CN111686305B - 促进骨愈合与再生的凝胶组合物的制备方法 - Google Patents

促进骨愈合与再生的凝胶组合物的制备方法 Download PDF

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CN111686305B
CN111686305B CN202010549738.9A CN202010549738A CN111686305B CN 111686305 B CN111686305 B CN 111686305B CN 202010549738 A CN202010549738 A CN 202010549738A CN 111686305 B CN111686305 B CN 111686305B
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stem cells
differentiation
mesenchymal stem
gel composition
regeneration
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CN111686305A (zh
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芦慧颖
李婷婷
张怡
刘艳青
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Tianqing Stem Cell Co ltd
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Tianqing Stem Cell Co ltd
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Abstract

促进骨愈合与再生的凝胶组合物的制备方法，它涉及干细胞领域。它用于软骨损伤和骨缺损的修复。方法：一、sPL的制备；二、间充质干细胞趋向分化；三、凝胶组合物的制备。本发明制备出sPL后对所含生长因子含量进行检测，再调节生长因子浓度，保证PL的均一性。进行治疗前，先对间充质干细胞进行趋向分化，使干细胞向治疗所需要的方向分化，这样可以有效缩短治疗周期，加速修复、填补损伤部位所需种类细胞。可使细胞继续分化，提高治疗效果，节约治疗成本。可用于治疗骨缺损、骨不连、骨损伤，可促进骨的愈合与再生。对软骨损伤和骨缺损修复有显著效果，软骨表面趋于平滑，缺损部位骨全部修复。本发明应用于软骨损伤和骨缺损的修复。

Description

促进骨愈合与再生的凝胶组合物的制备方法
技术领域
本发明属于干细胞领域，具体涉及促进骨愈合与再生的凝胶组合物的制备方法。
背景技术
PL(血小板裂解液)来源于自体血液，具有无疾病传染及免疫排斥等优点，且取材方便、制备简便、费用合理、手术时间短等优势，自发现以来已逐渐应用于口腔、整形、骨科、耳鼻喉科、神经外科等领域的组织修复中，已有大量的试验数据证明其在骨损伤修复的作用。Assoian首先发现了血浆中提取的PRP(浓缩血小板血浆)中含有多种生长因子,为骨缺损的修复提供了广泛的应用前景。PL具有促进移植骨成活和再生的能力；可使骨形成速度增加2倍，骨密度增加20％；可使种植牙早期获得固位成功，促进皮肤和黏膜组织的创伤愈合，无抗原抗体的免疫反应，感染的危险性小。
PL复合技术是近年研究的热点，复合的细胞是一类具有自我复制更新且具分化潜力的细胞。间充质干细胞(Mesenchymal stem cells,MSCs)是成体干细胞的一种，来自胚胎发育的过程中，MSCs担负者向骨、软骨、脂肪、肌腱韧带等的发育分化功能。研究体外扩增的MSCs向软骨分化和软骨损伤的研究可以追溯到上个世纪八、九十年代。Oshton(1980)、Nuttall(1998)等分别以动物骨髓MSCs或人骨髓MSCs体外试验证实了其具有分化为骨、软骨、纤维组织的能力，Davis等有从单细胞克隆角度，证实了同一细胞来源的MSCs可以分别分化为骨和软骨，从而进一步确立了MSCs的多向分化潜能。
