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通讯作者:

章非敏,E-mail:fmzhang@njmu.edu.cn

中图分类号:R336

文献标识码:A

文章编号:1007-4368(2023)03-326-08

DOI:10.7655/NYDXBNS20230305

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目录contents

    摘要

    目的:研究负载γ-Fe2O3的壳聚糖多孔海绵对大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cell,rBMSC)增殖和成骨分化的影响。方法:使用冻干-交联法制备负载γ-Fe2O3浓度分别为1%、5%、10%和20%的壳聚糖海绵,并制备空白对照组。将rBMSC培养于海绵上,通过扫描电镜和共聚焦显微镜观察细胞黏附及增殖情况,通过CCK-8法检测细胞第1、3、5、7天的增殖情况,通过碱性磷酸酶(alkaline phosphatases,ALP)染色及活性检测和荧光定量PCR检测第7、14天ALP活性和成骨指标 ALP、骨形态发生蛋白(bone morphogenetic protein 2,Bmp2)、胶原蛋白(collagen Ⅰ,Col1)和Runt相关转录因子2(core binding factor alphal 1,Runx2)的表达;使用茜素红定量法评估第21、28天细胞外基质矿化情况。结果:CCK-8结果显示rBMSC均能在材料上持续增殖,添加γ-Fe2O3对rBMSC增殖有促进作用;ALP染色及活性检测结果和PCR结果显示添加γ-Fe2O3能提高ALP活性并促进成骨指标表达;茜素红定量结果显示添加浓度为5%和10%时,矿化物形成量高于对照组(P < 0.05)。结论:负载γ- Fe2O3的壳聚糖海绵能够促进 rBMSC 的增殖和早期成骨分化,浓度为 5%和 10%时对 rBMSC 成骨分化晚期矿化物形成有促进作用。

    Abstract

    Objective:This study aims to observe the effects of γ-Fe2O3-loaded chitosan porous sponge on the proliferation and early osteogenic differentiation of rat bone marrow mesenchymal stem cells(rBMSC). Methods:Chitosan sponges loaded with γ- Fe2O3 at concentrations of 1%,5%,10% and 20% were prepared by freeze -drying and cross -linking,and blank control group was prepared. Scanning electron microscopy and confocal microscopy were used to evaluate the adhesion and proliferation of rBMSC on the sponges. Cell proliferation at 1,3,5 and 7 days was detected by CCK -8. The early osteogenic differentiation of rBMSC at 7 and 14 days was evaluated by alkaline phosphatases(ALP)staining and activity detection. Real - time fluorescence quantitative reverse transcription PCR was used to detect the expression of ALP,bone morphogenetic protein 2(Bmp2),collagen I(Col1)and Runt-related transcription factor 2(Runx2)after 7 and 14 days of osteogenic induction. The mineralization of extracellular matrix at 21 and 28 days was assessed by alizarin red staining quantitative method. Results:CCK-8 results showed each group added with γ-Fe2O3 promoted the proliferation of rBMSC. ALP staining and activity detection results showed the addition of γ-Fe2O3 can improve the activity of ALP. The results of real - time fluorescence quantitative reverse transcription PCR showed that the addition of γ - Fe2O3 can promote the expression of osteogenic indexes(ALP,Bmp2,Col1 and Runx2). The quantitative detection results of alizarin red staining showed that the amount of mineralization in the groups with γ-Fe2O3 added concentration of 5% and 10% was higher than that in the control group(P < 0.05).Conclusion:The chitosan porous sponge loaded with γ - Fe2O3 promoted the proliferation and the early indicators of osteogenic differentiation of rBMSC,and loading γ-Fe2O3 at concentration of 5% and 10% can promote the formation of mineralization in the late stage of osteogenic differentiation of rBMSC.

