CN114028616A - 一种用于牙槽骨缺损修复的组织工程化材料制备方法 - Google Patents
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Abstract
本发明公开了一种用于牙槽骨缺损修复的组织工程化材料制备方法,属于口腔医学和生物医疗技术领域,包括根尖牙***干细胞(SCAPs)和富血小板纤维蛋白(PRF),其操作步骤如下:1)SCAPs的分离、培养;2)SCAPs的免疫荧光法鉴定;3)PRF的制备;4)SCAPs/PRF复合体的构建及植入;本发明提供的一种用于牙槽骨缺损修复的组织工程化材料制备方法,可减少手术创伤,避免免疫排斥反应,可提高移植后患者的适应性,从而为临床上牙槽骨缺损修复提供一种新的综合治疗策略。
Description
技术领域
本发明涉及口腔医学和生物医疗技术领域,具体为一种用于牙槽骨缺损修复的组织工程化材料制备方法。
背景技术
牙槽骨缺损是临床上较为常见的疾病,常由炎症、创伤、肿瘤、先天畸形等因素所致,例如牙周炎导致的牙槽骨吸收缺损是牙周病发生、发展的病理基础,也是阻碍牙周病预后的主要瓶颈。而牙槽骨一旦发生破坏吸收、形成缺损后,很难自行修复重建,尤其是大面积的牙槽骨缺损尚缺少一种理想的修复方法。因此,牙槽骨的再生修复对牙周组织的恢复具有重要的临床意义。
目前临床上牙槽骨缺损修复的方法主要有自体骨移植、异体骨移植和人工合成材料,但是其都有一定的缺点:如①自体骨是指从患者自身获得的骨,如下颌骨外斜线、上颌结节等。但是由于取自体骨的时候会造成二次手术的创伤,手术时间长,移植后患者适应性差,受患者年龄及自身代谢的影响较大,供体相应部位存在发病率,及伦理道德问题等诸多原因,导致多数患者难以接受而放弃。②异体骨移植最常见问题是可能会有一定的免疫排斥反应,同时供体和受体移植过程中也可能存在疾病传播的问题;也因价格昂贵及异体来源问题,使部分患者仍然无法接受。③将人工种植材料移植到颌骨区,也会有受体对材料的适应性问题,可能会产生排斥和感染等问题,成骨及骨诱导性相对自体骨而言较差,且种植体修复需要大量的牙槽骨骨量。
发明内容
本发明的目的在于克服上述背景技术困难,提供了一种牙槽骨骨密度增加,骨形成区增大,破骨细胞数量减少,对牙槽骨缺损具有良好修复效果的一种用于牙槽骨缺损修复的组织工程化材料制备方法。
为达到上述目的,本发明采用的技术方案为:一种用于牙槽骨缺损修复的组织工程化材料制备方法,包括根尖牙***干细胞(SCAPs)和富血小板纤维蛋白(PRF),其操作步骤如下:
1)SCAPs的分离、培养,在无菌条件下,取出生一周内的SD乳鼠牙槽骨部分,用无菌磷酸盐缓冲液PBS洗去血污,用镊子拔出第三磨牙牙根,无菌剪刀剪取牙齿根部的根尖牙***组织;用枪头将剪下的牙***组织转移到含有Ⅰ、Ⅱ型混合胶原酶的离心管中并剪碎;置于37 ℃细胞培养箱中消化30 min;轻轻吹打消化后的组织,吸取一滴酶液在显微镜下观察,直至肉眼可见细胞团块掉落;加入DMEM/HIGH完全培养基终止消化,所述DMEM/HIGH完全培养基是往10 %的FBS种加入100 U/ml 青霉素和100 μg/ml 链霉素的双抗;然后按1200r/min的离心频率离心3 min;吸取上清后加入3 ml DMEM完全培养基重悬细胞,制成单细胞悬液,然后接种于6 cm细胞培养皿备用;
2)SCAPs的免疫荧光法鉴定,先将细胞铺板于96孔板,达80%融合后,更换为成骨诱导培养基,设立对照组和成骨诱导组,每3-4天换液1次;采用4%多聚甲醛固定孔板内各组细胞;其次先以PBS清洗3遍,每次5 min;在0.1% Triton X-100的条件下室温通透10 min;再以PBS清洗3遍,每次5 min;5 %FBS封闭,37℃,1h;加入根据抗体说明书进行稀释的一抗,在4℃条件下孵育过夜;然后以PBS清洗3遍,每次5 min;CY3标记的二抗,在37℃条件下孵育1h;最后以PBS漂洗3次,5min/次,室温避光条件下孵育Hoechst 10min,最后荧光显微镜下观察拍照,检测CD24呈阳性即为根尖牙***干细胞;
