CN107376017B - 3d打印的海藻酸钠-i型胶原-陶瓷复合支架、制备方法及应用 - Google Patents
3d打印的海藻酸钠-i型胶原-陶瓷复合支架、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种3D打印的海藻酸钠‑I型胶原‑陶瓷复合支架、制备方法及其应用。所述复合支架主要由海藻酸钠:I型胶原:钙镁硅酸盐以质量分数比(1‑6):2:(0‑10)构成,并以氯化钙作为交联剂,氯化钙占所述复合支架的质量分数为(1‑10)%;将钙镁硅酸盐粉体分散到去离子水中,充分搅拌后复合海藻酸钠、I型胶原形成混匀水凝胶状物,将水凝胶状物置入三维打印机中,用氯化钙交联,打印出多孔支架,得到所述复合支架。本发明复合支架能促进软骨细胞增殖与ALP活性;其生物活性优良,在组织工程中具有应用价值。
Description
技术领域
本发明涉及医用生物材料,特别涉及一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架、制备方法及应用。
技术背景
软骨缺损是骨科常见疾病,尤其是运动员,且软骨损伤通常合并了软骨下骨损伤。患者常有关节肿胀、疼痛,活动受限,影响日常生活与工作,给社会造成较大经济负担。软骨内无血管、无神经、无淋巴,损伤后自行修复能力差,治疗困难,一直是骨科的难题。而目前临床治疗手段如微骨折、骨软骨柱移植、软骨细胞移植等没有涉及骨软骨损伤的联合治疗,新生的软骨组织常常为纤维软骨而非透明软骨。纤维软骨力学性能差,无法承受膝关节日常活动中产生的巨大应力,易退变与磨损,因此无法保证远期疗效。
近年来,组织工程材料与3D打印技术的发展提供了新的方向。钙-硅基生物陶瓷被证明具有优良的生物活性和骨传导性,其中某些钙-硅基材料在接触体液后仅几分钟即发生生物活性反应;同时硅是人体必需的微量元素,与生物体粘多糖的合成密切相关,并在幼骨矿化区聚集。含镁的钙硅酸盐是一类典型的钙-硅基物质,根据其稳定性和形成条件的不同会形成化合物。我们利用仿生原理,使用有机-无机复合材料,在体外构建一种多孔支架以期能促进细胞增殖与ALP活性,为组织工程材料提供了新的思路。
发明内容
为了解决背景技术中存在的问题,本发明的内容是提供了一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架、制备方法及应用,所述复合支架具有良好的生物相容性,可促进细胞外基质分泌,体外实验证实其增强了细胞的矿化能力。可用于组织工程中。
为了构建组织工程支架,本发明采用的技术方案是以海藻酸钠为基础,通过复合I型胶原与掺镁硅灰石,经3D打印与氯化钙化学交联后成形。
本发明采用的技术方案是:
一、一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架:
所述复合支架主要由海藻酸钠:I型胶原:钙镁硅酸盐以质量分数比(1-6):2:(0-10)构成,并包含有氯化钙作为交联剂,氯化钙占所述复合支架的质量分数为(1-10)%。
本发明是以钙镁硅酸盐陶瓷与海藻酸钠、I型胶原复合,氯化钙交联而成。
所述的复合支架的长度或直径为5-15mm,孔道孔径尺度为50-500μm,孔隙率为20-80%。
所述的钙镁硅酸盐采用镁掺杂硅灰石,镁掺杂硅灰石中镁代替钙的摩尔百分数为4-15%。
所述复合支架呈长方体、立方体、圆柱体或圆台体,支架内相邻孔道相互贯通,所述的孔道形态为圆形、三角形、四边形、蜂窝形、多边形或者阿基米德螺旋弧形。
所述复合支架采用三维打印方法构建制成。
二、一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架的制备方法:
将钙镁硅酸盐粉体分散到去离子水中,充分搅拌后复合海藻酸钠、I型胶原形成混匀水凝胶状物,将水凝胶状物置入三维打印机中,打印出多孔支架,得到所述复合支架。
1)将钙镁硅酸盐粉体使用湿法球磨处理后获得颗粒度不超过5μm的超细粉体,将超细粉体分散到去离子水中,并在常温下搅拌1小时,再加入I型胶原粉末在常温下搅拌5分钟,最后再加入海藻酸钠在常温下搅拌15分钟,制成水凝胶状物作为复合材料墨水;
2)将水凝胶状物置入三维打印机中,使用三维打印机设备打印,打印过程中使用氯化钙喷雾交联,打印完毕后将支架浸泡于氯化钙溶液10分钟进一步交联,得到最终3D打印的复合支架。
所述步骤1)中氯化钙交联是采用气雾喷洒、液体浸泡或者两者的组合进行处理。
所述步骤2)是将复合材料墨水置入写入式三维打印机中,设定孔道形态、孔通道尺寸、支架尺寸和孔隙率,启动打印设备,进行三维打印,形成多孔复合支架。
本发明制成的所述复合支架应用于提高软骨细胞增殖与ALP活性。
本发明所说的3D打印复合支架,形状、尺寸无严格限制,以有利于方便植入和各部位力学特定需求。
本发明所说的3D打印复合支架,内部孔道尺度和孔隙率沿轴向没有严格限制,靠近软骨下骨孔道尺度和孔隙率提高可以加快新生软骨再生。
本发明所说的3D打印复合支架,对应用范围不存在严格限制,本发明采用膝关节为修复靶点,其他在髋关节、肩关节、踝关节等软骨、骨软骨再生重建均可运用。
本发明具有的有益效果体现在:
1)在组成上,本发明使用了高生物活性的钙镁硅酸盐与海藻酸钠、I型胶原的复合采用仿生理念,有效提高了软骨细胞增殖与ALP活性,具有优异的生物活性。
2)在微结构上,以三维打印工艺制备的复合支架,内部孔道有利于维持营养传输;支架内的孔隙率、孔尺度都可以广泛调节,并且相邻孔道完全贯通,非常有利于细胞粘附、增殖与迁移。
3)在可操作性上,三维打印制备复合支架,任意局部孔道尺度、孔隙率、形状、尺度控制上达到理想需求,降低尖锐棱角对组织的慢性炎症反应。
附图说明
图1是本发明的3D打印复合支架示意图。
图2是本发明的3D打印复合支架大体图片。
图3是本发明的钙镁硅酸盐材料XRD图谱。
图4是扫描电镜拍摄的本发明复合支架表面显微形貌图。
图5是3D打印支架与深层软骨细胞共培养过程中细胞增殖变化图。(纵坐标表示吸光度,横坐标表示天数),其中,(a)表示含钙镁硅酸盐支架,(b)表示含β-磷酸三钙支架,(c)表示加入透明质酸钠的钙镁硅酸盐支架。
图6是3D打印支架与深层软骨细胞共培养过程中碱性磷酸酶变化图。(纵坐标表示碱性磷酸酶含量,横坐标表示天数),其中,(a)表示含钙镁硅酸盐支架,(b)表示含β-磷酸三钙支架,(c)表示加入透明质酸钠的钙镁硅酸盐支架。
图7是3D打印支架与深层软骨细胞共培养后II型胶原表达western-blot条带图(β-actin为内参)。其中,(a)表示加入透明质酸钠的钙镁硅酸盐支架,(b)表示不含透明质酸钠的钙镁硅酸盐支架。
具体实施方式
下面结合附图和实施例对做本发明进一步说明。以下具体实施例用来解释说明本发明,但不应理解为对本发明的限制,在本发明的精神和权利要求的保护范围内,对本发明作出的任何修改和改变,都落入本发明的保护范围。所有实施例使用试剂不低于分析纯标准。
本发明的实施例如下:
实施例1:
1)将钙镁硅酸盐粉体使用湿法球磨处理后获得颗粒度不超过5μm的超细粉体,将超细粉体分散到去离子水中,并在常温下搅拌1小时,再加入I型胶原粉末在常温下搅拌5分钟,最后再加入海藻酸钠在常温下搅拌15分钟,制成水凝胶状物作为复合材料墨水;
其中海藻酸钠:I型胶原:钙镁硅酸盐的质量分数比为6:2:5。
所述的钙镁硅酸盐是镁掺杂硅灰石,镁掺杂硅灰石中镁代替钙的摩尔百分数为10%。
2)将水凝胶状物置入三维打印机中,使用三维打印机设备打印。支架的长度为10mm,孔道孔径尺度为300μm,孔隙率为43%。打印过程中使用质量分数10%的氯化钙喷雾交联,打印完毕后将支架浸泡于质量分数10%的氯化钙溶液10分钟进一步交联,得到最终3D打印的复合支架。