PL复合间充质干细胞技术在大量的动物模型试验已经表明在骨损伤治疗中的具有效果，但是，治疗的效果、均一性，损伤恢复时间，PL的浓缩率不确定及释放生长因子量不稳定，间充质干细胞是否在损伤环境中，定向分化成所需填充的细胞或分泌所需要的因子。
发明内容
本发明目的是提供促进骨愈合与再生的凝胶组合物的制备方法，用于软骨损伤和骨缺损的修复。
促进骨愈合与再生的凝胶组合物的制备方法，它按以下步骤实现：
一、超活性多细胞因子制剂(sPL)的制备：
a、枸橼酸钠抗凝管无菌采集新鲜人血液10～20ml，200g/min离心15min，收集血浆于浓缩管中，200g/min离心15min，收集上清，获得浓缩的血小板血浆，于-80℃保存至少24h；
b、取上述-80℃下保存的浓缩的血小板血浆，4℃融化，涡旋混合仪震荡5s，1000r/min离心3min，收集上清后采用0.22μm滤膜进行过滤，获得sPL；
二、间充质干细胞趋向分化：
c、培养完成的间充质干细胞进行成骨细胞趋向分化：按照1×104/ml密度将培养完成的间充质干细胞接种在成骨细胞趋向分化培养基中，第二天换液，以后每三天换液一次，培养3～5天，收集有趋向分化的成骨细胞，计数，冻存，备用；
d、培养完成的间充质干细胞进行软骨细胞趋向分化：按照5×105/ml密度将培养完成的间充质干细胞接种在成软骨细胞趋向分化培养基中，第二天换液，每三天换液一次，培养3-5天，收集有趋向分化的软骨细胞，计数，冻存，备用；
三、凝胶组合物的制备：
e、采用步骤b中所得sPL重悬步骤c中所得有趋向分化的成骨细胞或步骤d中所得有趋向分化的软骨细胞，重悬液的密度为(0.5～1)×106/ml，然后加入成骨治疗趋化因子或软骨治疗趋化因子，混匀，获得含有成骨趋向分化细胞的sPL溶液或含有软骨趋向分化细胞的sPL溶液；
f、采用10％CaCl2溶液配制浓度为100～200U/ml的凝血酶溶液；
g、将步骤e中所得含有成骨趋向分化细胞的sPL溶液或含有软骨趋向分化细胞的sPL溶液与凝血酶溶液按体积比1:1混匀，50～70s后形成凝胶，即完成促进骨愈合与再生的凝胶组合物的制备。
本发明的优点：
1、本发明制备出sPL后对所含生长因子含量进行检测，再调节生长因子浓度，保证PL的均一性。进行治疗前，先对间充质干细胞进行趋向分化，使干细胞向治疗所需要的方向分化，这样可以有效缩短治疗周期，加速修复、填补损伤部位所需种类细胞。而且使用PL软凝胶包裹细胞，并且在软凝胶中加入趋向因子，可使细胞继续分化，提高治疗效果，节约治疗成本。
2、本发明中制备的促进骨愈合与再生的凝胶组合物，可用于治疗骨缺损、骨不连、骨损伤，可促进骨的愈合与再生。本发明制备的凝胶组合物对软骨损伤的修复有显著效果，软骨表面趋于平滑；对骨缺损的修复有显著效果，缺损部位骨已经全部修复。
本发明应用于软骨损伤和骨缺损的修复。
附图说明
图1为实施例中未分化间充质干细胞的显微图；
图2为实施例中间充质干细胞向成骨细胞趋向分化的显微图；
图3为实施例中间充质干细胞向成骨细胞趋向分化QPCR检测图；
图4为实施例中间充质干细胞向成软骨细胞趋向分化的显微图；
图5为实施例中间充质干细胞向成软骨细胞趋向分化QPCR检测图；
图6为实施例中模型组关节软骨病理的显微图；
图7为实施例中PRP治疗组关节软骨病理的显微图；
图8为实施例中凝胶生物制剂治疗组关节软骨病理的显微图；
图9为实施例中模型组X光桡骨图；
图10为实施例中PRP治疗组X光桡骨图；
图11为实施例中凝胶生物制剂治疗组X光桡骨图。
具体实施方式
本发明技术方案不局限于以下所列举具体实施方式，还包括各具体实施方式间的任意组合。
具体实施方式一：本实施方式促进骨愈合与再生的凝胶组合物的制备方法，它按以下步骤实现：
一、超活性多细胞因子制剂(sPL)的制备：
a、枸橼酸钠抗凝管无菌采集新鲜人血液10～20ml，200g/min离心15min，收集血浆于浓缩管中，200g/min离心15min，收集上清，获得浓缩的血小板血浆，于-80℃保存至少24h；
b、取上述-80℃下保存的浓缩的血小板血浆，4℃融化，涡旋混合仪震荡5s，1000r/min离心3min，收集上清后采用0.22μm滤膜进行过滤，获得sPL；
二、间充质干细胞趋向分化：
