明胶/磷酸三钙复合纳米纤维膜的成骨诱导机制及骨修复应用研究
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摘要
骨缺损是临床实践中的常见病例。小范围的骨缺损可以通过机体自身的修复机制逐渐愈合完好并恢复其功能，而当骨缺损范围较大即达到“临界尺寸”时，则需要借助人工植入材料进行骨缺损的辅助治疗。自体骨移植是目前临床上最为有效的治疗方法，被称为骨修复的“金标准”，但存在取材来源有限、供区手术与功能障碍以及有感染的风险等缺陷而使其应用受到限制。为解决该问题，近年来广泛采用天然无机骨修复材料即异体移植骨治疗骨缺损，如小牛骨来源的Bio-Oss、猪骨来源的OsteoBiol。然而，这些异体骨存在加工费时、产品价格昂贵等缺陷。因此，众多学者将目光转移到人工合成材料上，尤其是有机/无机复合材料因其在组成和结构上类似天然骨细胞外基质而在骨修复研究中得到广泛应用，也取得了较好的成果。大量研究表明胶原与磷灰石复合材料在体内外均具有良好的生物学效应。本文就明胶与磷酸三钙（β-TCP）复合纳米纤维膜材料在骨修复中的应用以及对成骨分化的诱导机制进行了研究。
本研究采用静电纺丝技术成功获得不同磷酸三钙（β-TCP）含量（0，5，10和20wt%）的明胶/β-TCP复合纳米纤维膜材料，扫描电镜观察可见交联处理前的纳米纤维粗细均匀，呈现无规则排布，纤维之间互相交错形成很多孔隙，四种复合纳米纤维的纤维直径均在200-400nm之间，表明β-TCP的掺入不会影响纤维直径的变化。单纯明胶纳米纤维表面光滑，而明胶/β-TCP复合纳米纤维上可见突起于纤维表面的磷灰石颗粒，而且磷灰石颗粒在纤维表面的附着量随着β-TCP的含量增加而增加。透射电镜观察可见磷灰石颗粒被包裹在明胶纤维内部，这将为磷灰石溶解释放钙离子起到调节作用。交联后的纳米纤维之间发生融并，纤维直径增加，而纤维间的孔隙较交联处理前明显减小，从纳米纤维膜的侧面观可见膜材料内部的纤维也发生互相粘连，表明纳米纤维膜完全被交联，这将有利于避免用于细胞培养而使材料发生快速降解，但磷灰石颗粒仍然突起于材料表面，表明明胶/β-TCP复合纳米纤维膜具有一定的表面粗糙度，这将有利于细胞的黏附与增殖。结构与组分分析证实了β-TCP和明胶组分的存在。蛋白吸附实验结果显示复合纳米纤维膜材料的血清蛋白吸附量随着β-TCP含量的增加而增加，而且在24小时内蛋白吸附量呈现时间依赖性。钙离子释放实验结果显示，在不更换培养基的情况下，孵育1天后钙离子即开始释放，在第7天时含20wt%β-TCP的膜材料其钙离子释放浓度趋于饱和，而其它两组钙离子释放浓度在持续孵育第14天后达到饱和状态。在达到饱和浓度的20wt%β-TCP膜材料上表面可见大量磷灰石颗粒沉积，表明发生了钙离子再沉积现象。在定期更换培养基的条件下，20wt%β-TCP膜材料在第7天后仍然有钙离子的继续释放，表明该复合纳米纤维膜释放钙离子在一定时间内存在钙离子“溶解-再沉积-溶解”动态平衡状态。
将成骨细胞系MG-63和原代分离培养的大鼠骨髓间充质干细胞（rBMSCs）分别接种至四种不同β-TCP含量的纳米纤维膜材料上，培养1和7天后，扫描电镜观察细胞形貌以及细胞黏附率检测结果显示，四种纳米纤维膜材料均有利于MG-63细胞及rBMSCs的黏附与增殖，在磷灰石掺入的纳米纤维上细胞表面分泌大量的细胞外基质，且有许多细胞突形成，激光共聚焦显微镜观察细胞骨架显示细胞骨架肌动蛋白丝的分布及细胞铺展面积均与β-TCP的含量呈正相关。MG-63细胞在膜材料上的ELISA检测结果显示四种膜材料中以20wt%β-TCP膜材料上的成骨分化指标碱性磷酸酶的表达量为最高，以上结果表明20wt%β-TCP复合纳米纤维膜材料可以有效促进成骨细胞的黏附、增殖以及成骨分化。进一步在单纯明胶与20wt%β-TCP膜材料上接种第三代rBMSCs后的7，14和21天，实时荧光定量PCR检测结果显示含20wt%β-TCP膜材料不仅可促进BMSCs的成骨分化关键转录因子RUNX-2、成骨分化基因I型胶原、骨形态发生蛋白BMP-2以及骨钙素BGLAP的上调表达，而且还激活了钙敏感受体CaSR的上调表达，表明明胶/β-TCP复合纳米纤维膜诱导rBMSCs成骨分化与CaSR的激活以及BMP-2信号通路有关。
