CN113288521A - 一种3d打印多孔仿生膝关节软骨支架的制备方法 - Google Patents
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Abstract
本发明涉及一种移植关节支架,尤其涉及的是一种3D打印仿生膝关节软骨支架的制备方法。本发明是用CT扫描得到图像数据,根据CT图像数据建立适合移植关节的三维模型,以ECM、明胶、PLA作为基体材料,以体心立方体型多孔结构逐层打印,再加入透明质酸等,表面覆以生物陶瓷涂层,本发明打印出的软骨支架,有利于软骨组织的恢复与再生,促进生长因子等进入,可提升载药能力及缓释性能,并具有良好的力学性能及稳定性。
Description
技术领域
本发明设计一种医用移植假体,尤其涉及一种定制仿生膝关节处软骨支架的制备方法。
背景技术
关节软骨是一种缺乏血管、淋巴和神经的致密的结缔组织,一旦损伤自我再生和修复能力极为有限。目前单纯软骨损伤无有效的治疗方法,只能对症处理,而3D打印工程的出现为软骨的损伤再生修复带来了新的希望。半月板是构成膝关节的重要结构之一,是两个半月状纤维软骨,位于股骨髁与胫骨平台之间。其横断面呈三角形,外厚内薄,上面稍呈凹形,以便与股骨髁相吻合,下面为平的,与胫骨平台相接。这样的结构恰好使股骨髁在胫骨平台上形成一较深的凹陷,从而使球形的股骨髁与胫骨平台的稳定性增加。
由于关节软骨无血运和神经营养,系统性给药促使软骨修复和再生的能力极其有限。即使关节腔内给药,也并无直接和充足的证据表明软骨可以得到修 复,仅在一定程度上阻断炎症,缓解疼痛。微骨折术、软骨移植术、自体软骨细胞植入术等手术方式有助于软骨修复,但是再生的软骨和天然软骨仍有差异,并有各自的局限性。单髁置换术和关节置换术则是软骨病变严重者最后的选择,大多数是老年病人,但因创伤较大而面临诸多限制。
本发明打印出的半月板与传统方法相比,采用体心立方体型结构打印,优化了力学性能、抗疲劳性能及稳定性,脱细胞软骨细胞外基质(ECM) 可以模仿软骨细胞生长的天然微环境,适于内源性干细胞的黏附、增殖和成软骨分化已被广泛负载于骨科支架材料中,明胶和壳聚糖,具有良好的抗压性能和细胞黏附性,有利于软骨细胞的三维培养和细胞外基质的分泌,他们的加入提高了生长因子的接合和载荷,能够刺激骨髓间充质干细胞参与假体周围的骨修复,并且具备较强的抗菌功能,能够实现膝关节软骨的再生,避免二次手术的风险。
发明内容
本发明的目的是提供一种3D打印多孔仿生膝关节软骨支架的制备方法,通过该方法解决了人造软骨支架成分利用率低、支架的强度、韧性及耐磨性不足的问题,所用的多孔结构能够刺激骨细胞的修复与生长,使产品能满足人体骨骼亲和性的同时减少植入物的应力集中和应力遮挡,采用3D方法定制打印出来的产品更加符合人体的软骨特性,既保证了力学性能的同时也能实现骨骼的二次生长。
本发明是通过以下技术方案来实施:
用CT扫描人体两侧膝关节图像数据,根据CT图像通过镜像建立符合破损处膝关节的三维模型,以脱细胞软骨细胞外基质(ECM)、PLA和明胶作为3D打印的支架,乳酸-羟基乙酸共聚物,透明质酸作为支架的结合材料,将支架与结合材料以体心立方体型多孔结构分别进行分层打印基体,然后打印体表面覆以生物陶瓷涂层并抛光,其具体方案如下:
1.骨骼建模
通过CT扫描设备对膝关节及膝关节破损处进行三维扫描获取图像数据,根据破损的膝关节,利用三维软件重新设计出可以移植的尺寸及形态。
2.3D打印支架材料的制备
ECM浆料,采用差速离心法制备猪软骨ECM。具体方法为,提取新鲜软骨组织,加工成1*1*1mm的薄片,使用蒸馏水反复冲洗3-5次,进行杀菌处理打碎加入适量无菌三蒸水低温下粉碎均匀,在经过3次离心,去上清,即可得无细胞猪软骨ECM浆料。
取一定质量明胶粉末,溶解于 PBS 中 (pH=7.0),制备出 200 g/L 的明胶溶液,并将其与等体积 ECM 浆料混合,同时加入一定质量的PLA (终浓度 0.25%)。
3.透明质酸水凝胶制备
