CN107485730B - 一种改善生物衍生羟基磷灰石细胞相容性的方法 - Google Patents
一种改善生物衍生羟基磷灰石细胞相容性的方法 Download PDFInfo
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Abstract
本发明公开了一种改善生物衍生羟基磷灰石细胞相容性的方法，将猪四肢骨在‑176～‑18℃冷冻3个月后，去除骨组织表面的软组织，骨锯锯成松质骨块，不采用有机溶剂，纯化水超声清洗，去除其内骨髓成分，置于冷冻干燥机内冻干；置于超临界二氧化碳反应釜中，反应后取出置于碱溶液中，冷冻干燥，取出放于通气马弗炉渐进升降温处理；然后封装，钴‑60γ射线辐照灭菌制成。本发明采用超临界二氧化碳进行脱脂脱蛋白处理，无有机物质残留，能有效改善所制备材料的生物相容性和生物力学性能。
Description
技术领域
本发明属于生物移植材料技术领域，具体地说，涉及一种改善生物衍生羟基磷灰石细胞相容性的方法。
背景技术
骨移植是仅次于输血的第二大组织移植，目前常用的骨移植材料主要包括生物衍生类材料及人工合成类材料，生物衍生类材料由于与自体骨组织具有更为相近的物理结构和化学组成在临床得到广泛应用，但来源于同种异体或异种的骨组织具有较强的抗原性，移植后常易引起免疫排斥反应，导致移植失败，为有效降低这类材料的免疫原性，常采用脱细胞、去蛋白等方法去除抗原的载体，而当前对异种骨最常采用的去抗原方法为采用高温煅烧去除骨组织中的胶原蛋白等有机成分而制备的煅烧骨，其主要成分为羟基磷灰石，这类生物衍生羟基磷灰石虽然有效降低了抗原性，但却丧失了原有骨组织良好的细胞相容性，植入后不利于成骨细胞的贴附，从而影响成骨速度，同时其在制备过程中需采用大量有机溶剂去除蛋白质，易影响到骨组织的微观结构及导致有机溶剂残留，影响材料的生物相容性。
发明内容
本发明的目的在于提供一种改善生物衍生羟基磷灰石细胞相容性的方法，该方法采用超临界二氧化碳进行脱脂脱蛋白处理，并经碱超声联合冷冻干燥处理后进行重复渐进式升降温结合通气煅烧处理，所制备生物衍生羟基磷灰石无有机溶剂残留，能有效改善材料表面结构及生物相容性。其具体技术方案为：
一种改善生物衍生羟基磷灰石细胞相容性的方法，包括以下步骤：
步骤1、将猪四肢骨在-176～-18℃冷冻3个月后，去除骨组织表面的软组织，骨锯锯成5cm×5cm×5cm～5cm×5cm×20cm的松质骨块，超声清洗8～72小时，清洗温度为30～60℃，每1～4小时更换纯化水，并置于甩桶内离心5～15分钟，去除其内骨髓成分，置于冷冻干燥机内冻干至含水量小于5％；
步骤2、将步骤1骨粉或骨块置于超临界二氧化碳反应釜中，密封，然后将二氧化碳压进反应釜至反应釜内压力为15～30MPa，材料与二氧化碳体积比为1∶1～10，使反应釜内温度升至30～60℃，保持20～60分钟，然后以1～10MPa/min的速率放气，重复3～5次，打开反应釜，取出材料；
步骤3、将步骤2中骨粉或骨块置于0.1～1M的Ca(OH)2、KOH或NaOH溶液中，10～300g/L，10～40℃条件下超声10～600分钟，超声温度为30～60℃；
步骤4、将步骤3中材料取出后进行冷冻干燥至含水量为3～10％；
步骤5、将步骤4中材料放于通气马弗炉，200℃/小时渐进升温至600～1500℃，保持2～6小时，200℃/小时渐进退温，重复1～6次；
步骤6、然后封装，10～25kGy钴-60γ射线辐照灭菌制成。
优选地，步骤2中所述材料与二氧化碳体积比为1∶3。
优选地，步骤2中反应釜内温度升至37～40℃。
优选地，步骤3中骨粉或骨块置于0.5M的NaOH。
优选地，步骤4中所述含水量为5％。
优选地，步骤4中冷冻干燥后含水量的测定方法为：将水分测定仪设定在90℃，加一个天平秤盘，加热至自动停止，冷却至室温，迅速打开1袋待测产品，用镊子辅助将骨小块均匀铺洒在秤盘上，盖水分测定仪罩，待水分测定仪数据稳定后读取初始质量值W0，设定温度：90℃，时间：10～15分钟，并开启加热，单位样品的测定在小于设定时间内自动停止为有效，此时读取的R，值即为含水量。
优选地，步骤6中所述钴-60γ射线辐照灭菌过程中，辐射灭菌的辐射剂量为15～20kGy。
与现有技术相比，本发明的有益效果为：
1、本发明采用超临界二氧化碳进行脱脂脱蛋白处理，无有机物质残留，能有效改善所制备材料的生物相容性。
2、本发明在煅烧前采用碱超声处理联合冷冻干燥的方法，能显著提高材料的细胞相容性。
3、本发明采用重复渐进式升降温结合通气的方法进行煅烧处理，能有效改善煅烧过程中出现的裂纹对材料生物力学性能的影响。