  • 感染、创伤、肿瘤或先天性疾病等原因导致的口腔牙槽骨缺损的修复方法一直是医学难题[1-2]。随着人们对口腔问题的重视,口腔颌面部的骨修复需求正在迅速增长。目前,自体骨移植仍然是修复骨缺损最主要的方法[3-4],然而从自体获得的骨量有限,取骨后的局部血肿、新骨植入时的塑形等问题都值得关注[5-6]。近年来,骨组织工程的发展为解决这一难题开辟了新途径。

  • 骨组织工程包含三要素:干细胞或前体细胞、生物支架和生长因子。天然骨组织具有很强的再生能力,因为其内部细胞具有干细胞特性。骨髓间充质干细胞来源于中胚层,增殖能力强,具有自我更新及多向分化潜能,是骨组织工程理想的种子细胞[7]

  • 过去 30 年,人们发现水凝胶材料具有极为亲水的三维网络结构,可塑性强,是良好的多功能载体[8]。壳聚糖是一种天然多糖甲壳素脱除部分乙酰基的产物,具有与糖胺聚糖相同的结构,常被用于制作水凝胶[9]。壳聚糖溶解酶可以把壳聚糖降解成无毒可吸收的小分子氨基酸和多糖[10]。壳聚糖分子结构中的氨基基团反应活性强,使得该多糖具有优异的生物学功能并能进行化学修饰反应,壳聚糖的修饰(比如共价交联和硫化)可以改变其降解性质[11]。由于良好的生物相容性、生物降解性和抗菌作用,壳聚糖基支架已被广泛用于生物医学[12]。基于壳聚糖的纳米纤维支架可以模拟细胞外基质,有促进干细胞体外黏附、增殖和成骨分化的能力[13]

  • 近来人们对铁基纳米材料的研究发现,γ⁃Fe2O3 纳米粒子由于良好的生物相容性和特殊的磁学性能对骨修复有良好效果[14-17]。体外研究表明,磁场诱导下的γ⁃Fe2O3能够促进原代骨髓细胞向成骨细胞分化,加快细胞的成骨作用以促进骨修复[218]。也有研究考察了γ⁃Fe2O3对细胞成骨的直接生物学作用,在不施加磁场时,γ⁃Fe2O3能够通过激活丝裂原活化蛋白激酶(mitogen ⁃activated protein kinase,MAPK) 通路,促进间充质干细胞的成骨分化[19]

  • 因此,本研究通过制备负载γ⁃Fe2O3的壳聚糖多孔海绵支架,探究其在不同负载浓度下对大鼠骨髓间质干细胞(rat bone marrow mesenchymal stem cell,rBMSC)增殖及成骨分化的影响。

  • 1 材料和方法

  • 1.1 材料

  • 壳聚糖(脱乙酰度≥95%,上海 Macklin 公司),冰乙酸(纯度>99%,北京PSAITONG公司),聚乙二醇二缩水甘油醚(poly ethylene glycol diglycidyl ether,PEGDGE)(北京 Solarbio 公司);rBMSC(P2)、 rBMSC完全培养基及成骨诱导培养基(广州Cyagen 公司),0.25%胰酶⁃EDTA(Gibco公司,美国),磷酸盐缓冲液(phosphate buffered solution,PBS)(Hyclone 公司,美国);CCK⁃8试剂盒(合肥Biosharp公司),活死细胞染液(Invitrogen公司,美国),BCIP/NBT碱性磷酸酶(alkaline phosphatases,ALP)显色试剂盒(上海碧云天生物技术有限公司),ALP活性检测试剂盒 (南京建成生物工程研究所),BCA蛋白定量试剂盒 (北京雷根生物技术有限公司);茜素红染色液(广州Cyagen公司),氯化十六烷基吡啶(cetylpyridinium chloride,CPC)(合肥博美生物科技有限责任公司); RNA 提取试剂盒、RT ⁃PCR 逆转录试剂盒、SYBR Green Premix Ex Taq Ⅱ(TaKaRa公司,日本);5500R 万能试验机(Instron公司,美国),Spectra Max 190酶标仪(Molecular Device 公司,美国),MAIA3 场发射扫描电镜(scanning electron microscope,SEM)(TES⁃ CAN公司,捷克),LSM 710激光共聚焦显微镜(con⁃ focal laser scanning microscope,CLSM)(Carl Zeiss 公司,德国),QuantStudio7荧光定量PCR仪(ABI公司,美国)。γ⁃Fe2O3(纳米材料)由东南大学江苏省生物材料与设备重点实验室提供。

