CN114848895A - 3d打印钛合金多孔支架载双因子壳核微球缓释*** - Google Patents
3d打印钛合金多孔支架载双因子壳核微球缓释*** Download PDFInfo
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Abstract
本发明提供的3D打印钛合金多孔支架载双因子壳核微球缓释***,所述***的制备为:称取600mg明胶加入10ml去离子水中,在适温下充分搅拌溶解至溶液呈透明;将3D打印钛合金支架放入定制的聚四氟乙烯模具中,将壳核微球和明胶溶液分别注射入装有支架的聚四氟乙烯模具中,充分混匀,‑80℃预冻,冷冻干燥72小时;用京尼平溶液在37℃避光静置下交联;用无水乙醇润洗,干燥即得到3D打印钛合金多孔支架载双因子壳核微球缓释***。本***能够有效促进成骨分化和骨整合,能够有效促进成骨分化和骨整合。
Description
技术领域
本发明涉及医疗技术领域,尤其是指3D打印钛合金多孔支架载双因子壳核微球缓释***的制备。
背景技术
在目前临床可用的骨移植方案中,自体骨移植仍然被认为是骨移植的金标准,但其存在有效性差、供体部位损伤和手术时间较长等缺陷。异体骨或异种骨移植的应用也受到限制,因为其存在明显的缺陷,包括免疫反应并发症、疾病传播风险、缺乏成骨特性等,这些可能会导致骨吸收或骨不愈合。近年来,组织工程开发的各种生物材料已成为自体骨的替代物。羟基磷灰石(HA)、磷酸三钙(β-TCP)及其复合材料因其与正常骨成分相似而被广泛应用于骨科重建手术,是很有应用前景的生物材料。生物活性玻璃表现出独特的特征,形成矿化HA层,促进与骨骼的化学结合。由于其优越的生物学性能,这些生物材料已被应用于临床骨缺损修复。但是,目前这些生物材料由于其力学性能较差,仅局限用于非负重骨的临界尺寸缺陷。此外,由于骨缺损的变异,目前可用的生物材料无法桥接或填充丢失的骨组织的解剖形状和结构,因此无法满足较大的临界尺寸缺损的手术要求。传统的材料加工技术,包括相分离,冷冻干燥,溶剂铸造、气体发泡和静电纺丝,在各种组织工程支架制作中显示出了巨大的潜力。然而,这些传统支架加工技术不能精确地控制支架的孔径、几何形状和互联性。
发明内容
本发明通过计算机辅助设计(CAD)建模,通过3D打印SLM技术制备出钛合金多孔支架,并对其进行各项表征和生物力学检测,并进行体外生物相容性的检测,最终形成3D打印钛合金多孔支架载双因子壳核微球缓释***。
发明内容
为实现上述目的,本发明所提供的技术方案为:3D打印钛合金多孔支架载双因子壳核微球缓释***,所述***的制备包括有以下步骤:
1)、称取600mg明胶,加入10ml去离子水中,在适温下充分搅拌溶解至溶液呈透明;
2)、将3D打印钛合金支架放入定制的聚四氟乙烯模具中,
3)、将20mg VEGF/BMP-2壳核微球和500ul明胶溶液分别注射入装有支架的聚四氟乙烯模具中,充分混匀,-80℃预冻,冷冻干燥72 小时;
4)、用1%京尼平溶液在37℃避光静置下交联12小时;
5)、用无水乙醇润洗1小时,洗3次,干燥即得到3D打印钛合金多孔支架载双因子壳核微球缓释***。
所述3D打印钛合金支架的制备方法为:
(1)建模:通过Solid Works或CAD软件设计出3D打印钛合金多孔支架的三维数据模型,多孔支架的大体形状为圆柱体,设计成两种规格,一种直径10mm,高5mm,一种直径5mm,高10mm,内部孔隙率为 60%,孔径为500um;
(2)数据导入:将各种规格的模型参数导入金属激光熔融3D打印装置中;
(3)分层制造:根据层层加工的原理,金属激光熔融3D打印装置的内置控制软件将导入的三维数字模型分割成20um固定层厚的平面断层,在充有氮气的工作室中,大功率的激光束通过内置的激光控制***和振镜扫描***精准地逐层熔炼Ti6Al4V粉末,逐层冷却凝固后,最终制备出实体的钛合金多孔支架。
本发明将载VEGF/BMP-2双因子壳核缓释微球通过明胶水凝胶涂层负载到3D打印钛合金多孔支架上,成功制备出了3D打印钛合金多孔支架载双因子壳核微球缓释***。该***能够有效促进成骨分化和骨整合,能够达到VEGF与BMP-2的缓慢序贯释放。且能够有效促进成骨分化和骨整合。
附图说明
图1为本发明3D打印钛合金多孔支架载双因子壳核微球缓释***制备过程示意图。
图2为本发明具有镂空正方体(a)和镂空三棱柱(d)单元结构的两种多孔结构支架三维数字模型图(b、c和e、f),具有相同的空隙大小(500μm)和孔隙率(60%)。
图3为本发明有限元分析软件ANSYS模拟支架受力情况。