本实验在体外研究的基础上，进一步将单纯明胶纳米纤维膜与20wt%β-TCP膜材料分别植入直径为8mm的兔下颌骨临界尺寸缺损模型中，以商品胶原膜为对照，分别在术后4周和12周通过解剖学观察、Micro-CT形态学检测、H-E染色以及免疫组化分析显示，单纯明胶纳米纤维膜的骨修复效果在新生骨形貌、骨痂形成、骨密度、骨容积比例以及骨重塑程度等方面均接近商品胶原膜。相比较，明胶/β-TCP复合纳米纤维膜则具有更好的骨修复质量，且在术后12周与宿主骨形成了良好的骨整合，骨密度接近正常骨组织，表明明胶/β-TCP复合纳米纤维膜具有良好的引导骨再生作用。由此可见，本实验制备的明胶/β-TCP复合纳米纤维膜可以用于骨缺损的修复，同时本研究结果将有望对今后骨修复研究及骨缺损的临床治疗提供切实的理论指导。
Bone defects arising from damage and disease are common phenomenon in clinicalpractice. The smaller defects will heal spontaneously, while the larger bone defectsmeaning ‘critical sized defects’ requied the artificial implants to repair. Althoughautologous bone graft is considered the gold standard in clinical practice, limitedavailability, additional surgery, and potential donor site morbidity limited its widelyapplication. To solve the problem, xenogenic grafts are used to repair the bone defectsclinically due to their ready availability and good osteoconductivity as a result of theirorigin from natural bone tissues of animals, i.e., Bio-Oss derived from bovine femur,and OsteoBiol derived from porcine bone. Nevertheless, the disadvantage of theseproducts are their time-consuming manufacturing process and high price. Therefore,extensive research effort is focused on synthetic materials. Especially, the norganicand organic composite materials in composition and structure similar natural boneextracellular matrix have been widely applied in the bone repair and regenerativemedicine, and have achieved lots of good results. Numerous researches have shownthat collagen and apatite composite materials have good biological effects both invitro and in vivo. In this study, the application of composite nanofibers includinggelatin and thicalcium phosphate (β-TCP) in bone repair and their osteoinductionmechanisms were investigated.
