CN116236624A - 脱钙人牙基质材料及脱钙人牙基质制作方法 - Google Patents
脱钙人牙基质材料及脱钙人牙基质制作方法 Download PDFInfo
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Abstract
本发明公开了一种生物医用新材料,具体为一种脱钙人牙基质材料及脱钙人牙基质制作方法。主要为制备了改性再生丝素蛋白提取液和鹿筋胶原蛋白提取液,并将其与脱钙人牙基质复合,制成高度多孔的类似泡沫状的脱钙人牙基质胶原海绵材料。且该材料具有良好的表面活性,生物相容性及生物可降解性,同时能促进骨修复及伤口收敛,是理想的骨修复支架材料。
Description
技术领域
本发明属于生物医学技术领域,具体为一种生物医用新材料,特别涉及一种脱钙人牙基质材料及脱钙人牙基质制作方法。
背景技术
近年来,骨缺损及骨不连等骨病变成为医学研究中的一大难题。国内外开发出一系列来源于生物体的成骨材料,主要有自体骨材料和同种异体骨。自体骨无排异,有良好的骨诱导活性,是植骨材料的金标准。但自体骨的移植会给患者带来二次创伤,还会导致供骨区的功能缺失,另外自体骨治疗骨缺损对较大范围的缺损治疗有局限性,也受到患者体质和年龄的限制。同种异体骨虽然使用方便,但因为免疫原性问题较容易引起排异反应从而导致治疗失败,常面临二次手术的问题。研究发现,从人体及动物牙和骨组织中提取骨形态发生蛋白可以对骨缺损进行修复。
脱钙人牙基质(Decalcification Tooth Matrices DTM)复合胶原材料作为骨修复材料的可行性。脱钙人牙基质材料是以口腔临床治疗中拔除的废弃牙为原料,经过独特的灭活方法进行制备与提取,能够诱导骨生长、促进骨组织彬成、完成骨修复治疗,属于生物医学新材料类产品。该类材料现通过科学的方法适当的处理,得到了能够诱导骨生成、促进骨组织形成与修复一种新型的骨诱导材料,对骨不连、骨组织萎缩吸收、骨折愈合不良及大面积的骨缺损等病症的治疗具有良好的效果。研究表明脱钙人牙基质复合胶原材料具有骨修复材料的结构特点,且具有良好的细胞相容性及诱导成骨细胞增殖活性。但是,现有的脱钙人牙基质材料成品形态为10~100目之间的颗粒,在大面积颅骨损伤,关节损伤,以及严重粉碎性骨折的治疗中因缺乏支撑作用而无法有效利用,限制了该产品的临床应用范围。
梅花鹿鹿筋作为传统中药材具有强筋健骨、滋补肝肾、益阳祛风湿等功效,被广泛用于治疗骨代谢疾病。鹿筋富含胶原蛋及必须的氨基酸,是制备生物活性肽的优质资源。补充胶原蛋白肽可以促进骨重塑,改善骨质疏松。
蚕丝纤维是一种高级的纺织原料,也是一种宝贵的天然有机高分子资源。其包括桑蚕丝(又称家蚕丝)和非桑蚕丝(如柞蚕丝、蓖麻蚕丝、天蚕丝等)两大类。单根蚕丝纤维是由丝胶蛋白包裹着两根丝素单纤维共同组成。通过脱胶工艺去除蚕丝纤维表面的丝胶蛋白后,再进行溶解,可得到再生丝素蛋白水溶液。但是直接这样获得的再生丝素蛋白表面游离的氨基较少,其生物相容性也不能满足后续产品的需求。
因此,本发明制备了改性再生丝素蛋白提取液和鹿筋胶原蛋白提取液,并将其与脱钙人牙基质复合,制成高度多孔的类似泡沫状的脱钙人牙基质胶原海绵材料。该材料具有良好的表面活性,生物相容性及生物可降解性,同时能促进骨修复及伤口收敛,是理想的骨修复支架材料。
发明内容