图4为采用SEM观察3D打印钛合金多孔支架表面形态。
图5为3D打印钛合金多孔支架表面能谱分析图(EDS)。
图6为3D打印钛合金多孔支架X线衍射图(XRD)。
图7为DAPI染色结果。
图8为细胞粘附数量定量分析结果。
图9为多孔支架表面成骨细胞形貌SEM结果。
图10为采用活死细胞染色试剂盒测试细胞的活性情况。
图11为支架细胞增殖结果。
图12为支架细胞ALP定量结果。
图13为支架植入后股骨样本X线检查结果。
图14为Micro-CT三维重建结果。
图15为支架内Micro-CT扫描新生骨组织参数结果。
图16为钛合金多孔支架内新生骨组织荧光标记结果。
图17为钛合金多孔支架内新生骨组织VG染色结果。
图18为两组钛合金多孔支架内新生骨面积百分比。
图19为壳核结构微球镜下形态。
图20为壳核微球粒径(μm)。
具体实施方式
下面对本发明作进一步说明,本发明的较佳实施例为:参见所有附图,本实施例所述的3D打印钛合金多孔支架载双因子壳核微球缓释***,
第一步骤:3D打印钛合金多孔支架的制备:
(1)建模:通过Solid Works CAD软件,设计两种单元结构的 3D打印钛合金多孔支架的三维数据模型,多孔支架的大体形状为圆柱体,设计成两种规格,一种直径10mm,高5mm,一种直径5mm,高 10mm,内部孔隙率
为60%,孔径为500um;
(2)数据导入:将各种规格的模型参数导入金属激光熔融3D打印装置中;
(3)分层制造:根据层层加工的原理,金属激光熔融3D打印装置的内置控制软件将导入的三维数字模型分割成20um固定层厚的平面断层,在充有氮气的工作室中,大功率的激光束通过内置的激光控制***和振镜扫描***精准地逐层熔炼Ti6Al4V粉末,逐层冷却凝固后,最终制备出实体的钛合金多孔支架。
对上述钛合金多孔支架进行改性处理,其步骤为:
(1)清洗:从3D打印装置中取出多孔支架,用***吹去支架内未熔炼的金属粉末,并先后用丙酮、无水乙醇、去离子水超声清洗10分钟(清洗掉残留有毒金属颗粒);
(2)碱蚀:将多孔钛合金支架置于60℃的5mol/L NaOH溶液中 24小时,再用去离子水反复清洗后烘干;
(3)酸蚀:将多孔支架放入70℃的0.5mmol/L HCl溶液中24 小时,后再用去离子水反复清洗后烘干;
(4)退火:将多孔支架放入烧结炉中,以5℃/分钟的升温速度至 600℃,维持600℃,1小时常压退火。
上述步骤完成后进行3D打印钛合金多孔支架的有限元分析及生物力学检测,其步骤为:
(1)将3D打印钛合金多孔支架的三维数据模型导入ANSYS软件,完成有限元几何模型的创建,设置计算模型使用的材料属性,对有限元单元元素施加作用载荷,模拟支架受力情况。
(2)将多孔支架放入生物力学测试仪上对支架进行生物力学检测。
检测完成后对3D打印钛合金多孔支架的表面微观形貌观察,其方式为:将经表面改性处理后的支架样品先后用无水乙醇、去离子水超声清洗 10分钟,取出晾干,喷金,放置扫描电镜(SEM)下检测钛表面孔洞结构微观形貌,并用能谱分析(EDS)、X线衍射(XRD)分析材料表面元素构成。
将经表面改性处理后的的支架经丙酮、无水乙醇、去离子水分别超声清洗机清洗10分钟,取出材料晾干,经高温高压灭菌消毒后备用。
第二步骤:细胞悬液的制备
将MC3T3-E1成骨前体细胞悬液培养于含有10%胎牛血清和1%双抗 (链霉素和青霉素均为100U/ml)的α-MEM培养液中,置于37℃,5%C02 的细胞培养箱中培养,每2天换液一次,细胞消化传代,用细胞计数仪测量细胞数目,然后加培养液配成所需浓度的细胞悬液。
第三步骤:3D打印钛合金多孔支架表面细胞粘附
1)、将支架消毒后置于48孔板中,每组3个复样,加入少量完全培养基浸泡过夜,次日吸去培养基接种细胞;用细胞计数器测量细胞数目,然后加培养基配成2×106/ml浓度的细胞悬液,按每孔接种100ul 细胞悬液接种于材料表面,继续加入培养基700ul覆盖材料表面。
2)、将培养板置入置于37℃、5%CO2培养箱培养,细胞与支架共培养24小时后取出培养板,吸弃培养基,用PBS轻柔吹洗3遍,用显微镊将支架轻轻移入新的24孔板中,向每孔支架加入1ml的4%多聚甲醛溶液固定5分钟,吸弃固定液,PBS漂洗。用PBS漂洗后吸弃,向支架表面滴加50μg/ml的DAPI溶液对细胞核染色5分钟,染色结束后,加PBS溶液在摇床上振荡洗涤3次,每次10分钟,染色过程全程避光。染色结束后,将培养皿置于荧光显微镜下,随机选取5个视野,细胞计数定量。
用活死细胞活力/毒性检测试剂盒来检测3D打印钛合金多孔支架对成骨细胞活性的影响。
