仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原的制备及其细胞相容性

背景：目前骨组织工程常用的支架材料主要有无机材料、有机高分子材料及天然衍生材料等,上述材料各有优缺点,为了充分发挥各类材料的优势,弥补其不足,目前多采用联合材料制备复合支架。 目的：制备新型仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原,并观察其对骨髓间充质干细胞增殖、黏附及分化的影响。 方法：制备壳聚糖/纳米羟基磷灰石/胶原复合支架材料,扫描电镜观察支架材料表面微观形貌;采用真空吸附法将骨形态发生蛋白7多肽与支架材料复合,高效液相色谱仪检测骨形态发生蛋白7多肽在体外的释放规律;将骨髓间充质干细胞接种到复合骨形态发生蛋白7多肽的仿生支架材料上,以未复合多肽的支架材料作为对照,检测支架材料表面细胞增殖、黏附率、生长形态及碱性磷酸酶活性。 结果与结论：壳聚糖/纳米羟基磷灰石/胶原支架材料呈多孔状,孔径10~100 µm;骨形态发生蛋白7多肽可以从支架材料中缓慢释出;在复合多肽的仿生支架材料表面,骨髓间充质干细胞的黏附及向成骨细胞方向分化能力均明显强于对照组(P < 0.05),而增殖能力与对照组差异无显著性意义(P > 0.05)。说明新型仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原是一种理想的骨组织工程支架材料,具有良好的细胞相容性。     

纳米羟基磷灰石骨修复复合材料的研究进展

羟基磷灰石（Hydroxyapatite，HA）是一种性能良好的骨修复材料，但是由于脆性而限制了它在承力部位的应用。天然骨本身是纳米羟基磷灰石（Nanohydroxyapatite，n．HA）和胶原的复合材料，从仿生的角度看，n—HA与其它材料复合可以提高生物相容性和力学性能。目前研究的纳米羟基磷灰石复合材料可分为两类：非降解的纳米羟基磷灰石复合材料和可降解的纳米羟基磷灰石复合材料。前者包括n—HA/聚乙烯、n-HA／尼龙以及n—HA／聚丙烯酸。后者主要有n—HA与胶原、明胶、壳聚糖、聚乳酸和聚酸酐等的复合材料。本文详细综述了近年来纳米羟基磷灰石复合材料的制备、力学性能以及生物学性能的研究进展。     

胶原-纳米羟基磷灰石复合支架的细胞相容性

背景：观察成骨细胞在生物材料上的形态、增殖和分化等项目,可评估生物支架材料的生物相容性。 目的：观察复合支架材料纳米羟基磷灰石/胶原对成骨细胞增殖、分化的影响。 方法：取新生24 h内Wistar大鼠的颅盖骨,采用改良胶原酶消化法进行成骨细胞原代培养,取第3代细胞与纳米羟基磷灰石/胶原支架或普通羟基磷灰石材料体外复合培养。培养3,6,9 d后,观察材料周边的细胞形态及支架材料对细胞分化、增殖的影响。 结果与结论：纳米羟基磷灰石/胶原材料较普通的羟基磷灰石材料更有利于成骨细胞的黏附、生长、分化、增殖,证实其生物相容性更好,有望成为一种新型的骨组织工程支架材料。     

电纺纳米羟基磷灰石/聚酯酰胺复合支架材料的制备及其细胞相容性

背景：采用静电纺丝技术将功能性无机纳米微粒复合高分子超细纤维,形成类细胞外基质结构和功能的复合支架材料是骨组织工程支架领域一个新的研究方向。 目的：通过静电纺丝法构建纳米羟基磷灰石/脂肪族聚酯酰胺复合纤维支架材料,并初步考察其细胞相容性。 方法：以静电纺丝法制备纳米羟基磷灰石/脂肪族聚酯酰胺超细纤维支架材料,通过扫描电镜、原子能谱等表面形貌的物相分析,进行细胞在复合材料上的形态学观察。 结果与结论：通过静电纺丝法成功制备出纳米羟基磷灰石/脂肪族聚酯酰胺超细纤维复合材料,成骨细胞直接培养于材料上呈现良好生长行为,初步证实了复合支架材料的细胞相容性。说明静电纺丝技术在构建类骨细胞外基质结构和功能的仿生复合材料方面具有独特优势,电纺超细纤维复合材料有望成为新型的骨组织工程支架。     

多孔碳酸钙陶瓷与干细胞源性成骨细胞的相容性

背景：国内外的研究证实普通碳酸钙陶瓷作为骨替代材料时具有细胞支架作用。 目的：观察多孔碳酸钙陶瓷与成骨细胞的相容性,及作为骨组织工程支架的可能性。 方法：SD大鼠骨髓基质干细胞经矿化诱导培养、扩增并检测证实其已具成骨细胞表型后,分别与多孔碳酸钙陶瓷支架、普通羟基磷灰石陶瓷支架体外复合培养。 结果与结论：骨髓基质干细胞经体外诱导形成成骨细胞,钙结节、Ⅰ型胶原和碱性磷酸酶免疫染色结果阳性。多孔碳酸钙陶瓷支架材料与羟基磷灰石陶瓷材料皆有细胞附着生长,但多孔碳酸钙陶瓷支架材料细胞的黏附能力、增殖活力及成骨活性均强于羟基磷灰石陶瓷材料。提示多孔碳酸钙陶瓷支架材料与SD大鼠骨髓基质干细胞源性成骨细胞有良好相容性。     