  • 1.2 方法

  • 1.2.1 材料制备

  • 将 0.1 g 壳聚糖溶于 8 mL 0.5%乙酸,磁力搅拌机混匀,分别加入 0.1、0.5、1.0、2.0 mL 浓度为 3 mg/mL的γ⁃Fe2O3溶液,并设空白对照组;加入壳聚糖实际重量10%的交联剂PEGDGE,并用去离子水补齐 10 mL,制成 5 组体积浓度分别为 0(空白对照)、1%、5%、10%、20%的溶液。搅拌均匀后按每孔 100 μL加入96孔板。置于-40℃冰箱冷冻8 h,冷冻干燥机冻干 24 h,放入 37℃烘箱交联 48 h,得到直径 5.5 mm、高度 6.0 mm 的圆柱体海绵样品:CH0、 CH1、CH5、CH10和CH20。环氧消毒后密封保存。

  • 1.2.2 材料性质鉴定

  • 使用冷冻干燥机将海绵冻干 2 h,喷金 2 min, SEM 下观察海绵结构,使用 X 射线能谱仪(energy dispersive spectrometer,EDS)分析元素分布和占比情况。

  • 每组取 20 块海绵冷冻干燥机冻干 8 h,测量海绵原质量;每组取160块海绵置于培养基中,37℃培养,每周每组各取 20 块海绵用 PBS、去离子水和无水乙醇分别清洗3遍,置于-40℃冰箱冷冻2 h,冷冻干燥机冻干 8 h,测量海绵总质量。降解率=(1-冻干后质量/原质量)×100%。

  • 使用万能试验机以0.1 mm/s的压缩速度测量压缩距离为5 mm时海绵的力学性能。

  • 1.2.3 细胞培养

  • 将 rBMSC 培养在 37℃、5% CO2 的恒温培养箱中,隔天换液,待其融合至约80%传代。第3~5代细胞用于后续细胞实验。

  • 1.2.4 细胞黏附检测

  • 将海绵置于48孔板中,每块海绵中接种3×103 个细胞,培养 0.5 h,镜下观察细胞基本贴壁,每孔加 500 μL培养基。培养24 h后,弃原培养液,PBS清洗 1遍,每孔加入 0.5 mL 4%多聚甲醛固定 30 min 后, PBS 清洗 1 遍。分别用浓度梯度 30%、50%、70%、 90%和 100%乙醇脱水 10 min,冻干 2 h,SEM 下观察。

  • 培养细胞至第5天,将海绵浸入活死细胞染液 15 min,PBS清洗3遍,CLSM下观察。

  • 1.2.5 细胞增殖检测

  • 每块海绵中接种 2×104 个细胞,同样方法分别培养至 1、3、5、7 天,弃原培养液,每孔加入 500 μL CCK⁃8工作液(CCK⁃8与完全培养基按1∶10混合), 37℃、5% CO2 恒温培养箱中孵育 2 h,每孔吸出 100 μL 液体至 96 孔板中,酶标仪 450 nm 波长下检测吸光度值。

  • 1.2.6 细胞成骨诱导及ALP检测

  • 每块海绵中接种3×104 个细胞,培养1 d后加入成骨诱导培养基,隔天换液。在成骨诱导7 d后,弃原培养基,PBS 清洗 1 次,每孔加入 0.5 mL 4%多聚甲醛固定30 min,配制BCIP/NBT ALP 显色工作液,每孔加入0.5 mL,室温避光染色30 min,PBS清洗1次,倒置显微镜下观察。

  • 成骨诱导至第 7、14 天,弃原培养基,PBS 清洗 1 次,每孔加入 200 μL 的 0.5% Triton X⁃100 溶液裂解20 min,超声,离心,收集上清液,ALP活性和总蛋白浓度分别按照 ALP 活性检测试剂盒和 BCA 蛋白检测说明书进行检测。

  • 1.2.7 茜素红染色

  • 成骨诱导至第21、28天,加入4%多聚甲醛固定 30 min,PBS清洗1次。将材料浸入茜素红染液,室温避光染色 5~10 min,每孔加入 500 μL CPC 液 (10% wt)溶解矿化物,酶标仪550 nm波长下检测吸光度值。