In the present study, gelatin/β-TCP composite nanofibrous membranes werefabricated via electrospinning with β-TCP nanoparticles content of0,5,10, and20wt%respectively. Scanning electron microscope (SEM) observation showed thatuncrosslinked nanofibers present a homogeneous fiber morphology with the range of200–400nm in diameter for all kinds of nanofibers. This indicated that theincorporation of β-TCP could not affects the fiber diameter. The random arrangedand interlaced fibers formed many pore. Pure gelatin nanofibers presented a smoothsurface morphology, while composite nanofibers incorporated β-TCP showedgranule-like surface appearance. Meanwhile, the apparent granule appearance on thesurface of composite nanofibers increased with the increasing amount of β-TCPnano-particles. Transmission electron microscope (TEM) image showed that islets ofβ-TCP nano-particles were enveloped into gelatin matrices. The diameter of fiber increased clearly distribute to fiber swelling in the procedure of crosslinking treatment,while the pore size decreased significantly in comparison with the as-electrospun one.A phenomenon was observed that thfebers were curled and conglutinat ed with eachother throughout the membrane after being crosslinked. This indicated that all thenanofibrous memberanes were crosslinked absolutely. The apatite particles wereremained on the surface of composite nanofibers. It suggested that gelatin/β-TCPcomposite nanofibrous membranes have some surface roughness, which will benefitcell adhesion and proliferation. The structure integrity and functional groups ofgelatin and β-TCP were retained during the procedure of electrospinning. The resultsof protein adsorption analysis showed that the amount of serum protein adsorptiononto the composite nanofibrous membranes increased with the amount ofincorporated β-TCP, and presented a manner of time dependence within24h.
The results of calcium ion release test showed that calcium ions released obviouslyafter incubation for one day when the culture medium was not changed. At the7thday, the concentration ofcalcium ions release in20wt%β-TCP containing compositenanofibrous membranes reached the saturation state, while the other two groupsreached the saturation concentration after14days. Moreover, numerous apatiteparticles deposited on the surface of20wt%β-TCP membrane materials from SEMimages. When the medium was changed regularly, calcium ions continue to releaseafter7days for20wt%β-TCP membrane materials. From this phenomenon, it couldbe inferred that a balance between dissolution of β-TCP and formation of mineralitedepositions was detected.
When MG-63osteoblast cell line and primary cultured rats bone marrowmesenchymal stem cells (rBMSCs) were seeded on the composite nanofibrousmembranes respectively for1and7days, SEM images and cell attachment efficiencyanalysis showed that four kinds of nanofibrous membranes are beneficial to celladhesion and proliferation. For the composite nanofibers incorporated β-TCP groups,plenty of extracellular matrix secreted on the surface of cells and formed apparentprotruding cellular processes. Laser confocal microscope observation showed thatcytoskeletal organization and cell spreading area on the composite nanofibrousmembranes increased with the amount of incorporated β-TCP. ELISA tests showedthat cells in20wt%β-TCP group exhibited highest ALP activity, indicating higheramount of incorporated β-TCP enhanced higher ability of osteogenic differentiation of MG-63cells. These results showed that composite nanofiber membranes with20wt%β-TCP could promote effectively the adhesion and proliferation of osteoblasts.Furthermore, the third generation of rBMSCs were cultured on the pure gelatin and20wt%β-TCP membrane materials respectively after7,14and21days. Real-timequantitative PCR detection results show that the composite nanofibrous membranescontaining20wt%β-TCP not only can promote the upregulation expression ofosteoblast differentiation key transcription factors RUNX-2, type I collagen, BMP-2and bone gamma-carboxyglutamate protein (BGLAP), but also activate the calciumsensitive receptor (CaSR) during the experiment period, suggesting the osteoinductionmechanisms of gelatin/β-TCP composite nanofibrous membrane were related to theactivity of CaSR and BMP-2signaling pathway.