实验原理:在活细胞内存在大量酯酶,钙黄素AM本身无荧光,但进入细胞后在酯酶活性的作用下会生成绿色荧光物质,具有强烈荧光信号。而对死细胞来说,碘化丙啶(PI)可以穿过其受损的细胞膜进入细胞,PI本身具有较弱的荧光信号,与核酸结合后会放大其荧光信号,产生较强的红色荧光信号。
操作步骤:将支架消毒后置于48孔板中,每组3个复样,加入少量完全培养基浸泡过夜,次日吸去培养基后接种细胞;用细胞计数器测量细胞数目,然后加培养基配成2×106/ml浓度的细胞悬液,按每孔接种100ul细胞悬液接种于材料表面,继续加入培养基700ul覆盖材料表面。共培养3天后进行活死细胞活力/毒性试剂染色:
(1)取出钙黄绿素AM和PI试剂原液,恢复室温。取装有10ml 的PBS的离心管,加入5ul PI原液(16mM),充分混匀后得到8uM的 PI溶液。再在稀释后10ml的PI溶液(8uM)中加入5ul钙黄素AM 原液(4mM),充分混匀后即得到工作液(8uM PI和2uM钙黄素AM),用于细胞染色;
(2)吸去孔板内培养基,用PBS漂洗三次;
(3)加入足量的上述工作液,保证没过细胞,室温下孵育45分钟;
(4)吸出工作液终止孵育,加入微量PBS;
(5)在荧光显微镜下观察标记细胞:
将支架消毒后置于48孔板中,每组3个复样,加入少量完全培养基浸泡过夜,次日吸去培养基接种细胞;用细胞计数器测量细胞数目,然后加培养基配成2×106/ml浓度的细胞悬液,按每孔接种100ul细胞悬液接种于材料表面,继续加入培养基700ul覆盖材料表面;
在1、3、5、7天各时间点,将CCK-8试剂与培养液按1:10 混合稀释,弃去每孔培养液,每孔加入稀释后的CCK-8试剂800ul,放入培养箱中继续培养4小时,小心收集每孔上清液转移至96孔板中,最后用酶标仪在波长为450nm下测量吸光度(OD值)并记录:
记录3D打印钛合金多孔支架对碱性磷酸酶(ALP)活性的影响:
将支架消毒后置于48孔板中,每组3个复样,取5×106/ml浓度的细胞悬液,按每孔接种100ul细胞悬液接种于材料表面,继续加入培养基700ul覆盖材料表面。培养1天后,将培养基换为成骨诱导培养基 (α-MEM+β-甘油磷酸钠+抗坏血酸),置入细胞培养箱中共培养,每3 天换液一次。共培养第12天,吸弃培养基,用PBS轻轻漂洗3次,每孔加入200ul 1%TritonX-100的PBS缓冲液裂解细胞,随后离心取上清液,用ALP检测试剂盒对ALP活性进行定量检测。
第四步骤:
实验3D打印钛合金多孔支架体内对兔股骨缺损修复的效果:
构建兔股骨髁缺损模型;
实验选用雄性新西兰兔作为实验动物,动物等级为普通级,体重均为 3kg
左右,兔龄均为5个月,购置于中南大学实验动物学部。所有动物均严格按照标准喂养,所有动物实验均通过中南大学实验动物福利伦理审查标准;
选取雄性新西兰兔12只,随机分为2组,每组6只,动物造模后植入相应组别的支架,各组支架的大小均为底面直径5mm,高10mm;
将新西兰兔称重后,通过耳缘静脉注射的方式用3%戊巴比妥钠 (1ml/Kg)
进行麻醉。待麻醉成功后,用小动物剃毛器剔除兔左侧股骨髁周围大部分毛发。将兔取右侧卧位平放于手术台上固定,常规消毒铺单,取左侧股骨远端纵向切口,切开皮肤后,依次逐层分离筋膜及肌肉,剥离骨膜直至完全显露股骨外侧髁,在距关节面约4mm处标记,装好医用电钻,依次用直径为2mm的克氏针、3mm、4mm、5mm的钻头逐级扩孔,制出直径为5mm,深度为10mm的圆柱状骨缺损区域。在钻孔过程用生理盐水冲洗降温,钻孔完成后再依次用双氧水和生理盐水依次冲洗骨缺损区域,洗净区域内的碎骨屑,将各组支架植入缺损区域,逐层缝合切口,碘酒消毒,术后连续3天注射头孢西丁。术后定期观察动物的一般情况和手术切口愈合情况。
术后取材前14天及4天给需处死的动物分别注射钙绿黄素溶液。将400mg钙绿黄素溶于50ml生理盐水中,后加入1g碳酸氢钠使钙绿黄素完全溶解,并过滤灭菌。术后4周、12周,采用静脉推注过量麻药的方法处死动物,取出左侧股骨髁,剔除标本表面软组织,行80%酒精固定,避光4℃保存,行X线、micro-CT检查及硬组织切片检测;
将各组标本行X线平片检查,然后行micro-CT扫描,电压 70KV,电流141uA,扫描范围360°,断层扫描厚度18um。使用Ctan 软件进行三维重建,利用软件扫描选择支架底面以内的区域设为感兴趣区域(Region of interest,ROI),通过灰度分析得到多孔支架孔隙内新生骨组织的数据,并计算以下参数(1)骨小梁厚度(Trabecular thickness,Tb.Th);(2)骨小梁数量(Trabecularnumber, Tb.N);(3)骨体积分数(BV/TV)=骨小梁体积(Bonevolume,BV)/ ROI体积(Total volume,TV)。