经检测,支架尺寸约为10mm*10mm,孔道尺寸约270μm(见附图2)。钙镁硅酸盐XRD分析图谱见附图3,支架表面微结构见附图4。
实施例2:
1)将β-磷酸三钙粉体使用湿法球磨处理后获得颗粒度不超过5μm的超细粉体,将超细粉体分散到去离子水中,并在常温下搅拌1小时,再加入I型胶原粉末在常温下搅拌5分钟,最后再加入海藻酸钠在常温下搅拌15分钟,制成水凝胶状物作为复合材料墨水;
其中海藻酸钠:I型胶原:β-磷酸三钙的质量分数比为6:2:5。
2)将水凝胶状物置入三维打印机中,使用三维打印机设备打印。支架的长度为10mm,孔道孔径尺度为300μm,孔隙率为43%。打印过程中使用质量分数10%的氯化钙喷雾交联,打印完毕后将支架浸泡于质量分数10%的氯化钙溶液10分钟进一步交联,得到最终3D打印的复合支架。
经检测,支架尺寸约为10mm*10mm,孔道尺寸约270μm(见附图2)。钙镁硅酸盐XRD分析图谱见附图3,支架表面微结构见附图4。
针对实施例1和2的实验测试
测试1:
将实施例1所得含钙镁硅酸盐复合支架和实施例2所得的含β-磷酸三钙复合支架,放入体外标准条件化无菌培养的深层软骨细胞培养瓶中共培养14天,使用MTT检测细胞增殖。
经检测,细胞随时间推移增殖明显,14天时含钙镁硅酸盐支架的细胞增殖率比含β-磷酸三钙支架高16%,表明经含钙镁硅酸盐支架具有优良的生物相容性(见附图5中a,b组)。
测试2:
将实施例1所得含钙镁硅酸盐复合支架和实施例2所得的含β-磷酸三钙复合支架,放入体外标准条件化无菌培养的深层软骨细胞行共培养7天后对细胞内ALP检测。经检测,培养7天后,含钙镁硅酸盐支架组细胞内ALP活性明显上升,存在显著性差异,表明钙镁硅酸盐增强了细胞的具有优良的促进成骨活性(见附图6中a,b组)。
对比例1:
本发明申请人已自行设计含有透明质酸钠的含钙镁硅酸盐复合支架(余新宁等.基于三维打印的钙化层重建生物活性支架制备及其性能研究[J].浙江大学学报(医学版),2016,45(2):126-131.),按文中步骤制备:
取九水偏硅酸钠129.1g溶于1L去离子水;取四水硝酸钙96.5g、六水硝酸镁11.6g溶于1L去离子水,滴加浓氨水调节其酸碱度为10.0后滴入九水偏硅酸钠溶液中并搅拌12h后抽滤,用去离子水洗涤4次,再用无水乙醇洗涤1次,90度干燥24h,180度干燥8h,880度干燥150min后得到掺镁硅灰石粉末。按质量分数5%的比例加到10g 2%聚乙烯醇溶液内,均匀搅拌2h后加入0.20g I型胶原粉末静置溶解,再加入0.03g透明质酸钠和0.60g海藻酸钠粉末后搅拌为均匀溶液后放入37度培养箱溶胀1h后置于2度冰箱保存待用。
使用1ml针筒作为墨盒,选用400μm直径喷头,设定线宽400μm、层高320μm、孔隙宽度470μm,将墨盒内掺镁硅灰石混合溶液在电机驱动下使其水平移动并挤出溶液,逐层打印后使线条堆积形成三维状,利用海藻酸钠与10%氯化钙气雾化学交联成型原理,打印后向支架滴加5滴10%氯化钙以提高交联度,再滴加3-5滴无水乙醇冲洗。
实施例1和对比例1的对比:
对比测试1:
将实施例1所得不含透明质酸钠的钙镁硅酸盐复合支架和对比例1所得的加入透明质酸钠的含钙镁硅酸盐复合支架,放入体外标准条件化无菌培养的深层软骨细胞培养瓶中共培养14天,使用MTT检测细胞增殖。
经检测,细胞随时间推移增殖明显,14天时不含透明质酸钠的钙镁硅酸盐复合支架的细胞增殖率比加入透明质酸钠的含钙镁硅酸盐复合支架高30%,表明去除透明质酸钠组分后支架生物相容性增加(见附图5中b,c组)。
对比测试2:
将实施例1所得不含透明质酸钠的钙镁硅酸盐复合支架和对比例1所得的加入透明质酸钠的含钙镁硅酸盐复合支架,放入体外标准条件化无菌培养的深层软骨细胞行共培养7天后对细胞内ALP检测。经检测,培养7天后,含钙镁硅酸盐支架组细胞内ALP活性明显上升,存在显著性差异,表明去除透明质酸钠组分后支架引起细胞ALP活性增加、矿化能力增强(见附图6中b,c组)。
对比测试3:
将实施例1所得不含透明质酸钠的钙镁硅酸盐复合支架和对比例1所得的加入透明质酸钠的含钙镁硅酸盐复合支架,放入体外标准条件化无菌培养的深层软骨细胞行共培养7天后检测细胞II型胶原表达。经检测,培养7天后,不含透明质酸钠的钙镁硅酸盐复合支架组引起细胞II型胶原表达下调,表明去除透明质酸钠组分后,所拥有的促细胞成软骨作用被明显减弱(见附图7)。
透明质酸本身具有良好的可降解性和生物相容性,有保护软骨、润滑关节作用,在本实施例1的技术方案相比对比例1省去透明质酸后,不仅未降低生物相容性、细胞活性和矿化能力,反而增加了支架生物相容性、细胞活性和矿化能力,具有其突出显著的技术效果。
Claims (7)
1.一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架,其特征在于:所述复合支架由海藻酸钠:I型胶原:钙镁硅酸盐以质量分数比(1-6):2:(5-10)构成,并以氯化钙作为交联剂,氯化钙占所述复合支架的质量分数为(1-10)%;所述的钙镁硅酸盐采用镁掺杂硅灰石,镁掺杂硅灰石中镁代替钙的摩尔百分数为4-15%。
2.根据权利要求1所述的一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架,其特征在于:所述的复合支架的长度或直径为5-15mm,孔道孔径尺度为50-500μm,孔隙率为20-80%。
3.根据权利要求1所述的一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架,其特征在于:所述复合支架呈长方体、立方体、圆柱体或圆台体,支架内相邻孔道相互贯通,所述的孔道形态为圆形、三角形、四边形、蜂窝形或者阿基米德螺旋弧形。
4.根据权利要求1所述的一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架,其特征在于:所述复合支架采用三维打印方法构建制成。
5.如权利要求1所述的一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架的制备方法,其特征在于包括以下步骤:将钙镁硅酸盐粉体分散到去离子水中,充分搅拌后复合海藻酸钠、I型胶原形成混匀水凝胶状物,将水凝胶状物置入三维打印机中,打印出多孔支架,得到所述复合支架;
方法具体为:
1)将钙镁硅酸盐粉体使用湿法球磨处理后获得颗粒度不超过5μm的超细粉体,将超细粉体分散到去离子水中,并在常温下搅拌1小时,再加入I型胶原粉末在常温下搅拌5分钟,最后再加入海藻酸钠在常温下搅拌15分钟,制成水凝胶状物作为复合材料墨水;
2)将水凝胶状物置入三维打印机中,使用三维打印机设备打印,打印过程中使用氯化钙喷雾交联,打印完毕后将支架浸泡于氯化钙溶液10分钟进一步交联,得到最终3D打印的复合支架。
6.根据权利要求5所述的一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架的制备方法,其特征在于:
所述步骤2)是将复合材料墨水置入写入式三维打印机中,设定孔道形态、孔通道尺寸、支架尺寸和孔隙率,启动打印设备,进行三维打印,形成多孔复合支架。
7.权利要求1-4任一所述的一种3D打印的海藻酸钠-I型胶原-陶瓷复合支架或者权利要求5-6任一所述方法制成的复合支架的应用,其特征在于:所述复合支架应用于提高软骨细胞增殖与ALP活性。
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