c、培养完成的间充质干细胞进行成骨细胞趋向分化：按照1×104/ml密度将培养完成的间充质干细胞接种在成骨细胞趋向分化培养基中，第二天换液，以后每三天换液一次，培养3～5天，收集有趋向分化的成骨细胞，计数，冻存，备用；
d、培养完成的间充质干细胞进行软骨细胞趋向分化：按照5×105/ml密度将培养完成的间充质干细胞接种在成软骨细胞趋向分化培养基中，第二天换液，每三天换液一次，培养3-5天，收集有趋向分化的软骨细胞，计数，冻存，备用；
三、凝胶组合物的制备：
e、采用步骤b中所得sPL重悬步骤c中所得有趋向分化的成骨细胞或步骤d中所得有趋向分化的软骨细胞，重悬液的密度为(0.5～1)×106/ml，然后加入成骨治疗趋化因子或软骨治疗趋化因子，混匀，获得含有成骨趋向分化细胞的sPL溶液或含有软骨趋向分化细胞的sPL溶液；
f、采用10％CaCl2溶液配制浓度为100～200U/ml的凝血酶溶液；
g、将步骤e中所得含有成骨趋向分化细胞的sPL溶液或含有软骨趋向分化细胞的sPL溶液与凝血酶溶液按体积比1:1混匀，50～70s后形成凝胶，即完成促进骨愈合与再生的凝胶组合物的制备。
具体实施方式二：本实施方式与具体实施方式一不同的是，步骤c中所述成骨细胞趋向分化培养基的成分：在含10％FBS的α-MEM中加入50μM/ml抗坏血酸、10mm/mlβ-磷酸甘油和100nm/ml地塞米松。其它步骤及参数与具体实施方式一相同。
具体实施方式三：本实施方式与具体实施方式二不同的是，步骤d中所述成软骨细胞趋向分化培养基的成分：在α-MEM中加入1mmol/L丙酮酸钠、0.1mmol/L左旋维生素C、0.1nmol/L地塞米松、10ng/mL TGF-beta 3、6.25μ/mL重组人胰岛素、6.25μg/mL转铁蛋白、6.25ng/mL亚硒酸、1.25ng/mL牛血清白蛋白、5.35μg/mL亚油酸。其它步骤及参数与具体实施方式二相同。
具体实施方式四：本实施方式与具体实施方式一至三之一不同的是，步骤c和d中所述培养完成的间充质干细胞，参照申请号为201310313363.6的专利中记载内容进行操作。其它步骤及参数与具体实施方式一至三之一相同。
具体实施方式五：本实施方式与具体实施方式一至四之一不同的是，步骤c和d中所述培养的温度均为37℃。其它步骤及参数与具体实施方式一至四之一相同。
具体实施方式六：本实施方式与具体实施方式一至五之一不同的是，步骤e中所述成骨治疗趋化因子由抗坏血酸、β-磷酸甘油和地塞米松组成。其它步骤及参数与具体实施方式一至五之一相同。
具体实施方式七：本实施方式与具体实施方式一至六之一不同的是，步骤e中所述软骨治疗趋化因子由丙酮酸钠、左旋维生素C、地塞米松、TGF-beta 3、重组人胰岛素、转铁蛋白、亚硒酸、牛血清白蛋白和亚油酸组成。其它步骤及参数与具体实施方式一至六之一相同。
具体实施方式八：本实施方式与具体实施方式一至七之一不同的是，步骤g中每1ml的促进骨愈合与再生的凝胶组合物中含有50～100μmol抗坏血酸、10～50mmolβ-磷酸甘油和100-200nmol地塞米松。其它步骤及参数与具体实施方式一至七之一相同。
具体实施方式九：本实施方式与具体实施方式一至八之一不同的是，步骤g中每1ml的促进骨愈合与再生的凝胶组合物中含有1～5μmol丙酮酸钠、0.1～0.5μmol左旋维生素C、0.1～0.5pmol地塞米松、10～50ng TGF-beta 3、5～10U重组人胰岛素、5～10μg转铁蛋白、5～10ng亚硒酸、1～5ng牛血清白蛋白和3～8μg亚油酸。其它步骤及参数与具体实施方式一至八之一相同。
通过以下实施例验证本发明的有益效果：
实施例：
促进骨愈合与再生的凝胶组合物的制备方法，它按以下步骤实现：
一、超活性多细胞因子制剂(sPL)的制备：
a、枸橼酸钠抗凝管无菌采集新鲜人血液15ml，200g/min离心15min，收集血浆于浓缩管中，200g/min离心15min，收集上清，获得浓缩的血小板血浆，于-80℃保存至少24h；
b、取上述-80℃下保存的浓缩的血小板血浆，4℃融化，涡旋混合仪震荡5s，1000r/min离心3min，收集上清后采用0.22μm滤膜进行过滤，获得sPL；
二、间充质干细胞趋向分化：