(1)待完善micro-CT检查后,将所有样本放入10%***溶液中浸泡24小时,70%酒精浸2小时,95%酒精浸20小时,100%酒精浸2小时,然后在减压下干燥6小时;
(2)经上述凝固蛋白、防腐、脱水、干燥等处理后,把样品浸入盛有聚甲基丙烯酸甲酯单体的玻璃管中,在低真空下浸渗30分钟(此时无气饱冒出),置于27℃水浴中固化。为防止聚甲基丙烯酸甲酯单体在固化时炸裂,应特别注意固化温度的均匀。固化好的样品为透明而坚硬的圆柱体样坯,镶嵌在其中的试样清晰可见;
(3)利用硬组织切片机将样本切成约200μm的样本切片后,利用速干胶粘于树脂片上,将树脂片上的样本切片利用磨片机磨至50μm后抛光;
(4)放置激光共聚焦显微镜(CLSM)下观察骨组织荧光标记情况。
硬组织切片样品经CLSM观察后,进行VG染色,具体步骤如下:
(1)0.1%甲酸处理3分钟,流水冲洗;
(2)20%甲醇处理2小时,流水冲洗:
(3)60℃下亚甲基蓝溶液染色5分钟,60℃双蒸水清洗、干燥;
(4)室温下苦味酸品红液染色15分钟;
(5)无水乙醇清洗、干燥。
染色完成后,在光学显微镜下观察并采集图像,采用Image J软件对支架内新生骨组织面积进行半定量分析,计算方法如下,每个视野的新生骨面积(BoneArea Fraction)百分比(%)=新生骨面积/视野区总面积×100%。
所有资料选用采用SPSS24.0***对实验数据进行分析,数据均表示为均值±标准差(X±S),组间对比分析采用独立样本“t”检验,计算 P值,当P<0.05为差异有统计学意义。
所有资料选用采用SPSS24.0***对实验数据进行分析,数据均表示为均值±标准差(X±S),组间对比分析采用独立样本“t”检验,计算 P值,当P<0.05为差异有统计学意义。
通过有限元分析软件ANSYS模拟支架受力情况,由有限元模拟计算发现支架的压缩模量为116.91±0.0015MPa,与人体皮质骨的压缩模量 (89-164MPa)相近,有利于避免内植物的应力屏蔽效应。
通过生物力学检测仪检测支架轴向受力,结果为174.29±2.21 MPa,同样与人体皮质骨的压缩模量(89-164MPa)相近。
如图附图4所示,SEM观察3D打印钛合金多孔支架表面形态,与设计的三维模型相比,经SLM制备的多孔支架表面形态并不规整,孔洞周边存在突起,孔洞内壁有大量钛粉颗粒粘附,孔径的大小与模型存在一定的误差。镂空立方体单元结构的表面形态与设计的正方形存在差异,更接近圆形(图附图4A),其孔径大小为441.3±63.4um。镂空三棱柱单元结构的表面形态与设计的三角形基本一致(图附图4B),其孔径大小为453.6±38.2um。微观下(图附图4C)可见多孔支架表面可见一层不规则的微褶皱网状结构,即钛氧化层。如图附图5所示,EDS检测3D 打印钛合金多孔支架表面元素组成,O元素的存在说明支架表面有钛氧化层的形成。而XRD结果(图6)显示,钛的衍射峰均为α-钛,而 Ti6Al4V中的钛以α-钛为主。
图7为DAPI染色结果,支架接种细胞共培养24h后,从图中可以看出A组支架粘附的细胞数多于B组。图8为细胞粘附数量定量分析,随机选取5个视野,经过细胞计数定量统计分析显示,A组支架粘附细胞数明显多于B组(P<0.05)。
图9为多孔支架表面成骨细胞形貌SEM结果,在支架接种细胞共培养48h后,两组支架表面细胞均与钛氧化层紧密粘附,铺展良好并延伸出很多丝状伪足。
如图10所示,采用活死细胞染色试剂盒测试细胞的活性情况。染色后荧光显微镜下显示,两组均无明显死细胞,提示两组支架对细胞无毒性,生物相容性良好,A组细胞多于B组。
两组支架细胞增殖CCK-8结果如图11所示,随着共培养时间的增加,两组支架细胞数均持续增长,在各个时间点,A组与B组之间细胞数值均无明显差异(P>0.05)。
支架细胞ALP活性定量结果如图12所示,共培养后第12天,A 组细胞ALP活性高于B组(P<0.05),提示A组支架促成骨能力强于 B组。
在支架植入后第4周与12周分别获取兔股骨样本,经固定后行X 线平面检查,如图13,我们可以看到,所有植入的支架均位置良好,无松动、脱落、位移,周围组织无明显炎症反应和感染。