将200 mg透明质酸粉末溶解于15 mL 2-(N-吗啉)乙磺酸-水合物溶液中,加入285-290mg N-羟基琥珀酸亚胺,再加入95 mg炔丙基胺,透析2 h,冻干形成海绵状固体。取出海绵状固体 30-40mg 溶解于2-3mL双蒸水,叠氮-聚乙二醇-叠氮30mg溶解于30μL二甲基亚砜。将以上两种溶液混合,加入硫酸铜溶液10μL,混合均匀,再加入抗坏血酸溶液 10 μL,混合均匀制备成透明质酸水凝胶。
4.利用三维打印技术打印
将ECM、PLA和明胶与乳酸-羟基乙酸共聚物,透明质酸分别按质量分数比为9:1进行充分混合,将模拟出的三维模型导入3D打印机中,在辅助支架上打印出在模拟软件中已经做好的模型”,喷头直径设置为500μm,设置打印速度为5-8mm/s,打印的结构形态设为体心立方体型多孔结构,孔隙尺寸设置在600-650μm,孔隙率设置在45%-55%,支柱直径设计在1000-1200μm,打印完成后,使用超声振动变幅杆,振动频率80-100kHz,振幅20-150μm,工具头距激光头轴线40-50mm进行表面优化处理,至此完成的半成品,将半成品从辅助支架上取下,再将HA、β磷酸三钙粉末用电子天平按以5:3分别称量,并将两种粉末混合用球磨机研磨充分,HA的粉末粒度为30-50nm,纯度为99%,β磷酸三钙粉末粒度为30-50nm,纯度为99%,采用0.1%的异丙醇作为粘结剂混合搅拌5min,将上述混合粉末涂满至支架表面,预置层粉末厚度为1-1.3mm,在室温条件下静置12h后,真空干燥45-50℃中干燥2h,,采用激光熔覆的方法将与膏体结合,设置激光功率P=0.8KW,扫描速度V=5mm/s,光斑直径为1-1.5mm,搭接率为40%,氩气流速为10L/min,制备出生物陶瓷涂层,最后对成型产品进行抛光处理,降低材料表面粗糙度。
附图说明:
图1为3D打印膝关节软骨支架的结构示意图
具体实施方式:
第一步:用CT扫描人体两侧膝关节图像数据,根据CT图像通过镜像建立符合破损处膝关节的三维模型。
第二步:3D打印支架材料的制备,ECM浆料,采用差速离心法制备猪软骨ECM。具体方法为,提取新鲜软骨组织,加工成1*1*1mm的薄片,使用蒸馏水反复冲洗3-5次,进行杀菌处理打碎加入适量无菌三蒸水低温下粉碎均匀,在经过3次离心,去上清,即可得无细胞猪软骨ECM浆料。
第三步:取一定质量明胶粉末,溶解于 PBS 中 (pH=7.0),制备出 200 g/L 的明胶溶液,并将其与等体积 ECM 浆料混合,同时加入一定质量的PLA (终浓度 0.25%)。
第四步:透明质酸水凝胶制备,将200 mg透明质酸粉末溶解于15 mL 2-(N-吗啉)乙磺酸-水合物溶液中,加入285-290mg N-羟基琥珀酸亚胺,再加入95 mg炔丙基胺,透析2h,冻干形成海绵状固体。取出海绵状固体 30-40mg 溶解于2-3mL双蒸水,叠氮-聚乙二醇-叠氮30mg溶解于30μL二甲基亚砜。将以上两种溶液混合,加入硫酸铜溶液10μL,混合均匀,再加入抗坏血酸溶液 10 μL,混合均匀制备成透明质酸水凝胶。
第五步:将ECM、PLA和明胶与乳酸-羟基乙酸共聚物,透明质酸分别按质量分数比为9:1进行充分混合,将模拟出的三维模型导入3D打印机中,在辅助支架上打印出在模拟软件中已经做好的模型”,喷头直径设置为500μm,设置打印速度为5-8mm/s,打印的结构形态设为体心立方体型多孔结构,孔隙尺寸设置在600-650μm,孔隙率设置在45%-55%,支柱直径设计在1000-1200μm,打印完成后,使用超声振动变幅杆,振动频率80-100kHz,振幅20-150μm,工具头距激光头轴线40-50mm进行表面优化处理,至此完成的半成品,将半成品从辅助支架上取下。