3)PRF的制备,采用动脉抽血液10ml,置于无菌采血管中;立即将试管进行离心15min,离心频率为3000r/min;静置后,管内出现3层,由底层到上层分别为红细胞层、白色凝胶状PRF层、血清层,用无菌镊子夹取中间的PRF层备用;
4)SCAPs/PRF复合体的构建及植入,无菌条件下将PRF制备成2mm*3mm大小后置于24孔板内用DMEM培养基预湿,紫外线消毒45min;调整细胞浓度至6*107/ml,每块接种20µl,每块PRF上约有1.2*106个细胞,加1ml培养液进行孵育培养,孵育培养温度为37℃、饱和湿度为50ml/LCO2条件下隔日换液,每日在显微镜下观察并培养,培养至第3天时植入骨缺损区,于3周和6周后收取牙槽骨组织样本。
进一步,所述步骤1)中DMEM/HIGH完全培养基是往10 %的FBS种加入100 U/ml 青霉素和100 μg/ml 链霉素的双抗。
进一步,所述步骤2)SCAPs的免疫荧光法鉴定是先将细胞铺板于96孔板,达80%融合后,更换为成骨诱导培养基,设立对照组和成骨诱导组,每3-4天换液1次;采用4%多聚甲醛固定孔板内各组细胞;其次先以PBS清洗3遍,每次5 min;在0.1% Triton X-100的条件下室温通透10 min;再以PBS清洗3遍,每次5 min;5 %FBS封闭,37℃,1h;加入一抗,在4℃条件下孵育过夜;然后以PBS清洗3遍,每次5 min;CY3标记的二抗,在37℃条件下孵育1h;最后以PBS漂洗3次,5min/次,室温避光条件下孵育Hoechst 10min。
进一步,所述一抗根据抗体说明书进行稀释。
进一步,所述步骤3)中试管离心15min,离心频率为3000r/min。
进一步,所述步骤4)孵育培养温度为37℃、饱和湿度为50ml/LCO2条件下隔日换液,每日在显微镜下观察并培养。
本发明提供的一种用于牙槽骨缺损修复的组织工程化材料制备方法,具备以下有益效果:
本发明通过研究发现SCAPs在体外具有良好的成骨分化能力,经成骨诱导后其成骨相关基因ALP和OPN的表达显著增加;SCAPs/PRF复合体植入大鼠牙槽骨缺损部位后,能够提高牙槽骨新骨的形成,减少牙槽骨的吸收,促进伤口愈合,并表现出良好的生物相容性,使牙槽骨骨密度增加,骨形成区增大,破骨细胞数量减少,对牙槽骨缺损具有良好的修复效果,本发明提供的一种用于牙槽骨缺损修复的组织工程化材料制备方法,可减少手术创伤,避免免疫排斥反应,可提高移植后患者的适应性,从而为临床上牙槽骨缺损修复提供一种新的综合治疗策略。
附图说明
图1为SCAPs的间充质干细胞干性鉴定示意图。
图2为茜素红染色法鉴定SCAPs的体外成骨分化能力的示意图。
图3为实时荧光定量PCR法(q-RT-PCR)检测SCAPs中成骨相关基因ALP和OPN的表达的示意图。
图4为牙槽骨组织体式的示意图。
图5为牙槽骨组织X线片的示意图。
图6为牙槽骨组织HE染色的示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,其示例表示在附图中。下面的描述涉及附图时,除非另有表示,不同附图中的相同数字表示相同或同种要素。
显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
如图1,一种用于牙槽骨缺损修复的组织工程化材料制备方法,包括根尖牙***干细胞(SCAPs)和富血小板纤维蛋白(PRF),其操作步骤如下:
1)SCAPs的分离、培养,在无菌条件下,取出生一周内的SD乳鼠牙槽骨部分,用无菌磷酸盐缓冲液PBS洗去血污,用镊子拔出第三磨牙牙根,无菌剪刀剪取牙齿根部的根尖牙***组织;用枪头将剪下的牙***组织转移到含有Ⅰ、Ⅱ型混合胶原酶的离心管中并剪碎;置于37 ℃细胞培养箱中消化30 min;轻轻吹打消化后的组织,吸取一滴酶液在显微镜下观察,直至肉眼可见细胞团块掉落;加入DMEM/HIGH完全培养基终止消化,然后按1200 r/min的离心频率离心3 min;吸取上清后加入3 ml DMEM完全培养基重悬细胞,制成单细胞悬液,然后接种于6 cm细胞培养皿备用;
2)SCAPs的免疫荧光法鉴定,荧光显微镜下观察拍照,检测CD24呈阳性即为根尖牙***干细胞;