将micro-CT扫描结果用Ctan软件进行三维重建,观察支架内部骨长入的情况,如图14所示,白色部分为钛合金多孔支架,黄色部分为长入的新生骨组织,通过多孔支架底面三维图可以看出,在第4周,A组与B组支架孔洞周围及内部均有少量骨长入,第12周,两组支架的骨量较第4周明显增多,但A组与B组之间无明显差异。
Tb.Th指骨小梁平均厚度,在骨小梁数量一定的情况下,该值越大,提示骨量越多。Tb.N指骨小梁数量,该值越大,提示骨量越多。BV/TV 指新生骨小梁体积占ROI体积的百分比,该值越大,骨量越多。从这三项骨组织参数结果可以看出,4周和12周,A组和B组之间骨组织参数均无明显统计学差异(P>0.05),但是第12周,A组数据均高于B 组。
所有标本进行硬组织切片后置于CLSM下观察骨组织荧光标记情况, 如图16所示。绿色为钙黄绿素标记骨组织形成,黑色为钛合金材料。第 4周时,两组的荧光亮度均整体较弱,仅有少量荧光亮度较高,表明并无明显的成熟新生骨形成。第12周时,两组的荧光亮度较第4周时明显增强,根据荧光的分布情况,可以看到A组荧光分布致密,有明显成型的成熟新生骨,且与材料结合处荧光较强,而B组荧光分布均匀分散,新生骨无明显成型,与材料结合处荧光较弱,提示相比B组支架,A组支架有更好的骨整合能力。
所有硬组织切片在CLSM下观察完毕后均行VG染色,置于光学显微镜下观察,结果如图17所示。红色为成熟的新生骨组织,灰褐色为骨髓组织,黑色为钛合金材料。第4周,镜下可见两组支架内部的孔隙内长入了大量褐灰色的骨髓组织,其中少量组织已成红色,可以看到A组支架内的骨髓组织分布更为致密。第12周,可见两组支架内部孔隙中成熟骨组织明显增多,A组支架内部孔隙内的骨组织与材料表面结合紧密,而B组支架内部材料表面与骨组织接触并不紧密,多处可见缝隙存在。支架内新生骨面积百分比结果如图18所示,第4周,两组支架内新生骨面积百分比无明显统计学差异(P>0.05),第12周,A组支架内新生骨面积百分高于B组(P<0.05)。
本发明选择性激光熔炼(SLM)利用聚焦和计算机控制的激光束提供的热能,通过有选择地将金属粉末逐层熔化,从而生成复杂的三维金属部件。近年来研究表明,SLM可以制备出形态和力学性能具有高度可控性和可重复性的多孔生物惰性Ti6Al4V结构。此外,已有研究表明,SLM产生的Ti6Al4V多孔结构具有生物相容性,能骨整合中起到积极作用。
钛及其合金具有良好的生物相容性、优异的耐腐蚀性和极低的组织反应性,而且钛合金几乎不可降解,结构性能稳定,在体内能长期保存,适合作为硬组织的替代物。
制备多孔支架而非实心支架的原因除了保证细胞的渗透、营养物质的扩散、废物的清除、以及血管组织和新骨的长入以外,还为了能降低实心金属的压缩模量,从而避免应力屏蔽效应。人皮质骨的压缩模量为89- 164MPa,而实心TC4钛合金的弹性模量高达110GPa。选择制备多孔支架能显著降低材料的弹性模量。
本发明对制备出来的3D打印钛合金多孔支架进行有限元分析和生物力学检测,其压缩模量分别为116.91±0.0015Mpa和174.29±2.21 Mpa,不仅较实心钛合金明显下降,还接近人体皮质骨的压缩模量。
在体外生物相容性的实验中,两组不同单元结构的多孔支架均无明显细胞毒性,生物相容性良好,细胞铺展形态也良好,两组支架细胞增殖结果无明显差异。
而在体内对兔股骨缺损修复的实验中,Micro-CT三维图与新生骨长入定量结果显示两组的新生骨量虽然并无明显统计学差异。
同时,从硬组织切片荧光标记以及VG染色结果可以看出,A组支架内部新生骨与支架的骨整合能力高于B组,而VG染色新生骨面积百分比结果显示A组支架促成骨分化能力要高于B组。说明多孔支架的镂空立方体单元结构比三棱柱单元结构具有更好的促成骨分化与骨整合能力。
从体外和体内的实验结果可以看出,多孔支架的孔隙形状对细胞活性和增殖并无明显影响,而立方体单元结构比三棱柱单元结构具有更好的促成骨分化与骨整合能力,这可能是因为误差和空间分辨率的原因,导致立方体单元结构的表面形状更接近圆形,使梁与梁之间的夹角呈圆弧状,更有利于细胞的附着与铺展,且其更大的表面积可以增加细胞粘附的面积,使细胞接受更多的机械刺激。
第五步骤:VEGF/BMP-2壳核缓释微球的制备
空白微球制备溶液:在50ml的蓝瓶中分别加入各500mg的PLGA (分子量4万)和PLLA(分子量9万)。PLGA用10ml乙酸乙酯 (EA)溶解,PLLA用10ml二氯甲烷(DCM)溶解。置于恒温振荡器进行溶解1-1.5小时,直至PLGA和PLLA完全溶解。待PLLA在DCM中充分溶解后,加入微量香豆素-6使其呈浅绿色。