为解决上述问题,本发明利用新兴技术提取了鹿筋胶原蛋白液,通过等离子体处理获得了改性再生丝素蛋白提取液,并将其与脱钙人牙基质复合制成高度多孔的类似泡沫状的脱钙人牙基质胶原海绵材料。具体合成步骤如下:
S1、天然丝素蛋白不溶于水,因此本发明需要通过水盐溶剂***提取得到再生丝素蛋白:将100-300g的家蚕丝在0.05-0.5 mol/L的Na2CO3或NaHCO3中煮沸20-80 min,分别用去离子水和无水乙醇冲洗10-15min,在30-40℃条件下干燥10-15h。然后,将蚕丝溶于新配制浓度为5-8mol/L LiBr或NaBr溶液中,在65-80℃的烘箱中保温8-10h确保丝素蛋白充分溶解。
S2、改性再生丝素蛋白提取液:将步骤S1制备的溶液转移到截留分子量为3300-3800的透析袋中,溶液体积不超过透析袋最大容量的3/5,用密封夹封口后置于离子水中持续80-100h。换水间隔为5-8h。透析后得到的丝素蛋白原液转移至离心管内,以4800-5200 r/ min的离心速率进行离心分离。向上层液中加入0.3-0.5mol/L的NaCl溶液,并将其通入等离子体设备中,让再生丝素蛋白清液在整个放电管的外套和水槽间循环,通入氮气N2,气体流量控制为0.02-5m3 / h,当气流稳定后开始放电2-8h。该步骤通过等离子处理,增加了再生丝素蛋白的表面游离氨基的数目,大大提高其生物相容性。另,该步骤加无机的盐目的是提高外电极的电导率,使放电更容易发生。最后,等离子处理所得的再生丝素蛋白水溶液在5℃环境中储存。
S3、将20-90 g鹿筋加入浓度为3-8 mol/L的柠檬酸溶液中,浸泡10-30h后置于沸水中煮化,等水溶液温度降到20-30℃时用8000-10000 r/min离心10-30 min,离心后向上清液中加3-5 ml胃蛋白酶、风味蛋白酶或木瓜蛋白酶,放置在20-40℃摇床酶解,酶解后得到胶原蛋白粗液,然后将胶原蛋白粗液加入到微波超声加热仪中,超声加热到80-90℃,保持15-30 min,使胃蛋白酶、风味蛋白酶或木瓜蛋白酶失活,然后离心收集鹿筋胶原蛋白层清液,并在5℃环境中储存,该步骤中用微波超声处理胶原蛋白粗液除了使酶失活以外,更重要的是微波辅助可以使鹿筋胶原蛋白变的更细腻。
S4、取80-180ml步骤S2制备的改性再生丝素蛋白提取液和120-220ml步骤S3制备的鹿筋胶原蛋白液,用搅拌机发泡后,加入湿重40-60g的脱钙人牙骨基质材料(骨又生),混合均匀,倒入不锈钢托盘中,真空冷冻干燥后于90-100℃下交联固定3-5h,形成脱钙人牙基质胶原海绵,经紫外光照射,灭菌后即可获得脱钙人牙基质复合胶原骨修复材料。
优选地:所述步骤S1中家养蚕丝用量为300g,浸泡在0.5 mol/L的Na2CO3溶液中煮沸60 min。
优选地:所述步骤S2中等离子体处理通入氮气N2,气体流量控制为3m3 / h。
优选地:所述步骤S3中选用风味蛋白酶进行酶解。
优选地:所述步骤S3中胶原蛋白粗液在微波超声加热仪中,超声加热到90℃,保持20 min。
优选地:所述步骤S4选用的脱钙人牙骨基质材料为来源于深圳光明创博生物制品发展有限公司生产的产品,国食药监械(准)字2005 第 3461182 号,呈白色颗粒状。
本发明的有益效果在于:
1、单纯的骨形态发生蛋白在体内扩散迅速,且容易被蛋白酶降解,作用于靶细胞的时间非常有限,难以充分发挥其诱导活性,骨形态发生蛋白可以与本发明制备的脱钙人牙基质复合胶原骨修复材料复合,诱导骨生成长。