附图说明
图1是未经脱脂的材料的扫描电镜图；
图2是经过超临界二氧化碳脱脂的扫描电镜图；
图3是未经超临界二氧化碳脱脂的材料煅烧的扫描电镜观察图；
图4是经过超临界二氧化碳脱脂的材料煅烧的扫描电镜观察图；
图5是未经碱处理组材料与MC3T3-E1细胞复合培养扫描电镜观察图，其中，图5a：材料表面可见死亡细胞；图5b：材料孔隙内可见死亡细胞；
图6是经胶原包被组材料与MC3T3-E1细胞复合培养扫描电镜观察图，其中，图6a：包被胶原表面可见大量死亡细胞；图6b：材料表面可见细胞贴附，但存在大量死亡细胞；
图7是NaOH处理组材料与MC3T3-E1细胞复合培养扫描电镜观察图，其中，图7a：材料表面及孔隙内均可见大量细胞贴附；图7b：材料表面贴附细胞形态良好。
具体实施方式
下面结合附图和具体实施例对本发明的技术方案作进一步详细地说明。
实施例1
超临界二氧化碳脱脂效果观察，将经过超临界二氧化碳脱脂与未经脱脂的骨材料进行扫描电镜观察，结果显示未经脱脂的材料表面有大量脂滴存在，而经超临界二氧化碳脱脂处理的材料表面干净，脂肪去除彻底，同时可去除细胞成份。
由图1可见未经脱脂的材料有大量脂滴存在，脂肪清洗不彻底；图2可见经超临界二氧化碳脱脂骨组织表面未见脂肪颗粒，可见去除细胞后的细胞陷窝。
实施例2
对本发明制备的生物衍生类羟基磷灰石材料进行肉眼和扫描电镜观察，结果显示：未经超临界二氧化碳脱脂的材料煅烧后颜色呈黄白色，表明前期脂肪去除不彻底，对后续羟基磷灰石材料的制备有较显著影响；经超临界二氧化碳脱脂的材料煅烧后颜色呈象牙白，显微结构观察表明传统煅烧方法材料表面易产生大量的微裂痕，而采用本方法制备的煅烧骨表面基本无微裂痕产生。
实施例3为表明本发明制备的生物衍生类羟基磷灰石材料生物力学性能，测定了传统方法制备羟基磷灰石与本法制备羟基磷灰石的生物力学性能，具体如下：
将制备的复合材料修剪5×5×5mm的正方体，每组5块，用RGT-20A微机控制万能试验机测定其抗压缩强度，加载速率为1mm/s，并计算弹性模量。具体结果见表1。表明本法所制备羟基磷灰石由于降低了煅烧时产生的微裂痕，有效提高了材料的生物力学性能。
表1传统煅烧方法与本法制备的生物衍生类羟基磷灰石材料抗压强度和弹性模量
实施例4
为表明本发明制备的生物衍生类羟基磷灰石材料的细胞相容性，做了体外细胞相容性试验，具体如下：
煅烧骨的制备分为3组：1、未经碱处理组：将脱脂处理后的异种骨未经NaOH处理后渐进式升降温700℃4h，重复3次；2、NaOH处理组，置于0.5M NaOH溶液中超声1小时，(40℃)置马弗炉渐进升降温700℃4h，重复3次。3、胶原处理组：未经碱处理组采用传统胶原包被改善细胞相容性的方法，即将材料浸泡于0.2％胶原蛋白溶液中进行包被，冷冻干燥备用。
材料与成骨细胞复合培养试验：将上述3组材料修剪成3mm×3mm×3mm大小，放置于6孔培养板内；将传代至第3代的MC3T3-E1细胞以1×104/ml接种于已放置材料的培养板内，每孔500ul，加入DMEM高糖培养基至完全淹没材料，分别于接种后第1、3、5d倒置相差显微镜观察细胞生长及与材料贴附情况，并于第5d取出材料，2.5％戊二醛4℃固定4h，PBS缓冲液冲洗三遍，冷冻干燥机进行冻干处理后，离子溅射仪进行表面喷金处理，扫描电镜观察细胞贴附情况。
结果如图5至图7所示，未经碱处理组材料表面无细胞贴附生长，可见大量死亡细胞；胶原包被处理组在有胶原包被的地方有较多细胞贴附生长，而无胶原包被的地方，无细胞生长，且可见大量死亡细胞；NaOH处理组材料表面及孔洞内有大量细胞贴附生长，细胞排列密集，细胞形态良好。表明NaOH处理能有效改善材料的细胞亲和性。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (1)
1.一种改善生物衍生羟基磷灰石细胞相容性的方法，其特征在于，包括以下步骤：
步骤1、将猪四肢骨在-176～-18℃冷冻3个月后，去除骨组织表面的软组织，骨锯锯成5cm×5cm×5cm～5cm×5cm×20cm的松质骨块，超声清洗8～72小时，清洗温度为30～60℃，每1～4小时更换纯化水，并置于甩桶内离心5～15分钟，去除其内骨髓成分，置于冷冻干燥机内冻干至含水量小于5％；
步骤2、将步骤1骨粉或骨块置于超临界二氧化碳反应釜中，密封，然后将二氧化碳压进反应釜至反应釜内压力为15～30MPa，材料与二氧化碳体积比为1∶3，使反应釜内温度升至37～40℃，保持20～60分钟，然后以1～10MPa/min的速率放气，重复3～5次，打开反应釜，取出材料；