载因子微球制备溶液:两种因子溶液均加入5%海藻糖作为稳定剂,将VEGF溶于去离子水制成水相(10ug/ml),取PLGA溶于EA(5%,w/ v)制成有机相,水相-有机相按体积比为1:10进行混合后置于超声震荡仪中进行超声乳化(低温超声震荡1分钟),直至形成均匀的乳化液。按同样的方法将BMP-2溶于去离子水制成水相(100ug/ml)时,PLLA 溶于DCM(5%,w/V)制成有机相,同样水相-有机相按体积比仍为1: 10。
在配制溶液时,使用微量香豆素-6将PLLA/DCM溶液染成浅绿色,在制备壳核微球过程中利用载玻片在喷雾区域接收少量微球,并在激光共聚焦扫描显微镜(CLSM)下观察微球是否有明显的壳核结构。选择普通光和蓝色激光通道,观察所制备微球的粒径及壳核分布情况(香豆素-6 在蓝色激光下被激活呈现绿色)。此外,将冷冻干燥后的微球进行喷金60秒,在扫描电镜下(SEM)下观察壳核结构微球的表面形貌以及粒径大小。
为了测定每种生长因子在不同组别壳核微球中的包封率,称取每组载生长因子壳核微球组样本各3份,每份取20mg微球置于1ml DCM中,低温下超声震荡溶解。使微球的壳核结构载体PLGA与PLLA均完全溶解后,加入1ml PBS涡流旋转10分钟,然后置于低温高速离心机中以 14000rmp离心3分钟,离心完毕后小心吸取水相层液体。上述步骤重复两次,以最大限度回收VEGF与BMP-2。然后用酶联免疫吸附(ELISA) 试剂盒测定回收的各组微球VEGF和BMP-2浓度(按试剂盒说明书步骤进行操作)。
包封率(EE)=(样品中生长因子实际总量/理论总量)×100%。
载生长因子壳核微球体外释放实验:
称取A-E组载生长因子微球每组各20mg,将微球置于透析袋中,然后将透析袋置于装有10.0ml PBS的离心管中,再将离心管放置于120 rpm的恒温(37℃)轨道摇床中。定期在各个时间点(1、2、4、6、 8、11、14、18、22、27天)吸出离心管中的PBS,立即再用5ml 新鲜PBS替换,保持恒温轨道摇床的参数不变。将回收的PBS置于- 20℃冰箱保存,所有时间点回收完毕后进行检测,用ELISA试剂盒测定每个时间点回收的BMP-2和VEGF的浓度,计算出释放量,进一步计算不同时间点释放量占理论总量的百分比,绘制出释放曲线。
第六步骤:细胞悬液的制备:
将MC3T3-E1 Subclone 14成骨前体细胞系细胞悬液培养于含有10%胎牛血清和1%双抗(链霉素和青霉素均为100U/ml)的α-MEM培养液中,置于37℃,5%C02的细胞培养箱中培养,每2天换液一次。细胞消化传代,用细胞计数仪测量细胞数目,然后加培养液配成所需浓度的细胞悬液。
测试载因子壳核缓释微球对成骨细胞增殖的影响:
取24孔Trans-well板(孔径为8um),将消毒后的6组微球每孔20mg置于上室,将2×104个细胞种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。每2天换液一次。在1、3、5、 7天各时间点,将CCK8试剂与培养液按1:10混合稀释,弃去每孔培养液,每孔加入稀释后的CCK-8试剂800ul,放入培养箱中继续培养4 小时,小心收集每孔上清液转移至96孔板中,最后用酶标仪在波长为 450nm下测量吸光度(OD值)并记录。
上述原理为:在活细胞内存在大量酯酶,钙黄素AM本身无荧光,但进入细胞后在酯酶活性的作用下会生成绿色荧光物质,具有强烈荧光信号。而对死细胞来说,碘化丙啶(PI)可以穿过其受损的细胞膜进入细胞,PI本身具有较弱的荧光信号,与核酸结合后会放大其荧光信号,产生较强的红色荧光信号。
操作步骤:取24孔Trans-well板(孔径为8um),将消毒后的 6组微球每孔20mg置于上室,将2×104个细胞种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。每2天换液一次。培养 3天后进行活死细胞活力/毒性试剂染色。
1.取出钙黄绿素AM和PI试剂原液,恢复室温。取装有10ml的 PBS的离心管,加入5ul PI原液(16mM),充分混匀后得到8uM的PI 溶液。再在稀释后10ml的PI溶液(8uM)中加入5ul钙黄素AM原液 (4mM),充分混匀后即得到工作液(8uM PI和2uM钙黄素AM),用于细胞染色。
2.弃去孔板上室,吸去培养基,用PBS漂洗三次。
3.加入足量的上述工作液,保证没过细胞,室温下孵育45分钟。
4.吸出工作液终止孵育,加入微量PBS。
5.在荧光显微镜下观察标记细胞。
ALP活性检测:
碱性磷酸酶(ALP)活性是检测成骨细胞早期分化的重要指标。