2、本发明制备的具有材料多孔的结构,比表面较大,有利于充分接触体液,加快材料降解.同时,多孔性也为成骨细胞的生长提供了较大的粘附面,有利于细胞粘附及血管组织向内生长。
3、本发明用等离子体制备改性的再生丝素蛋白的表面游离氨基的数目,大大提高其生物相容性。
4、本发明中使用用微波超声处理胶原蛋白粗液,使鹿筋胶原蛋白变的更细腻。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1和对比例1-3制备的再生丝素蛋白提取液细胞毒性检测结果统计柱状图。
图2为本发明实施例2制备再生丝素蛋白的扫描电子显微镜SEM图。
图3为本发明实施例3制备再生丝素蛋白的扫描电子显微镜SEM图。
图4为本发明实施例4和对比例6制备鹿筋胶原蛋干预MC3T3细胞24后的增殖活性图。
图5为本发明实施例4制备的脱钙人牙基质复合胶原骨修复材料的SEM图
实施方式
为了使本发明专利所要解决的技术问题、技术方案及有益效果更加清楚明白,以下结合附图及实施例,对本发明专利进行进一步详细说明。应当理解,此处所描述的具体实施例仅用以解释本发明内容,并不用于限定本发明专利。
实施例
S1、蚕丝纤维是一种高级的纺织原料,也是一种宝贵的天然有机高分子资源。将100g的家蚕丝在0.05mol/L的Na2CO3中煮沸20 min,分别用去离子水和无水乙醇冲洗10min,在30℃条件下干燥10h。然后,将蚕丝溶于新配制浓度为5mol/L LiBr溶液中,在65℃的烘箱中保温8h确保丝素蛋白充分溶解。
S2、将步骤S1制备的溶液转移到截留分子量为3300的透析袋中,溶液体积不超过透析袋最大容量的3/5,用密封夹封口后置于离子水中持续80h。换水间隔为5h。透析后得到的丝素蛋白原液转移至离心管内,以4800r / min的离心速率进行离心分离。向上层液中加入3ml的0.3mol/L的NaCl溶液,并将其通入等离子体设备中,让再生丝素蛋白清液在整个放电管的外套和水槽间循环,通入氮气N2,气体流量控制为0.02 m3 / h,当气流稳定后开始放电2h。该步骤通过等离子处理,增加了再生丝素蛋白表面游离氨基的数目,大大提高其生物相容性。该步骤加无机的盐目的是提高外电极的电导率,使放电更容易发生。最后,等离子处理所得的再生丝素蛋白水溶液在5℃环境中储存。
S3、将20g鹿筋加入浓度为3mol/L的柠檬酸溶液中,浸泡10h后置于沸水中煮化,等水溶液温度降到20℃时用8000 r/min离心10 min,离心后向上清液中加3 ml胃蛋白酶,放置在20℃摇床酶解,酶解后得到胶原蛋白粗液,然后将胶原蛋白粗液加入到微波超声加热仪中,超声加热到80℃,保持15 min,使胃蛋白酶失活,然后离心收集鹿筋胶原蛋白层清液,并在5℃环境中储存。
S4、取80ml步骤S2制备的改性再生丝素蛋白提取液和220ml步骤S3制备的鹿筋胶原蛋白液,用搅拌机发泡后,加入20g的脱钙人牙骨基质材料,混合均匀,倒入不锈钢托盘中,真空冷冻干燥后于90℃下交联固定3h,形成脱钙人牙基质胶原海绵,经紫外光照射,灭菌后即可获得脱钙人牙基质复合胶原骨修复材料。
对比例1:除步骤S1中用柞蚕丝代替家蚕丝以外,其余均与实施例1相同。
对比例2:除步骤S1中用蓖麻蚕丝代替家蚕丝以外,其余均与实施例1相同。
对比例3:除步骤S1中用天蚕丝代替家蚕丝以外,其余均与实施例1相同。