  • 1.2.8 实时荧光定量PCR

  • 成骨诱导第7、14天,弃原培养基,胰酶消化后离心,使用 RNA 提取试剂盒收集细胞并分离总 RNA,逆转录为 cDNA。采用 SYBR Premix Ex Taq Ⅱ试剂盒配成10 μL反应体系并进行实时荧光定量 PCR,检测成骨指标 ALP、骨形态发生蛋白(bone morphogenetic protein 2,Bmp2)、胶原蛋白(collagen I,Col1)和 Runt 相关转录因子 2(core binding factor alphal1,Runx2)基因的表达。引物由上海生工生物工程有限公司合成,管家基因GAPDH设为内参。

  • 1.3 统计学方法

  • 采用 SPSS 21.0 统计分析软件,实验数据以均数±标准差(x-±s)表示,组间比较采用单因素方差分析,两两比较采用LSD⁃t检验,P <0.05为差异有统计学意义。

  • 2 结果

  • 2.1 材料表征

  • 扫描电镜下,壳聚糖海绵表面疏松多孔(图1A)。海绵孔隙率为(91.61± 0.70)%,各组差异无统计学意义(P >0.05)。

  • EDS元素分析示Fe元素均匀弥散分布(图1B、 C)。CH0、CH1、CH5、CH10 和 CH20 组 Fe 元素占比分别约为0、0.1%、0.5%、0.9%和1.9%。

  • 第8周时海绵降解率约为49.14%(图1D),各组间差异无统计学意义(P >0.05)。

  • 使用万能力学系统测量海绵力学性能,压缩距离为 5 mm 时,海绵压缩弹性模量(自动杨氏)为 (125.53 ± 0.52)MPa(图1E),各组间差异无统计学意义(P >0.05)。

  • 2.2 细胞黏附

  • rBMSC 接种第 1 天,扫描电镜下见细胞紧密黏附于海绵表面(图2)。rBMSC接种第5天,活死细胞染色后,活细胞散在分布在海绵表面,未见死亡细胞(图3A)。CLSM示各组细胞均能很好地生长于海绵上,生长形态未见明显差异(图3B)。

  • 2.3 细胞增殖

  • CCK⁃8 结果显示,细胞数量在 7 d 内均逐渐增加,呈稳定增殖状态(图4)。第1天,各实验组的吸光度值均高于 CH0 组,CH5、CH10 和 CH20 组的吸光度值高于 CH1 组;第 3、5、7 天,CH5、CH10 和 CH20 组的吸光度值均高于 CH0 和 CH1 组(P <0.05)。这表明负载γ⁃Fe2O3浓度为 5%、10%和 20% 的壳聚糖海绵对rBMSC的增殖有促进作用。

  • 2.4 ALP活性

  • ALP活性检测结果显示,各组细胞ALP活性在第 7 和 14 天均有增加。第 7 天,CH1、CH5、CH10、 CH20组ALP活性均高于CH0组,CH10组高于CH1 组;第14天,CH5、CH10和CH20组ALP活性均高于 CH0 组和 CH1 组(P <0.05)。第 7 天 ALP 染色结果与ALP活性检测结果一致(图5)。镜下染色结果示 CH0组与CH1组蓝紫色反应物散在分布,CH5组蓝紫色反应物出现聚集,CH10与CH20组蓝紫色反应物连接成片状。

  • 2.5 成骨相关基因的表达

  • PCR结果显示,第7天,CH10和CH20组ALP表达高于CH0组;第14天,CH5、CH10和CH20组ALP 表达高于CH0组,CH10和CH20组ALP表达高于其他实验组。第 7 天,CH5、CH10 和 CH20 组 Bmp2 表达高于其他实验组;第14天,CH10组Bmp2表达高于其他实验组。第7和14天,CH5、CH10和CH20组 Col1表达均高于其他实验组,第14天CH5和CH10 组Col1表达高于CH20组。第7天,CH5和CH10组 Runx2表达均高于 CH0 和 CH1 组,第 14 天 CH10 组 Runx2 表达高于其他实验组。差异均有统计学意义(P <0.05,图6)。