3)PRF的制备,采用动脉抽血液10ml,置于无菌采血管中;立即将试管进行离心;静置后,管内出现3层,由底层到上层分别为红细胞层、白色凝胶状PRF层、血清层,用无菌镊子夹取中间的PRF层备用;
4)SCAPs/PRF复合体的构建及植入,无菌条件下将PRF制备成2mm*3mm大小后置于24孔板内用DMEM培养基预湿,紫外线消毒45min;调整细胞浓度至6*107/ml,每块接种20µl,每块PRF上约有1.2*106个细胞,加1ml培养液进行孵育培养,培养至第3天时植入骨缺损区,于3周和6周后收取牙槽骨组织样本。
本发明制备过程中主要是通过以下试剂及仪器进行以上操作,胎牛血清(浙江天杭生物),PCR引物(上海生工),M-MLV逆转录酶和RNase抑制剂(Promega),dNTP Mix(上海生工),RNAiso TM Plus(TaKaRa),q-RT-PCR试剂盒(Bio-Rad),FBS(Lonsera),DMEM培养基(Hyclone),青链双抗(上海生工),成骨诱导培养基(无锡菩禾),SD乳鼠(常州卡文斯),茜素红染色试剂盒(索莱宝)。
生物洁净工作台(安泰空气技术公司),二氧化碳培养箱(Gold-Sim西盟国际公司),倒置荧光显微镜(IX73,Olympus),PCR仪(Bio-Rad),低温台式离心机(德国Eppendorf),细胞培养箱(Thermo),光学显微镜(XDS-1A)。
本发明提供的一种用于牙槽骨缺损修复的组织工程化材料制备方法,鉴定上述步骤结果如下所述:
(1)SCAPs的分离、培养和鉴定
鉴定结果如图1所示,本发明的SCAPs体外分离培养后,经免疫荧光法鉴定结果显示,间充质干细胞特异性基因CD24的表达呈阳性,表明分离的SCAPs细胞具有间充质干细胞的干性。
(2)茜素红染色法鉴定SCAPs的体外成骨分化能力
成骨诱导培养液体外诱导SCAPs后,采用茜素红染色鉴定SCAPs的成骨分化能力,显微镜下观察显示成骨诱导后呈红褐色,SCAPs细胞排列紧密,细胞周围有大量红色矿化结节形成,鉴定结果如图2所示,图2左边为SCAPs非成骨诱导组鉴定,图2右边为SCAPs成骨诱导组鉴定;由图2鉴定结果对比可得SCAPs在体外具有较好的成骨分化能力。
(3)实时荧光定量PCR法(q-RT-PCR)检测SCAPs中成骨相关基因ALP和OPN的表达
成骨诱导培养液体外诱导SCAPs后,采用实时荧光定量PCR法检测SCAPs中成骨相关基因ALP和OPN的表达情况,结果显示,成骨诱导后SCAPs细胞中ALP、OPN的表达水平均显著升高,见图3所示(注:**表示与非诱导组相比,P<0.01),进一步证实了SCAPs在体外具有较好的成骨分化能力。
(4)SCAPs/PRF复合体植入后牙槽骨缺损部位的大体观察
构建大鼠牙槽骨缺损动物模型后,将SCAPs/PRF复合体植入牙槽骨缺损部位,于3周(3W)或6周(6W)后,用体式显微镜观察牙槽骨缺损部位大体修复情况,结果显示参照图4,与对照组相比,SCAPs组和PRF组牙槽骨缺损修复均有所增加,而SCAPs/PRF复合体组牙槽骨缺损修复增加更为显著。
(5)SCAPs/PRF复合体植入后牙槽骨骨密度的观察
取牙槽骨标本拍X线片以观察骨密度情况,结果显示参照图5,与对照组相比,SCAPs组和PRF组牙槽骨的骨密度均有所增加,而SCAPs/PRF复合体组牙槽骨的骨密度增加更为显著。
(6)SCAPs/PRF复合体植入后牙槽骨HE染色观察
苏木精-伊红(HE)染色结果显示参照图6,与对照组相比,SCAPs组、PRF组和SCAPs/PRF组牙槽骨骨壁更平整,骨形成区进一步增大,破骨细胞显著减少,其中SCAPs/PRF组牙槽骨恢复效果最为显著。