在本发明实施例1和对比例1-3制备的再生丝素蛋白提取液中接种人成骨样细胞MG-63并培养。继而进行细胞增殖实验,采用MTT比色法,通过测定吸光度,求得细胞存活率。采用CCK-8试剂盒对细胞毒性进行检查。图1为本发明实施例1和对比例1-3制备的再生丝素蛋白提取液细胞毒性检测结果,可以看出实施例1制备的再生丝素蛋白提取液对兔骨髓间充质干细胞没有细胞毒性,而且吸光度随时间延长有明显增加,证明本发明实施例1制备的再生丝素蛋白提取液有良好的细胞相容性。
实施例
S1、蚕丝纤维是一种高级的纺织原料,也是一种宝贵的天然有机高分子资源。将300g的家蚕丝在0.5 mol/L的NaHCO3中煮沸80 min,分别用去离子水和无水乙醇冲洗15min,在40℃条件下干燥15h。然后,将蚕丝溶于新配制浓度为8mol/L NaBr溶液中,在80℃的烘箱中保温10h确保丝素蛋白充分溶解。
S2、将步骤S1制备的溶液转移到截留分子量为3800的透析袋中,溶液体积不超过透析袋最大容量的3/5,用密封夹封口后置于离子水中持续100h。换水间隔为8h。透析后得到的丝素蛋白原液转移至离心管内,以5200r / min的离心速率进行离心分离。向上层液中加入10ml的0.5mol/L的NaCl溶液,并将其通入等离子体设备中,让再生丝素蛋白清液在整个放电管的外套和水槽间循环,通入氮气N2,气体流量控制为5m3 / h,当气流稳定后开始放电8h。该步骤通过等离子处理,增加了再生丝素蛋白的表面游离氨基的数目,大大提高其生物相容性。该步骤加无机的盐目的是提高外电极的电导率,使放电更容易发生。最后,等离子处理所得的再生丝素蛋白水溶液在5℃环境中储存。
S3、将90g鹿筋加入浓度为8 mol/L的柠檬酸溶液中,浸泡30h后置于沸水中煮化,等水溶液温度降到30℃时用10000 r/min离心30 min,离心后向上清液中加5 ml风味蛋白酶,放置在40℃摇床酶解,酶解后得到胶原蛋白粗液,然后将胶原蛋白粗液加入到微波超声加热仪中,超声加热到90℃,保持30 min,使风味蛋白酶失活,然后离心收集鹿筋胶原蛋白层清液,并在5℃环境中储存。
S4、取180ml步骤S2制备的改性再生丝素蛋白提取液和120ml步骤S3制备的鹿筋胶原蛋白液,用搅拌机发泡后,加入60g的脱钙人牙骨基质材料,混合均匀,倒入不锈钢托盘中,真空冷冻干燥后于100℃下交联固定5h,形成脱钙人牙基质胶原海绵,经紫外光照射,灭菌后即可获得脱钙人牙基质复合胶原骨修复材料。
对比例4:除步骤S2中不用等离子体处理再生丝素蛋白清液外,其余步骤均与实施例2相同。
对比例5:除步骤S2中用氩气代替氮气通入等离子体设备处,其余步骤均与实施例2相同。
游离氨基是再生丝素蛋白化学修饰和交联的重要基团,本发明采用茚三酮显色法检测不同溶解时间再生丝素蛋白的游离氨基。用甘氨酸配制氨基氮含量为 3-8 µmol/ml的标准氨基酸溶液。分别取各浓度的标准氨基酸溶液1ml,加入具塞试管中,再加入1.5mol/L的乙酸/乙酸钠缓冲液2 ml (量取2.5 mol/L 的乙酸钠溶液65ml,加入2.5 mol/L的乙酸12ml混合,调至pH为5)和茚三酮溶液 2 ml,混合均匀后盖住试管口,置沸水浴中加热15 min,取出,冷却。加入 60%乙醇稀释 20 倍,于570nm波长下测定,以吸光度为纵坐标、含氮量为横坐标,绘制标准曲线。结果表明,游离氨基浓度在0-5 µmol/ml范围内与吸光度线性关系良好,线性方程为吸光度=0.0953×含氮量 + 0.0539,R2=0.9997。将实施例2和对比例4、5制备的再生丝素蛋白冻干成粉后,用超纯水配制成0.01%的溶液,按上述操作,计算游离氨基含量,结果如表1所示。