取24孔Trans-well板(孔径为8um),将消毒后的6组微球每孔20mg置于上室,将5×104细胞种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。培养1天后,将培养基换为成骨诱导培养基(α-MEM+β-甘油磷酸钠+抗坏血酸),置入细胞培养箱中共培养,每3天换液一次。
共培养第12天,吸除培养基,用PBS润洗2-3次,加入4%多聚甲醛固定2分钟,吸除固定液,TBST润洗3次,吸除TBST溶液,加入足量新鲜配置的ALP染色工作液,室温避光放置20分钟。吸除染色液,PBS润洗1次,最后保存于PBS溶液中,显微镜下观察染色结果。
按上述相同方法接种细胞,共培养第12天,吸弃培养基,用PBS 轻轻漂洗3次,每孔加入200ul 1%Triton X-100的PBS缓冲液裂解细胞,随后离心取上清液,用碱性磷酸酶检测试剂盒对ALP活性进行定量检测。
通过Real-time PCR检测成骨相关基因:
取24孔Trans-well板(孔径为8um),将消毒后的6组微球每孔20mg置于上室,将5×104细胞种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。培养1天后,将培养基换为成骨诱导培养基(α-MEM+β-甘油磷酸钠+抗坏血酸),置入细胞培养箱中共培养,每3天换液一次。共培养第14天,吸去孔板内培养基,加入PBS 轻轻洗涤3次后,用RT-qPCR技术对成骨相关基因进行检测。步骤如下:
(1)RNA提取
1.向培养板中加入Trizol,每孔1ml,吹打混匀,室温裂解5分钟;
2.加入三氯甲烷,每孔200ul,剧烈振荡,室温静置3分钟;
3.离心15分钟(4℃,12000rpm),取上层液,转移到新的 RNase-Free离心管中;
4.加入异丙醇(等量),混匀后室温静置10分钟;
5.离心15分钟(4℃,12000rpm),去上清,加入75%乙醇,每孔 1ml;
6.沉淀溶解后再次离心3分钟(12000rpm,4℃),去上清;
7.空气干燥5~10分钟。加入无菌无酶水,每孔20ul;
8.沉淀溶解后用紫外分光光度计测定浓度,在260nm与280nm处测其吸光度值,并计算其浓度跟纯度。
制备RNA的琼脂糖凝胶电泳:
1.配制1%琼脂糖凝胶;
2.取提取的RNA 5ul,与6*loading buffer混匀(比例1: 5),140V恒压电泳,停止电泳后在凝胶成像***下观察。
茜素红染色
钙结节测定是检测成骨细胞晚期分化的重要指标。
取24孔Trans-well板(孔径为8um),将消毒后的6组微球每孔20mg置于上室,将5×104细胞种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。培养1天后,将培养基换为成骨诱导培养基(α-MEM+β-甘油磷酸钠+抗坏血酸),置入细胞培养箱中共培养,每3天换液一次。
共培养第21天,吸弃培养基,用PBS润洗3次,用4%多聚甲醛固定30分钟,吸弃固定液,用PBS润洗3次,加入茜素红染液在 37℃下反应30分钟,吸弃茜素红染液,用去离子水反复漂洗直至无色,去除非特异性茜素红沉积,放置显微镜下观察染色结果并拍照。
为定量测定钙结节含量,向每孔加入氯代十六烷基吡啶(10%),室温反应下待茜素红完全溶解后,加入96孔板内于620nm波长处测OD 值。
Western blot检测成骨相关蛋白
取24孔Trans-well板(孔径为8um),将消毒后的6组微球每孔20mg置于上室,将5×104细胞种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。培养1天后,将培养基换为成骨诱导培养基(α-MEM+β-甘油磷酸钠+抗坏血酸),置入细胞培养箱中共培养,每3天换液一次。
共培养第21天,吸去孔板内培养基,加入PBS轻轻洗涤3次后,用Westernblot技术对成骨相关蛋白进行检测。
检测载因子壳核缓释微球对血管内皮细胞增殖的影响:
将人脐静脉细胞株(ECs)EA.hy926细胞株悬液培养于含有10%胎牛血清和1%双抗(链霉素和青霉素均为100U/ml)的高糖DMEM培养基中,置于37℃,5%C02的细胞培养箱中培养,每2天换液一次。细胞消化传代,用细胞计数仪测量细胞数目,然后加培养液配成所需浓度的细胞悬液。取消毒后的6组微球每孔20mg置于24孔Trans-well板(孔径为8um)的上室,将2×104细胞种入下室,另取空白孔种植相同数量细胞作为对照组。每组
3个复孔。每2天换液一次。在1、3、5、7天各时间点,同上述成骨细胞CCK-8检测法对血管内皮细胞进行检测。