综上所述,本发明提供的一种用于牙槽骨缺损修复的组织工程化材料制备方法,包括根尖牙***干细胞(SCAPs)和富血小板纤维蛋白(PRF);本发明通过研究发现SCAPs还可以促进CD4+CD25效应T细胞向CD4+CD25+Foxp3+调节性T细胞的转化,进一步揭示了SCAPs具有较强的免疫调节能力,SCAPs在体外具有良好的成骨分化能力、成骨诱导后其成骨相关基因ALP和OPN的表达显著增加;其中PRF具有立体网状结构,能够提供适宜的微环境和支撑支架,以供种子细胞附着和生长,同时进行气体交换和废料的排出,有利于伤口的愈合,PRF能够促进间充质干细胞的迁移和增殖,利用骨髓间充质干细胞(BMSCs)作为种子细胞,形成的BMSCs/PRF复合体能够提高牙槽骨新骨的形成、减少牙槽骨的吸收、促进伤口愈合,并表现出良好的生物相容性;SCAPs/PRF复合体植入大鼠牙槽骨缺损部位后,牙槽骨骨密度增加,骨形成区增大,破骨细胞数量减少,对牙槽骨缺损具有良好的修复效果;本发明提供的一种用于牙槽骨缺损修复的组织工程化材料制备方法,可减少手术创伤,避免免疫排斥反应,可提高移植后患者的适应性,从而为临床上牙槽骨缺损修复提供一种新的综合治疗策略。
本发明的保护范围不仅限于上述公开的具体技术方案,以上所述仅为本实发明的较佳实施方式,并不能以此来限制本发明,凡是依据本发明的技术实质对以上具体技术方案所作的任何细微修改、等同替换和改进,均应包含在本发明技术方案的保护范围之内。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (6)
1.一种用于牙槽骨缺损修复的组织工程化材料制备方法,其特征在于,包括根尖牙***干细胞(SCAPs)和富血小板纤维蛋白(PRF),其操作步骤如下:
1)SCAPs的分离、培养,在无菌条件下,取出生一周内的SD乳鼠牙槽骨部分,用无菌磷酸盐缓冲液PBS洗去血污,用镊子拔出第三磨牙牙根,无菌剪刀剪取牙齿根部的根尖牙***组织;用枪头将剪下的牙***组织转移到含有Ⅰ、Ⅱ型混合胶原酶的离心管中并剪碎;置于37℃细胞培养箱中消化30 min;轻轻吹打消化后的组织,吸取一滴酶液在显微镜下观察,直至肉眼可见细胞团块掉落;加入DMEM/HIGH完全培养基终止消化,然后按1200 r/min的离心频率离心3 min;吸取上清后加入3 ml DMEM完全培养基重悬细胞,制成单细胞悬液,然后接种于6 cm细胞培养皿备用;
2)SCAPs的免疫荧光法鉴定,荧光显微镜下观察拍照,检测CD24呈阳性即为根尖牙***干细胞;
3)PRF的制备,采用动脉抽血液10ml,置于无菌采血管中;立即将试管进行离心;静置后,管内出现3层,由底层到上层分别为红细胞层、白色凝胶状PRF层、血清层,用无菌镊子夹取中间的PRF层备用;
4)SCAPs/PRF复合体的构建及植入,无菌条件下将PRF制备成2mm*3mm大小后置于24孔板内用DMEM培养基预湿,紫外线消毒45min;调整细胞浓度至6*107/ml,每块接种20µl,每块PRF上约有1.2*106个细胞,加1ml培养液进行孵育培养,培养至第3天时植入骨缺损区,于3周和6周后收取牙槽骨组织样本。
2.根据权利要求1所述的用于牙槽骨缺损修复的组织工程化材料制备方法,其特征是:所述步骤1)中DMEM/HIGH完全培养基是往10 %的FBS种加入100 U/ml 青霉素和100 μg/ml链霉素的双抗。
3.根据权利要求1所述的用于牙槽骨缺损修复的组织工程化材料制备方法,其特征是:所述步骤2)SCAPs的免疫荧光法鉴定是先将细胞铺板于96孔板,达80%融合后,更换为成骨诱导培养基,设立对照组和成骨诱导组,每3-4天换液1次;采用4%多聚甲醛固定孔板内各组细胞;其次先以PBS清洗3遍,每次5 min;在0.1% Triton X-100的条件下室温通透10 min;再以PBS清洗3遍,每次5 min;5 %FBS封闭,37℃,1h;加入一抗,在4℃条件下孵育过夜;然后以PBS清洗3遍,每次5 min;CY3标记的二抗,在37℃条件下孵育1h;最后以PBS漂洗3次,5min/次,室温避光条件下孵育Hoechst 10min。
4.根据权利要求3所述的用于牙槽骨缺损修复的组织工程化材料制备方法,其特征是:所述一抗根据抗体说明书进行稀释。
5.根据权利要求1所述的用于牙槽骨缺损修复的组织工程化材料制备方法,其特征是:所述步骤3)中试管离心15min,离心频率为3000r/min。
6.根据权利要求1所述的用于牙槽骨缺损修复的组织工程化材料制备方法,其特征是:所述步骤4)孵育培养温度为37℃、饱和湿度为50ml/LCO2条件下隔日换液,每日在显微镜下观察并培养。
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