  • 图1 材料表征及力学性能

  • Figure1 Material characterization and mechanical properties

  • 图2 扫描电镜下rBMSC第1天在各组海绵上的黏附情况(×5 000)

  • Figure2 Adhesion of rBMSC on the sponges of each group on the 1st day under scanning electron microscope(×5 000)

  • 图3 激光共聚焦显微镜下rBMSC第5天的生长情况

  • Figure3 Growth of rBMSC on the 5th day under laser confocal microscope

  • 图4 CCK8检测rBMSC增殖结果

  • Figure4 CCK8 detection results of rBMSC proliferation

  • 2.6 细胞外基质矿化物形成量

  • 茜素红可检测rBMSC成骨晚期细胞外基质的钙结节情况。成骨诱导第 21 和 28 天,定量检测结果显示:第 21 天,CH1、CH5 和 CH10 组矿化物形成量高于CH0组;第28天,CH5和CH10组矿化物形成量高于 CH0 组与 CH1 组,差异有统计学意义(P <0.05,图7)。

  • 3 讨论

  • 骨组织工程使用支架、生长因子和干细胞进行骨再生[20]。支架为细胞附着、增殖和分化提供位点。目前,骨组织工程中用作支架的生物材料可分为以下几类:陶瓷、天然或合成聚合物和去细胞外基质。生物活性陶瓷的不可降解性和较差的机械性能限制了其临床应用[21];高分子聚合物材料便于塑形,然而其降解产物在临床上会引起强烈的炎症反应,如聚乙醇酸 (polyglycolic acid,PGA)降解后产生的乙醇酸和其他酸性产物[22];在去细胞化细胞外基质(decellularized extracellular matrix,dECM)中接种成骨细胞的细胞外基质构建体虽然表现出了较强的骨再生能力[23-24],然而去细胞化技术、组织处理方法、组织来源和储存条件等方面需要更多的研究改进[25]

  • 本研究选择天然聚合物壳聚糖制备多孔海绵支架,与上述材料相比,天然聚合物通常具有良好的生物相容性和降解性,而且取材方便,来源广泛。与其他天然聚合物相比,壳聚糖本身便具有良好的骨诱导性。壳聚糖无论游离还是合成支架,都可以影响成骨相关信号通路的激活,通过上调 Runx2、ALP 和成骨细胞特异性转录因子表达来刺激小梁骨的产生,增加骨桥蛋白(osteopontin,Opn) 和 Col1 的表达,并减少破骨细胞生成,促进间充质干细胞中的成骨细胞增殖和成骨[26-28]

  • 本研究添加γ⁃Fe2O3纳米粒子改进用于骨组织工程的壳聚糖水凝胶支架,增强其促成骨能力,使用冻干法将其制备成多孔海绵,扫描电镜及EDS元素分析检测海绵表面疏松多孔,Fe元素均匀弥散分布,材料孔隙率较高,具有充足的用于新生血管及成骨的空间,培养于其中的rBMSC能够紧密黏附在海绵表面。CCK⁃8结果显示,细胞数量在7 d内均逐渐增加,γ⁃Fe2O3负载浓度为5%、10%和20% 的壳聚糖海绵对 rBMSC 增殖有促进作用。ALP 是成骨细胞功能性酶,一种由成骨细胞分泌的非胶原蛋白,是干细胞成骨分化的早期标志之一[29],对骨矿化至关重要。Bmp2 是新骨形成的关键因子,是调节典型成骨细胞分化和骨形成的转录因子[30-31]。一旦被激活,它就会易位到细胞核中诱导骨基质蛋白(如 Runx2、ALP和Col1)的表达[32]。Col1通常在成骨细胞分化和矿化阶段表达增加,Runx2是成骨细胞分化的关键转录因子,是骨形成或再生的最早和最关键的生物标志物,其表达随着细胞发育成未成熟的成骨细胞而增加[33-34]。本研究分别在成骨培养第7、 14 天检测 ALP 活性及这些关键的成骨调节基因的表达水平,CH5、CH10和CH20组ALP活性及成骨调节基因的表达高于其他实验组,这提示γ⁃Fe2O3负载浓度为5%、10%和20%的壳聚糖海绵对rBMSC早期成骨分化有促进作用。第21、28天茜素红染色定量检测结果显示:CH5 和 CH10 组矿化物形成量高于空白对照组,表明γ⁃Fe2O3负载浓度为 5%和 10%的壳聚糖海绵能够促进rBMSC成骨晚期矿化物形成。