表1
分析表1的数据可以知,本发明用氮气等离子体处理再生丝素蛋白提取液,增加了再生丝素蛋白的表面游离氨基的数目,大大提高其生物相容性。氨基的增多也有利于材料胶原骨的修复。图2为本发明实施例2制备再生丝素蛋白的扫描电子显微镜SEM图,从图中可以看出为均为片状结构,在做骨修复时可以提供很好的支撑。
实施例
S1、蚕丝纤维是一种高级的纺织原料,也是一种宝贵的天然有机高分子资源。将220g的家蚕丝在0.3 mol/L的Na2CO3中煮沸60 min,分别用去离子水和无水乙醇冲洗13min,在34℃条件下干燥10-15h。然后,将蚕丝溶于新配制浓度为6 mol/L LiBr溶液中,在70℃的烘箱中保温9h确保丝素蛋白充分溶解。
S2、将步骤S1制备的溶液转移到截留分子量为3600的透析袋中,溶液体积不超过透析袋最大容量的3/5,用密封夹封口后置于离子水中持续90h。换水间隔为7h。透析后得到的丝素蛋白原液转移至离心管内,以4900r / min的离心速率进行离心分离。向上层液中加入8ml的0.4mol/L的NaCl溶液,并将其通入等离子体设备中,让再生丝素蛋白清液在整个放电管的外套和水槽间循环,通入氮气N2,气体流量控制为3 m3 / h,当气流稳定后开始放电7h。该步骤通过等离子处理,增加了再生丝素蛋白纳米粒的表面游离氨基的数目,大大提高其生物相容性。该步骤加无机的盐目的是提高外电极的电导率,使放电更容易发生。最后,等离子处理所得的再生丝素蛋白水溶液在5℃环境中储存。
S3、将70 g鹿筋加入浓度为7 mol/L的柠檬酸溶液中,浸泡20h后置于沸水中煮化,等水溶液温度降到25℃时用9000 r/min离心18 min,离心后向上清液中加4ml木瓜蛋白酶,放置在38℃摇床酶解,酶解后得到胶原蛋白粗液,然后将胶原蛋白粗液加入到微波超声加热仪中,超声加热到89℃,保持19 min,使胃蛋白酶、风味蛋白酶或木瓜蛋白酶失活,然后离心收集鹿筋胶原蛋白层清液,并在5℃环境中储存。
S4、取110ml步骤S2制备的改性再生丝素蛋白提取液和190ml步骤S3制备的鹿筋胶原蛋白液,用搅拌机发泡后,加入35g的脱钙人牙骨基质材料,混合均匀,倒入不锈钢托盘中,真空冷冻干燥后于95℃下交联固定4h,形成脱钙人牙基质胶原海绵,经紫外光照射,灭菌后即可获得脱钙人牙基质复合胶原骨修复材料。
图3为本发明实施例3制备再生丝素蛋白的扫描电子显微镜SEM图,从图中可以看出为均为片状结构,与实施例2制备的片状略有差异,但均为片状结构,符合本发明的设计理念。
测定实施例3制备的脱钙人牙基质复合胶原骨修复材料的生物相容性:用含8%的猪血清的RPMI1640培养基,在36℃环境中培养MCT3T-E1细胞,每5天更换一次培养液,待细胞生长至对数期,调节细胞浓度至5×105个/ml;将脱钙人牙基质复合胶原材料预先用含血清的培养基预浸泡18h;接种5×104的细胞至材料表面。继续培养3天后,改用含有48μg/ml的左旋维他命C和8mM甘油磷酸酯的分化培养基培养,每4天更换一次培养液,同时设1%琼脂糖对照组。