On the basis of in vitro studies, pure gelatin nanofibrous membranes and compositenanofibrous membranes with20wt%β-TCP were further implanted into rabbitmandibular critical size defect models (8mm in diameter) respectively. Thecommercial collagen membranes were used as control. At4and12weekspost-operation, anatomical observation, Micro-CT detection, H&E staining andimmunohistochemical analysis showed that the bone repair efficiency of pure gelatinnanofibrous membranes was close to that of commercial collagen membranesregarding new bone morphology, callus formation, bone mineral density, bone volumeratio, and bone remodeling degree. By comparison, gelatin/β-TCP compositenanofibrous membranes had better bone repair quality, and formed good boneintergration with host bone at12weeks post-operation. Bone mineral density ofgelatin/β-TCP composite nanofibrous membranes group was close to that of normalbone tissue, sugessting the gelatin/β-TCPcomposite nanofibrous membranes had wellguid bone regeneration effects. These results demonstrated that electrospungelatin/β-TCP composite nanofibrous membranes could be used to bone repairtreatment, and the results would provide the feasible and theoretical guidance forfurther bone regeneration research.
本研究采用静电纺丝技术成功获得不同磷酸三钙（β-TCP）含量（0，5，10和20wt%）的明胶/β-TCP复合纳米纤维膜材料，扫描电镜观察可见交联处理前的纳米纤维粗细均匀，呈现无规则排布，纤维之间互相交错形成很多孔隙，四种复合纳米纤维的纤维直径均在200-400nm之间，表明β-TCP的掺入不会影响纤维直径的变化。单纯明胶纳米纤维表面光滑，而明胶/β-TCP复合纳米纤维上可见突起于纤维表面的磷灰石颗粒，而且磷灰石颗粒在纤维表面的附着量随着β-TCP的含量增加而增加。透射电镜观察可见磷灰石颗粒被包裹在明胶纤维内部，这将为磷灰石溶解释放钙离子起到调节作用。交联后的纳米纤维之间发生融并，纤维直径增加，而纤维间的孔隙较交联处理前明显减小，从纳米纤维膜的侧面观可见膜材料内部的纤维也发生互相粘连，表明纳米纤维膜完全被交联，这将有利于避免用于细胞培养而使材料发生快速降解，但磷灰石颗粒仍然突起于材料表面，表明明胶/β-TCP复合纳米纤维膜具有一定的表面粗糙度，这将有利于细胞的黏附与增殖。结构与组分分析证实了β-TCP和明胶组分的存在。蛋白吸附实验结果显示复合纳米纤维膜材料的血清蛋白吸附量随着β-TCP含量的增加而增加，而且在24小时内蛋白吸附量呈现时间依赖性。钙离子释放实验结果显示，在不更换培养基的情况下，孵育1天后钙离子即开始释放，在第7天时含20wt%β-TCP的膜材料其钙离子释放浓度趋于饱和，而其它两组钙离子释放浓度在持续孵育第14天后达到饱和状态。在达到饱和浓度的20wt%β-TCP膜材料上表面可见大量磷灰石颗粒沉积，表明发生了钙离子再沉积现象。在定期更换培养基的条件下，20wt%β-TCP膜材料在第7天后仍然有钙离子的继续释放，表明该复合纳米纤维膜释放钙离子在一定时间内存在钙离子“溶解-再沉积-溶解”动态平衡状态。
将成骨细胞系MG-63和原代分离培养的大鼠骨髓间充质干细胞（rBMSCs）分别接种至四种不同β-TCP含量的纳米纤维膜材料上，培养1和7天后，扫描电镜观察细胞形貌以及细胞黏附率检测结果显示，四种纳米纤维膜材料均有利于MG-63细胞及rBMSCs的黏附与增殖，在磷灰石掺入的纳米纤维上细胞表面分泌大量的细胞外基质，且有许多细胞突形成，激光共聚焦显微镜观察细胞骨架显示细胞骨架肌动蛋白丝的分布及细胞铺展面积均与β-TCP的含量呈正相关。MG-63细胞在膜材料上的ELISA检测结果显示四种膜材料中以20wt%β-TCP膜材料上的成骨分化指标碱性磷酸酶的表达量为最高，以上结果表明20wt%β-TCP复合纳米纤维膜材料可以有效促进成骨细胞的黏附、增殖以及成骨分化。进一步在单纯明胶与20wt%β-TCP膜材料上接种第三代rBMSCs后的7，14和21天，实时荧光定量PCR检测结果显示含20wt%β-TCP膜材料不仅可促进BMSCs的成骨分化关键转录因子RUNX-2、成骨分化基因I型胶原、骨形态发生蛋白BMP-2以及骨钙素BGLAP的上调表达，而且还激活了钙敏感受体CaSR的上调表达，表明明胶/β-TCP复合纳米纤维膜诱导rBMSCs成骨分化与CaSR的激活以及BMP-2信号通路有关。
本实验在体外研究的基础上，进一步将单纯明胶纳米纤维膜与20wt%β-TCP膜材料分别植入直径为8mm的兔下颌骨临界尺寸缺损模型中，以商品胶原膜为对照，分别在术后4周和12周通过解剖学观察、Micro-CT形态学检测、H-E染色以及免疫组化分析显示，单纯明胶纳米纤维膜的骨修复效果在新生骨形貌、骨痂形成、骨密度、骨容积比例以及骨重塑程度等方面均接近商品胶原膜。相比较，明胶/β-TCP复合纳米纤维膜则具有更好的骨修复质量，且在术后12周与宿主骨形成了良好的骨整合，骨密度接近正常骨组织，表明明胶/β-TCP复合纳米纤维膜具有良好的引导骨再生作用。由此可见，本实验制备的明胶/β-TCP复合纳米纤维膜可以用于骨缺损的修复，同时本研究结果将有望对今后骨修复研究及骨缺损的临床治疗提供切实的理论指导。