第六步:将HA、β磷酸三钙粉末用电子天平按以5:3分别称量,并将两种粉末混合用球磨机研磨充分,HA的粉末粒度为30-50nm,纯度为99%,β磷酸三钙粉末粒度为30-50nm,纯度为99%,采用0.1%的异丙醇作为粘结剂混合搅拌5min,将上述混合粉末涂满至支架表面,预置层粉末厚度为1-1.3mm,在室温条件下静置12h后,真空干燥45-50℃中干燥2h,,采用激光熔覆的方法将与膏体结合,设置激光功率P=0.8KW,扫描速度V=5mm/s,光斑直径为1-1.5mm,搭接率为40%,氩气流速为10L/min,制备出生物陶瓷涂层,最后对成型产品进行抛光处理,降低材料表面粗糙度。
Claims (3)
1.一种3D打印多孔仿生膝关节软骨支架的制备方法,其特征在于:用CT扫描得到图像数据,根据CT图像数据建立适合移植关节软骨支架的三维模型, ECM、明胶、PLA作为基体材料,以体心立方体型多孔结构逐层打印,再加入透明质酸等,表面覆以生物陶瓷涂层,通过该方法制得的具有优异的再生、稳定性及力学性能,并具有良好的骨再生诱导修复作用,所述制备的具体工艺步骤为:
a:用CT扫描人体两侧膝关节图像数据,根据CT图像通过镜像建立符合破损处膝关节的三维模型;
b:3D打印支架材料的制备,ECM浆料,采用差速离心法制备猪软骨ECM;
具体方法为,提取新鲜软骨组织,加工成1*1*1mm的薄片,使用蒸馏水反复冲洗3-5次,进行杀菌处理打碎加入适量无菌三蒸水低温下粉碎均匀,在经过3次离心,去上清,即可得无细胞猪软骨ECM浆料;
c:取一定质量明胶粉末,溶解于 PBS 中 (pH=7.0),制备出 200 g/L 的明胶溶液,并将其与等体积 ECM 浆料混合,同时加入一定质量的PLA (终浓度 0.25%);
d:透明质酸水凝胶制备,将200 mg透明质酸粉末溶解于15 mL 2-(N-吗啉)乙磺酸-水合物溶液中,加入285-290mg N-羟基琥珀酸亚胺,再加入95 mg炔丙基胺,透析2 h,冻干形成海绵状固体;
取出海绵状固体 30-40mg 溶解于2-3mL双蒸水,叠氮-聚乙二醇-叠氮30mg溶解于30μL二甲基亚砜;
将以上两种溶液混合,加入硫酸铜溶液10μL,混合均匀,再加入抗坏血酸溶液 10 μL,混合均匀制备成透明质酸水凝胶;
e:将ECM、PLA和明胶与乳酸-羟基乙酸共聚物,透明质酸分别按质量分数比为9:1进行充分混合,将模拟出的三维模型导入3D打印机中,在辅助支架上打印出在模拟软件中已经做好的模型”,喷头直径设置为500μm,设置打印速度为5-8mm/s,打印的结构形态设为体心立方体型多孔结构,孔隙尺寸设置在600-650μm,孔隙率设置在45%-55%,支柱直径设计在1000-1200μm,打印完成后,使用超声振动变幅杆,振动频率80-100kHz,振幅20-150μm,工具头距激光头轴线40-50mm进行表面优化处理,至此完成的半成品,将半成品从辅助支架上取下;
f:将HA、β磷酸三钙粉末用电子天平按以5:3分别称量,并将两种粉末混合用球磨机研磨充分,HA的粉末粒度为30-50nm,纯度为99%,β磷酸三钙粉末粒度为30-50nm,纯度为99%,采用0.1%的异丙醇作为粘结剂混合搅拌5min,将上述混合粉末涂满至支架表面,预置层粉末厚度为1-1.3mm,在室温条件下静置12h后,真空干燥45-50℃中干燥2h,,采用激光熔覆的方法将与膏体结合,设置激光功率P=0.8KW,扫描速度V=5mm/s,光斑直径为1-1.5mm,搭接率为40%,氩气流速为10L/min,制备出生物陶瓷涂层,最后对成型产品进行抛光处理,降低材料表面粗糙度。
2.根据权利要求1所述一种3D打印多孔仿生膝关节软骨支架的制备方法,其特征在于打印中设计了软骨最佳孔距和孔隙率,3D打印的多孔膝关节软骨支架结构,满足人体软骨的力学性能,拥有更好的抗疲劳性能,更是提高了生物相容性,提升手术的成功率。
3.根据权利要求1所述一种3D打印多孔仿生膝关节软骨支架的制备方法,其特征在于在所用的材料配方上进行改进,由于不同材料的加入使得支架不仅能够实现降解功能,还能够刺激充质干细胞尽快完成骨修复,避免了二次手术的发生,实现软骨再生。
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