分别于5、10、15、20天收集细胞,用Hoechst dye 33258 kit对细胞总DNA进行荧光定量分析。用Alkaline Phosphatase Fluorescence Assay Kit对培养物的细胞碱性磷酸酶活性进行荧光定量分析。本发明利用MCT3T-E1细胞系与实施例3制备的脱钙人牙基质复合胶原骨修复材料混合培养,随着培养时间的延长,实施例3和1%琼脂糖对照组的总DNA含量在增加,培养5天后,细胞增殖进入对数生长期,实施例3和1%琼脂糖对照组细胞总DNA差异不大。碱性磷酸酶活性实验结果显示,培养至30天时,实施例3组中碱性磷酸酶活性显著高于1%琼脂糖对照组,说明该材料具有较好的生物相容性,同时具有诱导成骨细胞增殖的能力。
实施例
S1、蚕丝纤维是一种高级的纺织原料,也是一种宝贵的天然有机高分子资源。将210g的家蚕丝在0.4 mol/L的NaHCO3中煮沸50 min,分别用去离子水和无水乙醇冲洗13min,在37℃条件下干燥11h。然后,将蚕丝溶于新配制浓度为6.5mol/L NaBr溶液中,在77℃的烘箱中保温8.6h确保丝素蛋白充分溶解。
S2、将步骤S1制备的溶液转移到截留分子量为3700的透析袋中,溶液体积不超过透析袋最大容量的3/5,用密封夹封口后置于离子水中持续84h。换水间隔为7.3h。透析后得到的丝素蛋白原液转移至离心管内,以5100r / min的离心速率进行离心分离。向上层液中加入7ml的0.4mol/L的NaCl溶液,并将其通入等离子体设备中,让再生丝素蛋白清液在整个放电管的外套和水槽间循环,通入氮气N2,气体流量控制为0.1m3 / h,当气流稳定后开始放电6.2h。该步骤通过等离子处理,增加了再生丝素蛋白纳米粒的表面游离氨基的数目,大大提高其生物相容性。该步骤加无机的盐目的是提高外电极的电导率,使放电更容易发生。最后,等离子处理所得的再生丝素蛋白水溶液在5℃环境中储存。
S3、将70 g鹿筋加入浓度为5 mol/L的柠檬酸溶液中,浸泡22h后置于沸水中煮化,等水溶液温度降到28℃时用9800 r/min离心29 min,离心后向上清液中加3.6 ml木瓜蛋白酶,放置在36℃摇床酶解,酶解后得到胶原蛋白粗液,然后将胶原蛋白粗液加入到微波超声加热仪中,超声加热到88℃,保持17 min,使木瓜蛋白酶失活,然后离心收集鹿筋胶原蛋白层清液,并在5℃环境中储存,
S4、取99ml步骤S2制备的改性再生丝素蛋白提取液和135ml步骤S3制备的鹿筋胶原蛋白液,用搅拌机发泡后,加入55g的脱钙人牙骨基质材料,混合均匀,倒入不锈钢托盘中,真空冷冻干燥后于96℃下交联固定4.2h,形成脱钙人牙基质胶原海绵,经紫外光照射,灭菌后即可获得脱钙人牙基质复合胶原骨修复材料。
对比例6:除步骤S3中用水浴加热代替微波超声外,其余均与实施例4相同。
图4为本发明实施例4和对比例6制备鹿筋胶原蛋干预MC3T3细胞24后的增殖活性图。本发明利用CCK-8试剂检测鹿筋胶原在不同浓度下干预MC3T3细胞24h后的增殖活性,从图中可以看出用实施例6制备的鹿筋胶原蛋白液使细胞存活活性高。因此可以证明本发明使用微波超声加热的重要性。本发明制备的脱钙人牙基质复合胶原骨修复材料呈白色,有规则形状物体大小均匀,分布均匀的材料颗粒被胶原和再生丝素蛋白连接在一起,形成一种多孔疏松的材料。图5为本发明实施例4制备的脱钙人牙基质复合胶原骨修复材料的SEM图,可以看到絮层状的多孔隙结构,孔隙间有互相贯通的微孔。