Bone defects arising from damage and disease are common phenomenon in clinicalpractice. The smaller defects will heal spontaneously, while the larger bone defectsmeaning ‘critical sized defects’ requied the artificial implants to repair. Althoughautologous bone graft is considered the gold standard in clinical practice, limitedavailability, additional surgery, and potential donor site morbidity limited its widelyapplication. To solve the problem, xenogenic grafts are used to repair the bone defectsclinically due to their ready availability and good osteoconductivity as a result of theirorigin from natural bone tissues of animals, i.e., Bio-Oss derived from bovine femur,and OsteoBiol derived from porcine bone. Nevertheless, the disadvantage of theseproducts are their time-consuming manufacturing process and high price. Therefore,extensive research effort is focused on synthetic materials. Especially, the norganicand organic composite materials in composition and structure similar natural boneextracellular matrix have been widely applied in the bone repair and regenerativemedicine, and have achieved lots of good results. Numerous researches have shownthat collagen and apatite composite materials have good biological effects both invitro and in vivo. In this study, the application of composite nanofibers includinggelatin and thicalcium phosphate (β-TCP) in bone repair and their osteoinductionmechanisms were investigated.
In the present study, gelatin/β-TCP composite nanofibrous membranes werefabricated via electrospinning with β-TCP nanoparticles content of0,5,10, and20wt%respectively. Scanning electron microscope (SEM) observation showed thatuncrosslinked nanofibers present a homogeneous fiber morphology with the range of200–400nm in diameter for all kinds of nanofibers. This indicated that theincorporation of β-TCP could not affects the fiber diameter. The random arrangedand interlaced fibers formed many pore. Pure gelatin nanofibers presented a smoothsurface morphology, while composite nanofibers incorporated β-TCP showedgranule-like surface appearance. Meanwhile, the apparent granule appearance on thesurface of composite nanofibers increased with the increasing amount of β-TCPnano-particles. Transmission electron microscope (TEM) image showed that islets ofβ-TCP nano-particles were enveloped into gelatin matrices. The diameter of fiber increased clearly distribute to fiber swelling in the procedure of crosslinking treatment,while the pore size decreased significantly in comparison with the as-electrospun one.A phenomenon was observed that thfebers were curled and conglutinat ed with eachother throughout the membrane after being crosslinked. This indicated that all thenanofibrous memberanes were crosslinked absolutely. The apatite particles wereremained on the surface of composite nanofibers. It suggested that gelatin/β-TCPcomposite nanofibrous membranes have some surface roughness, which will benefitcell adhesion and proliferation. The structure integrity and functional groups ofgelatin and β-TCP were retained during the procedure of electrospinning. The resultsof protein adsorption analysis showed that the amount of serum protein adsorptiononto the composite nanofibrous membranes increased with the amount ofincorporated β-TCP, and presented a manner of time dependence within24h.
