,其特征在于,该负载SDF-1-α和CGPR因子的nHA/SF人工骨膜是在以高分子溶液为溶剂的高分子材料溶液中,加入负载CGRP因子的GPTMS改性nHA悬液,然后通过静电纺丝法制得负载CGRP因子的GPTMS-HAp-SF膜,再将膜在多巴胺中改性,后置于含有生物活性因子SDF-1α的溶液中浸泡后,制得负载SDF-1α和CGRP因子的HAp-SF人工骨膜;
所述负载SDF-1-α和CGPR因子的nHA/SF人工骨膜的纤维具有无规则排列或定向排列的纳米纤维结构。
2.根据权利要求1所述负载SDF-1α/CGRP的HAp-SF人工骨膜的制备方法,其特征在于,所述负载SDF-1α和CGRP因子的HAp-SF人工骨膜厚度为500~1000μm,纤维直径为0.5-1.5μm。
3.根据权利要求1所述的负载SDF-1α/CGRP的HAp-SF人工骨膜,其特征在于,所述高分子材料为可降解天然高分子材料或可降解合成高分子材料。
4.根据权利要求3所述的负载SDF-1α/CGRP的HAp-SF人工骨膜,其特征在于,所述可降解天然高分子材料为丝素蛋白。
5.根据权利要求1所述的负载SDF-1α/CGRP的HAp-SF人工骨膜,其特征在于,羟基磷灰石HAp占人工骨膜质量的2%。
6.根据权利要求1所述的负载SDF-1α/CGRP的HAp-SF人工骨膜,其特征在于,所述高分子溶液为六氟异丙醇。
7.根据权利要求1所述的负载SDF-1α/CGRP的HAp-SF人工骨膜,其特征在于,所述丝素蛋白高分子材料占人工骨膜质量的7-8%。
8.根据权利要求1所述的负载SDF-1α/CGRP的HAp-SF人工骨膜,其特征在于,人工骨膜中SDF-1-α的负载量,质量百分比:1:2*105~1:4*105。
9.根据权利要求1所述的负载SDF-1α/CGRP的HAp-SF人工骨膜,其特征在于,人工骨膜中CGRP的负载量,质量百分比:1:100~1:200。
10.根据权利要求1所述的负载SDF-1α/CGRP的HAp-SF人工骨膜,其特征在于,包括以下步骤:
1)丝素提取,采用碱脱胶法对天然蚕丝进行脱胶
取去蛹蚕茧100-120g浸入0.02-0.04mol/L的Na2CO3溶液中,加热至95℃,不断搅拌30-60分钟,捞出丝素,用去离子水冲洗3-5次,置于60-80℃烘箱中烘干,得到丝素;
2)丝素蛋白溶液的配置
首先,配置丝素溶液,取出脱胶后干燥过的丝素蛋白,溶于摩尔比为1:2:8的CaCl2,CH3CH2OH,H2O三元溶剂中,溶解过程中加热至70-80℃,不断搅拌直到丝素完全溶解,用脱脂纱布过滤溶解后的丝素溶液除去不溶解的杂质,得到蚕丝蛋白溶液;
之后冷却溶液,将丝素蛋白溶液装入3500分子量的透析袋中,将透析袋置于去离子水中,并于4℃冰箱中低温透析,4-6h更换一次透析液,直至完全去除溶剂中的无机盐离子,透析结束后将蚕丝蛋白溶液倒出,冷冻干燥,得到丝素蛋白;
最后,将冻干后的丝素蛋白按质量体积比为8:100-10:100的比例溶于六氟异丙醇(HFIP)中,室温下用磁力搅拌子以1000-1200转/分的速度搅拌,搅拌24-48h,得到丝素蛋白溶液;
3)羟基磷灰石(HAp)表面改性处理
将HAp与浓度为2-2.5%v/v的GPTMS(-γ(2,3-环氧丙氧基)丙基三甲氧基硅烷)乙醇溶液混合,60-70℃下以100-120rpm搅拌2-4h;
在80-90℃下继续加热2-4h,使乙醇在对流炉中蒸发;然后将GPTMS-HAp在120-130℃真空烘箱中干燥24-48h;将GPTMS改性的HAp悬浮于蒸馏水中,得到浓度为20-30wt%的悬浮液;
4)羟基磷灰石(HAp)悬浮液负载CGRP因子
将所得20-30wt%的GPTMS-HAp悬液超声处理5-8min,加入CGRP因子,继续搅拌6-8h,得到负载CGRP因子的GPTMS-HAp悬浮液;
5)静电纺丝溶液配置
将所得负载CGRP因子的GPTMS-Hap悬浮液与所得丝素蛋白溶液按质量比1:10-1.5:10的比例混合,搅拌6-8h,得到静电纺丝GPTMS-HAp-SF溶液;
6)静电纺丝负载CGRP因子的GPTMS-HAp-SF膜
采用19号静电纺丝针头,调节接收距离为10cm,加正高压12kV,负高压1kV;调节液体流速0.5mL/h的速度,5mL注射器,接收滚筒转速600r//min;得到负载CGRP的GPTMS-HAp-SF膜;
7)将上述膜浸入多巴胺溶液中(0.2mg/mL,10mMTris-HCl缓冲液,pH 8.5)中室温浸泡10min,取出负载CGRP的GPTMS-HAp-SF膜,用去离子水清洗,去除残留多巴胺,然后置于含有生物活性因子SDF-1α的溶液中浸泡10min后取出,冷冻干燥,即得到负载SDF-1α/CGRP的HAp-SF人工骨膜。
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Application publication date: 20220301 |