以上所述实施例仅表达了本发明的具体实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (6)
1.一种脱钙人牙基质材料及脱钙人牙基质制作方法,其特征在于:具体制作步骤如下:
S1、将100-300g的家蚕丝在0.05-0.5 mol/L的Na2CO3或NaHCO3中煮沸20-80 min,分别用去离子水和无水乙醇冲洗10-15min,在30-40℃条件下干燥10-15h;然后,将蚕丝溶于新配制浓度为5-8mol/L LiBr或NaBr溶液中,在65-80℃的烘箱中保温8-10h确保丝素蛋白充分溶解;
S2、改性再生丝素蛋白提取液:将步骤S1制备的溶液转移到截留分子量为3300-3800的透析袋中,溶液体积不超过透析袋最大容量的3/5,用密封夹封口后置于离子水中持续80-100h;换水间隔为5-8h;透析后得到的丝素蛋白原液转移至离心管内,以4800-5200 r /min的离心速率进行离心分离;向上层液中加入0.3-0.5mol/L的NaCl溶液,并将其通入等离子体设备中;让再生丝素蛋白清液在整个放电管的外套和水槽间循环,通入氮气N2,气体流量控制为0.02-5m3 / h,当气流稳定后开始放电2-8h;最后,等离子处理所得的再生丝素蛋白水溶液在5℃环境中储存;
S3、将20-90 g鹿筋加入浓度为3-8 mol/L的柠檬酸溶液中,浸泡10-30h后置于沸水中煮化,等水溶液温度降到20-30℃时用8000-10000 r/min离心10-30 min,离心后向上清液中加3-5 ml胃蛋白酶、风味蛋白酶或木瓜蛋白酶,放置在20-40℃摇床酶解,酶解后得到胶原蛋白粗液,然后将胶原蛋白粗液加入到微波超声加热仪中,超声加热到80-90℃,保持15-30 min,使胃蛋白酶、风味蛋白酶或木瓜蛋白酶失活,然后离心收集鹿筋胶原蛋白层清液,并在5℃环境中储存;
S4、取80-180ml步骤S2制备的改性再生丝素蛋白提取液和120-220ml步骤S3制备的鹿筋胶原蛋白液,用搅拌机发泡后,加入湿重40-60g的脱钙人牙骨基质材料,混合均匀,倒入不锈钢托盘中,真空冷冻干燥后于90-100℃下交联固定3-5h,形成脱钙人牙基质胶原海绵,经紫外光照射,灭菌后即可获得脱钙人牙基质复合胶原骨修复材料。
2.根据权利要求1所述的一种脱钙人牙基质材料及脱钙人牙基质制作方法,其特征在于:所述步骤S1中家养蚕丝用量为300g,浸泡在0.5 mol/L的Na2CO3溶液中煮沸60 min。
3.根据权利要求1或2所述的一种脱钙人牙基质材料及脱钙人牙基质制作方法,其特征在于:所述步骤S1中家养蚕丝用量为100g,浸泡在0.5 mol/L的NaHCO3溶液中煮沸80 min。
4.根据权利要求1所述的一种脱钙人牙基质材料及脱钙人牙基质制作方法,其特征在于:所述步骤S2中等离子体处理通入氮气N2,气体流量控制为3m3 / h。
5.根据权利要求1所述的一种脱钙人牙基质材料及脱钙人牙基质制作方法,其特征在于:所述步骤S3中选用风味蛋白酶进行酶解。
6.根据权利要求5所述的一种脱钙人牙基质材料及脱钙人牙基质制作方法,其特征在于:所述步骤S3中胶原蛋白粗液在微波超声加热仪中,超声加热到90℃,保持20 min。
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