The results of calcium ion release test showed that calcium ions released obviouslyafter incubation for one day when the culture medium was not changed. At the7thday, the concentration ofcalcium ions release in20wt%β-TCP containing compositenanofibrous membranes reached the saturation state, while the other two groupsreached the saturation concentration after14days. Moreover, numerous apatiteparticles deposited on the surface of20wt%β-TCP membrane materials from SEMimages. When the medium was changed regularly, calcium ions continue to releaseafter7days for20wt%β-TCP membrane materials. From this phenomenon, it couldbe inferred that a balance between dissolution of β-TCP and formation of mineralitedepositions was detected.
When MG-63osteoblast cell line and primary cultured rats bone marrowmesenchymal stem cells (rBMSCs) were seeded on the composite nanofibrousmembranes respectively for1and7days, SEM images and cell attachment efficiencyanalysis showed that four kinds of nanofibrous membranes are beneficial to celladhesion and proliferation. For the composite nanofibers incorporated β-TCP groups,plenty of extracellular matrix secreted on the surface of cells and formed apparentprotruding cellular processes. Laser confocal microscope observation showed thatcytoskeletal organization and cell spreading area on the composite nanofibrousmembranes increased with the amount of incorporated β-TCP. ELISA tests showedthat cells in20wt%β-TCP group exhibited highest ALP activity, indicating higheramount of incorporated β-TCP enhanced higher ability of osteogenic differentiation of MG-63cells. These results showed that composite nanofiber membranes with20wt%β-TCP could promote effectively the adhesion and proliferation of osteoblasts.Furthermore, the third generation of rBMSCs were cultured on the pure gelatin and20wt%β-TCP membrane materials respectively after7,14and21days. Real-timequantitative PCR detection results show that the composite nanofibrous membranescontaining20wt%β-TCP not only can promote the upregulation expression ofosteoblast differentiation key transcription factors RUNX-2, type I collagen, BMP-2and bone gamma-carboxyglutamate protein (BGLAP), but also activate the calciumsensitive receptor (CaSR) during the experiment period, suggesting the osteoinductionmechanisms of gelatin/β-TCP composite nanofibrous membrane were related to theactivity of CaSR and BMP-2signaling pathway.
On the basis of in vitro studies, pure gelatin nanofibrous membranes and compositenanofibrous membranes with20wt%β-TCP were further implanted into rabbitmandibular critical size defect models (8mm in diameter) respectively. Thecommercial collagen membranes were used as control. At4and12weekspost-operation, anatomical observation, Micro-CT detection, H&E staining andimmunohistochemical analysis showed that the bone repair efficiency of pure gelatinnanofibrous membranes was close to that of commercial collagen membranesregarding new bone morphology, callus formation, bone mineral density, bone volumeratio, and bone remodeling degree. By comparison, gelatin/β-TCP compositenanofibrous membranes had better bone repair quality, and formed good boneintergration with host bone at12weeks post-operation. Bone mineral density ofgelatin/β-TCP composite nanofibrous membranes group was close to that of normalbone tissue, sugessting the gelatin/β-TCPcomposite nanofibrous membranes had wellguid bone regeneration effects. These results demonstrated that electrospungelatin/β-TCP composite nanofibrous membranes could be used to bone repairtreatment, and the results would provide the feasible and theoretical guidance forfurther bone regeneration research.
引文
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