步骤3、将步骤2中骨粉或骨块置于0.5M的NaOH溶液中10-300g/L，超声10-600分钟，超声温度为30～60℃；
步骤4、将步骤3中材料取出后进行冷冻干燥至含水量为5％；
冷冻干燥后含水量的测定方法为：将水分测定仪设定在90℃，加一个天平秤盘，加热至自动停止，冷却至室温，迅速打开1袋待测产品，用镊子辅助将骨小块均匀铺洒在秤盘上，盖水分测定仪罩，待水分测定仪数据稳定后读取初始质量值W0，设定温度：90℃，时间：10-15分钟，并开启加热，单位样品的测定在小于设定时间内自动停止为有效，此时读取的R值即为含水量；
步骤5、将步骤4中材料放于通气马弗炉，200℃/小时渐进升温至600～1500℃，保持2～6小时，200℃/小时渐进退温，重复1～6次；
步骤6、然后封装，10～20kGy钴-60γ射线辐照灭菌制成。
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1126619A (zh) * | 1995-01-11 | 1996-07-17 | 韩巽 | 动物骨制生物相容性人工义眼台 |
| CN1579562A (zh) * | 2003-08-01 | 2005-02-16 | 中国人民解放军第四军医大学口腔医学院 | 一组同类不同结构的陶瓷化骨的制备方法 |
| CN102008749A (zh) * | 2010-11-26 | 2011-04-13 | 邓旭亮 | 一种骨修复支架材料及其制备方法 |
| WO2013057720A2 (pt) * | 2011-10-19 | 2013-04-25 | Universidade Católica Portuguesa - Ucp | Composições e método de obtenção de composições à base de apatite a partir de fonte natural, materiais relacionados e seus usos |
| CN103638557A (zh) * | 2013-10-24 | 2014-03-19 | 陕西佰傲再生医学有限公司 | 一种脱抗原生物骨及其制备方法 |
| CN104174067A (zh) * | 2013-05-22 | 2014-12-03 | 烟台正海生物技术有限公司 | 一种天然无机骨基质及其制备方法 |
| CN104689372A (zh) * | 2015-03-26 | 2015-06-10 | 北京鑫康辰医学科技发展有限公司 | 一种同种骨超临界二氧化碳脱脂处理的方法 |
-
2016
- 2016-12-02 CN CN201611114596.3A patent/CN107485730B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1126619A (zh) * | 1995-01-11 | 1996-07-17 | 韩巽 | 动物骨制生物相容性人工义眼台 |
| CN1579562A (zh) * | 2003-08-01 | 2005-02-16 | 中国人民解放军第四军医大学口腔医学院 | 一组同类不同结构的陶瓷化骨的制备方法 |
| CN102008749A (zh) * | 2010-11-26 | 2011-04-13 | 邓旭亮 | 一种骨修复支架材料及其制备方法 |
| WO2013057720A2 (pt) * | 2011-10-19 | 2013-04-25 | Universidade Católica Portuguesa - Ucp | Composições e método de obtenção de composições à base de apatite a partir de fonte natural, materiais relacionados e seus usos |
| CN104174067A (zh) * | 2013-05-22 | 2014-12-03 | 烟台正海生物技术有限公司 | 一种天然无机骨基质及其制备方法 |
| CN103638557A (zh) * | 2013-10-24 | 2014-03-19 | 陕西佰傲再生医学有限公司 | 一种脱抗原生物骨及其制备方法 |
| CN104689372A (zh) * | 2015-03-26 | 2015-06-10 | 北京鑫康辰医学科技发展有限公司 | 一种同种骨超临界二氧化碳脱脂处理的方法 |
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