c、培养完成的间充质干细胞进行成骨细胞趋向分化：按照1×104/ml密度将培养完成的间充质干细胞接种在成骨细胞趋向分化培养基中，第二天换液，以后每三天换液一次，培养3～5天，收集有趋向分化的成骨细胞，计数，冻存，备用；
d、培养完成的间充质干细胞进行软骨细胞趋向分化：按照5×105/ml密度将培养完成的间充质干细胞接种在成软骨细胞趋向分化培养基中，第二天换液，每三天换液一次，培养3-5天，收集有趋向分化的软骨细胞，计数，冻存，备用；
三、凝胶组合物的制备：
e、采用步骤b中所得sPL重悬步骤c中所得有趋向分化的成骨细胞或步骤d中所得有趋向分化的软骨细胞，重悬液的密度为0.5×106/ml，然后加入成骨治疗趋化因子或软骨治疗趋化因子，混匀，获得含有成骨趋向分化细胞的sPL溶液或含有软骨趋向分化细胞的sPL溶液；
f、采用10％CaCl2溶液配制浓度为200U/ml的凝血酶溶液；
g、将步骤e中所得含有成骨趋向分化细胞的sPL溶液或含有软骨趋向分化细胞的sPL溶液与凝血酶溶液按体积比1:1混匀，60s后形成凝胶，即完成促进骨愈合与再生的凝胶组合物的制备。
本实施例步骤c中所述成骨细胞趋向分化培养基的成分：在含10％FBS的α-MEM中加入50μM/ml抗坏血酸、10mm/mlβ-磷酸甘油和100nm/ml地塞米松。
本实施例步骤d中所述成软骨细胞趋向分化培养基的成分：在α-MEM中加入1mmol/L丙酮酸钠、0.1mmol/L左旋维生素C、0.1nmol/L地塞米松、10ng/mL TGF-beta 3、6.25μ/mL重组人胰岛素、6.25μg/mL转铁蛋白、6.25ng/mL亚硒酸、1.25ng/mL牛血清白蛋白、5.35μg/mL亚油酸。
本实施例步骤c和d中所述培养完成的间充质干细胞，参照申请号为201310313363.6的专利中记载内容进行操作。
本实施例步骤c和d中所述培养的温度均为37℃。
本实施例步骤e中所述成骨治疗趋化因子由抗坏血酸、β-磷酸甘油和地塞米松组成。
本实施例步骤e中所述软骨治疗趋化因子由丙酮酸钠、左旋维生素C、地塞米松、TGF-beta 3、重组人胰岛素、转铁蛋白、亚硒酸、牛血清白蛋白和亚油酸组成。
本实施例步骤g中每1ml的促进骨愈合与再生的凝胶组合物中含有50～100μmol抗坏血酸、10～50mmolβ-磷酸甘油和100-200nmol地塞米松。
本实施例步骤g中每1ml的促进骨愈合与再生的凝胶组合物中含有1～5μmol丙酮酸钠、0.1～0.5μmol左旋维生素C、0.1～0.5pmol地塞米松、10～50ng TGF-beta 3、5～10U重组人胰岛素、5～10μg转铁蛋白、5～10ng亚硒酸、1～5ng牛血清白蛋白和3～8μg亚油酸。
本实施例步骤b中所得sPL进行生长因子含量检测及对比：
用Elisa方法检测PRP、PL、sPL中PDGF、TGF-β的因子含量，对比其生长因子浓度变化，如表1所示，调节sPL中生长因子，可长期保存，待使用时进行制备。
表1
PRP PL sPL
PDGF(ng/ml) 41.87±3.53 90±5.10 206.59±21.09
TGF-β(ng/ml) 95.12±10.57 180±12.68 396.43±34.23
本实施例步骤c中培养3～5天，细胞形态与未分化组对比稍有变化即可，参见附图1和2，QPCR检测趋向分化效果，参见附图3，可见细胞已经开始进入分化状态，可以补充组织所需修复细胞。
本实施例步骤d中培养3～5天，细胞形态与未分化组对比稍有变化即可，参见附图1和4，QPCR检测趋向分化效果，参见附图5，可见细胞已经开始进入分化状态，可以补充组织所需修复细胞。
本实施例中制备所得凝胶生物制剂对软骨损伤的修复作用：
用机械损伤法将大鼠左后肢膝关节软骨损伤，将模型动物分成三组，第一组为模型组，第二组为PRP治疗组，第三组为凝胶生物制剂治疗组。模型组损伤后直接缝合，不做任何治疗；治疗组损伤后分别使用制备好的PRP和凝胶生物制剂进行治疗，然后再进行缝合。每天观察动物生活情况，一周后处死动物，取膝关节，进行病理样本制备，HE染色后观察病理情况。结果参见附图6、7、8，可见采用凝胶生物制剂在治疗大鼠软骨损伤模型有显著效果，病理检测结果显示，使用凝胶生物制剂后的动物，软骨损伤部位有明显的修复，软骨表面趋于平滑。