检测载因子壳核缓释微球对血管内皮细胞活性的影响:
取24孔Trans-well板(孔径为8um),将消毒后的6组微球每孔20mg置于上室,将2×104细胞(EA.hy926)种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。每2天换液一次。培养 3天后,同上述成骨细胞活死细胞染色法对血管内皮细胞进行染色。
检测载因子壳核缓释微球对血管内皮细胞NO分泌的影响:
取24孔Trans-well板(孔径为8um),将消毒后的6组微球每孔20mg置于上室,将2×104细胞(EA.hy926)种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。每2天换液一次。培养 3天后,收集每孔上清液,按照NO检测试剂盒说明书对NO分泌量进行检测。
Real-time PCR检测成血管相关基因:
取24孔Trans-well板(孔径为8um),将消毒后的6组微球每孔20mg置于上室,将5×104细胞(EA.hy926)种入下室,另取空白孔种植相同数量细胞作为对照组。每组3个复孔。每2天换液一次。培养 7天后,同qPCR检测成骨相关基因方法,对成血管相关基因VEGF,HIF-1α进行检测。
采用SPSS24.0***进行统计分析,数据均以“均值±SD”的形式表示。组间分析采用独立样本“t”检验,P<0.05表示有统计学差异。
当微球制备的PLGA与PLLA的浓度为5%,同轴针内外管溶液流速均为1.5ml/h,电压为10.0KV时,微球呈壳核结构,均一性良好,分散性良好,具有强的立体感,壳核界限清晰,表面光整,不出现褶皱,不出现孔隙。为在同轴静电喷雾制备微球过程中,用载玻片接收微球在普通光学显微镜下观察所示,可见微球壳核结构明显,形状圆润,均一性良好;图19的b-c为在CLSM下观察所示,内核的PLLA中加入了香豆素-6,在共聚焦蓝色激光激发下呈绿色,微球显示壳核结构清晰明显,微球的均一性和分散性良好;图d-f为在SEM下观察微球形貌,微球形态规则,具有强立体感,表面圆润光滑,无明显孔隙,均一性良好,未见明显微球粘连。
在SEM下观察并测量微球粒径,每组微球随机选取2个视野,每个视野随机计数10个(如图20)。载因子微球A组直径为
17.85±1.45μm,B组微球直径为17.52±1.06μm,C组直径为 17.16±1.10μm,D组直径为17.58±1.08μm,E组直径为 17.36±1.32μm,空白微球组F组直径为17.66±1.54μm。A-E组与F组间差异无统计学意义(P>0.05),表明空白微球与载因子微球间粒径无显著性差异;各载因子微球组间差异无统计学意义(P>0.05),提示所载生长因子对粒径大小无明显影响。
缓释复合体系能够将多种重要的生长因子与协同释放动力学相结合,通过模拟参与自然骨发育和自发愈合的时空信号级联,为增强骨再生提供了一种有前景的方法。
Claims (2)
1.3D打印钛合金多孔支架载双因子壳核微球缓释***,其特征在于:所述***的制备包括有以下步骤:
1)、称取600mg明胶,加入10ml去离子水中,在适温下充分搅拌溶解至溶液呈透明;
2)、将3D打印钛合金支架放入定制的聚四氟乙烯模具中,
3)、将20mg VEGF/BMP-2壳核微球和500ul明胶溶液分别注射入装有支架的聚四氟乙烯模具中,充分混匀,-80℃预冻,冷冻干燥72小时;
4)、用1%京尼平溶液在37℃避光静置下交联12小时;
5)、用无水乙醇润洗1小时,洗3次,干燥即得到3D打印钛合金多孔支架载双因子壳核微球缓释***。
2.根据权利要求1所述的3D打印钛合金多孔支架载双因子壳核微球缓释***,其特征在于:所述3D打印钛合金支架的制备方法为:
(1)建模:通过Solid Works或CAD软件设计出3D打印钛合金多孔支架的三维数据模型,多孔支架的大体形状为圆柱体,设计成两种规格,一种直径10mm,高5mm,一种直径5mm,高10mm,内部孔隙率为60%,孔径为500um;
(2)数据导入:将各种规格的模型参数导入金属激光熔融3D打印装置中;
(3)分层制造:根据层层加工的原理,金属激光熔融3D打印装置的内置控制软件将导入的三维数字模型分割成20um固定层厚的平面断层,在充有氮气的工作室中,大功率的激光束通过内置的激光控制***和振镜扫描***精准地逐层熔炼Ti6Al4V粉末,逐层冷却凝固后,最终制备出实体的钛合金多孔支架。
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