  • 图5 第7天ALP染色及第7、14天ALP活性定量检测结果(×100)

  • Figure5 ALP staining results on the 7th day and ALP activity quantitative detection results on the 7th and 14th days(×100)

  • 图6 第7和14天成骨相关基因ALP(A)、Bmp2(B)、Col1(C)和Runx2(D)表达结果

  • Figure6 Results of osteogenesis related gene expression of ALP(A),Bmp2(B),Col1(C)and Runx2(D)on the 7th and 14th days

  • 图7 第21和28天茜素红染色定量检测结果

  • Figure7 Quantitative detection results of Alizarin red staining on the 21st and 28th days

  • 研究表明添加γ⁃Fe2O3能使支架材料的微观结构发生变化,增加表面积,从而为蛋白质分子提供更多的黏附空间[35]。本研究中,壳聚糖海绵表面均匀分布的γ⁃Fe2O3可能直接影响细胞表面结合的蛋白质层,增加细胞识别和黏附位点。因此,壳聚糖海绵的纳米形貌可能是细胞黏附的影响因素,添加 γ⁃Fe2O3改善了壳聚糖海绵的性质,促进更多的蛋白质结合以及细胞黏附和扩散。

  • 本研究未对负载γ⁃Fe2O3的壳聚糖海绵施加磁场,也检测到其促进间充质干细胞成骨分化的能力。考虑到纳米材料对细胞的生物效应应当是系统化和多因素的,除了磁场刺激,其他物理和化学等因素的刺激也可能存在。在目前已有的研究中,机械刺激可以通过细胞内膜系统或细胞骨架传递到核区并导致核拉伸,从而立即引发染色体反应和随后的下游信号表达[36]。较高浓度的γ⁃Fe2O3在细胞早期增殖及成骨分化方面的促进作用较显著,因为促进成骨分化的机械应激信号可能在细胞膜和 γ⁃Fe2O3相互作用时便产生。研究表明,γ⁃Fe2O3对地塞米松诱导的成骨分化表现出抑制作用,并且归因于从γ⁃Fe2O3中进入细胞内的游离铁。研究表明, γ⁃Fe2O3对rBMSC的毒性作用与时间和剂量有关[19]。考虑这些结果,较高浓度的γ⁃Fe2O3可能在细胞成骨分化晚期表现出抑制作用。本研究发现γ⁃Fe2O3负载浓度为5%、10%和20%的壳聚糖海绵对rBMSC增殖和成骨分化早期指标均有促进作用,但浓度为 10%时与其余组差异显著,且能够促进rBMSC成骨晚期矿化物的形成,可能是促进成骨分化的合适浓度,可以作为未来研究γ⁃Fe2O3添加浓度的参考。

  • 综上,本研究通过交联γ⁃Fe2O3改进了用于骨组织工程的壳聚糖水凝胶支架,证明了负载γ⁃Fe2O3的壳聚糖多孔海绵对 rBMSC 的体外增殖及成骨分化具有一定促进作用,是用于骨组织工程治疗骨缺损很有前景的一项候选方案。但添加γ⁃Fe2O3并未明显改善壳聚糖多孔海绵的机械性能,未来仍需将其与生物材料复合以增强其机械性能。此外,rBMSC 在体内的分化与生长过程以及促进 rBMSC 体外增殖及成骨分化的机制还有待进一步研究。未来将通过动物实验来研究负载γ⁃Fe2O3的壳聚糖多孔海绵支架对体内骨再生的影响,研究在体内多因素影响的复杂环境中,新型负载γ⁃Fe2O3的壳聚糖多孔海绵刺激骨再生的功效。

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