本实施例中制备所得凝胶生物制剂对骨缺损的修复作用：
用截骨方法将大鼠右前肢桡骨截断3～4mm，将模型动物分成三组，第一组为模型组，第二组为PRP治疗组，第三组为凝胶生物制剂治疗组。模型组损伤后直接缝合，不做任何治疗；治疗组损伤后分别使用制备好的PRP和凝胶生物制剂进行治疗，然后再进行缝合。每天观察动物生活情况，八周后对动物进行X光拍照。参见说明书附图9、10、11，可见采用凝胶生物制剂在治疗大鼠骨缺损模型有显著效果，X光照射结果显示，使用凝胶生物制剂后的动物，缺损部位骨已经全部修复。

Claims (4)

1.促进骨愈合与再生的凝胶组合物的制备方法，其特征在于它按以下步骤实现：
一、超活性多细胞因子制剂的制备：
a、枸橼酸钠抗凝管无菌采集新鲜人血液10～20mL，200g/min离心15min，收集血浆于浓缩管中，200g/min离心15min，收集上清，获得浓缩的血小板血浆，于-80℃保存至少24h；
b、取上述-80℃下保存的浓缩的血小板血浆，4℃下融化，涡旋混合仪震荡5s，1000r/min离心3min，收集上清后采用0.22μm滤膜进行过滤，获得超活性多细胞因子制剂；
二、间充质干细胞趋向分化：
c、培养完成的间充质干细胞进行成骨细胞趋向分化：按照1×104/mL密度将培养完成的间充质干细胞接种在成骨细胞趋向分化培养基中，第二天换液，以后每三天换液一次，培养3～5天，收集有趋向分化的成骨细胞，计数，冻存，备用；所述成骨细胞趋向分化培养基的成分：在含10％FBS的α-MEM中加入50μM/mL抗坏血酸、10mm/mLβ-磷酸甘油和100nm/mL地塞米松；
d、培养完成的间充质干细胞进行软骨细胞趋向分化：按照5×105/mL密度将培养完成的间充质干细胞接种在成软骨细胞趋向分化培养基中，第二天换液，每三天换液一次，培养3-5天，收集有趋向分化的软骨细胞，计数，冻存，备用；所述成软骨细胞趋向分化培养基的成分：在α-MEM中加入1mmol/L丙酮酸钠、0.1mmol/L左旋维生素C、0.1nmol/L地塞米松、10ng/mL TGF-beta 3、6.25μ/mL重组人胰岛素、6.25μg/mL转铁蛋白、6.25ng/mL亚硒酸、1.25ng/mL牛血清白蛋白、5.35μg/mL亚油酸；
三、凝胶组合物的制备：
e、采用步骤b中所得超活性多细胞因子制剂重悬步骤c中所得有趋向分化的成骨细胞或步骤d中所得有趋向分化的软骨细胞，重悬液的密度为(0.5～1)×106/mL，然后加入成骨治疗趋化因子或软骨治疗趋化因子，混匀，获得含有成骨趋向分化细胞的超活性多细胞因子溶液或含有软骨趋向分化细胞的超活性多细胞因子溶液；所述成骨治疗趋化因子由抗坏血酸、β-磷酸甘油和地塞米松组成；所述软骨治疗趋化因子由丙酮酸钠、左旋维生素C、地塞米松、TGF-beta 3、重组人胰岛素、转铁蛋白、亚硒酸、牛血清白蛋白和亚油酸组成；
f、采用10％CaCl2溶液配制浓度为100～200U/mL的凝血酶溶液；
g、将步骤e中所得含有成骨趋向分化细胞的超活性多细胞因子溶液或含有软骨趋向分化细胞的超活性多细胞因子溶液与凝血酶溶液按体积比1:1混匀，50～70s后形成凝胶，即完成促进骨愈合与再生的凝胶组合物的制备。
2.根据权利要求1所述的促进骨愈合与再生的凝胶组合物的制备方法，其特征在于步骤c和d中所述培养的温度均为37℃。
3.根据权利要求1所述的促进骨愈合与再生的凝胶组合物的制备方法，其特征在于步骤g中每1mL的促进骨愈合与再生的凝胶组合物中含有50～100μmol抗坏血酸、10～50mmolβ-磷酸甘油和100～200nmol地塞米松。
4.根据权利要求1所述的促进骨愈合与再生的凝胶组合物的制备方法，其特征在于步骤g中每1mL的促进骨愈合与再生的凝胶组合物中含有1～5μmol丙酮酸钠、0.1～0.5μmol左旋维生素C、0.1～0.5pmol地塞米松、10～50ng TGF-beta 3、5～10U重组人胰岛素、5～10μg转铁蛋白、5～10ng亚硒酸、1～5ng牛血清白蛋白和3～8μg亚油酸。
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