纳米晶羟基磷灰石胶原复合骨髓间充质干细胞修复骨缺损(英文)

背景:临床上对大范围骨缺损还没有很有效的治疗手段,而纳米晶羟基磷灰石胶原复合材料与天然骨骼的结构类似,具有较好的生物相容性,可能为修复骨缺损提供新的途径。目的:观察纳米晶羟基磷灰石胶原材料复合骨髓间充质干细胞在修复骨缺损中的作用。方法:分离培养人骨髓间充质干细胞,与纳米晶羟基磷灰石胶原材料于体外联合培养;通过大体观察、组织学分析及电镜观察了解成骨情况,进一步临床应用于修复骨缺损。结果与结论:人骨髓间充质干细胞在体外可以大量扩增,复合细胞的材料植入骨缺损处后,X射线摄片动态观察可见骨缺损处连接良好。说明骨髓间充质干细胞具有成骨细胞作用,纳米晶羟基磷灰石胶原材料是一种很好的构建组织工程骨的支架材料。     

纳米羟基磷灰石/I型胶原复合人工骨组成结构及成骨活性的实验研究

按照仿生的方法合成纳米羟基磷灰石/I型胶原人工骨支架材料.采用扫描电镜、X线衍射分析、孔隙率测定的方法对人工骨支架材料进行分析.制作兔颅骨缺损模型,植入人工骨支架材料,组织切片观察支架材料在体内的反应.纳米羟基磷灰石/I型胶原人工骨支架材料呈疏松海绵状,具有100～300 μm的孔径和90%以上的孔隙率,具有类似天然骨的结构.植入兔颅骨缺损模型未出现急性或慢性的炎症反应,在4周左右人工骨支架内出现大量新生毛细血管,12周新生骨完全修复骨缺损,并形成成熟的骨小梁结构.纳米羟基磷灰石和I型胶原复合人工骨约3个月左右降解完全.     

聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架与兔软骨细胞的相容性

背景：传统的支架材料存在疏水性强,材料表面缺乏细胞表面受体特异结合的生物活性分子,材料的酸性降解产物易引发无菌性炎性反应等不足。根据仿生原理及软骨真实结构和构成来选择和制备组织工程软骨支架能够获得理想效果。 目的：制备聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架,评价其与兔膝关节软骨细胞的生物相容性,探讨其应用于关节软骨组织工程的可行性。 方法：采用二次相分离技术制备聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石复合支架,将第3代新西兰兔软骨细胞接种至复合支架材料上复合培养,倒置相差显微镜下观察细胞生长情况。细胞-支架复合物在24孔板中培养5 d以后,将其植入裸鼠皮下8周。 结果与结论：聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架材料经化学合成后,具有合适的三维多孔结构,孔隙率为90%,孔径300~450 μm;植入裸鼠皮下8周后Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色显示细胞-支架复合物中的软骨细胞可以像天然软骨一样分泌黏多糖和Ⅱ型胶原。提示生物材料聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石对于兔软骨细胞有良好的生物相容性,可作为生物组织工程支架。     

壳聚糖/纳米羟基磷灰石分层复合支架的生物相容性研究

制备壳聚糖/纳米羟基磷灰石(CS/nHA)分层复合支架,对其进行细胞毒性评价.分离培养大鼠软骨细胞接种于支架,相差显微镜和扫描电镜观察细胞的黏附及生长情况.动物皮下埋植试验观察其组织相容性.实验结果证实壳聚糖/纳米羟基磷灰石分层复合支架具有良好的生物相容性,有望成为较好的骨软骨组织工程支架.     

骨修复用纳米羟基磷灰石/聚酰胺66的体内外生物相容性(英文)

背景:采用基于纳米羟基磷灰石溶胶新方法制备纳米羟基磷灰石/聚酰胺66复合材料,该材料提高了纳米羟基磷灰石在聚酰胺66基体中的均匀分布和二者的有效键合,进而有利于改善材料的生物性能,有望成为新型骨修复材料。目的:评价纳米羟基磷灰石/聚酰胺66复合材料体内外生物相容性。方法:①将原代培养的成骨细胞与纳米羟基磷灰石/聚酰胺66及聚酰胺66材料复合培养,使用倒置相差显微镜和场发射扫描电子显微镜观察材料周围及表面的细胞形态。②将纳米羟基磷灰石/聚酰胺66复合材料植入兔右侧胫骨,将聚酰胺66作为对照组材料植入兔左侧胫骨。在术后2,8周,取材料周围骨组织进行病理组织切片观察。结果与结论:①纳米羟基磷灰石/聚酰胺66和聚酰胺66未表现出明显的细胞毒性,纳米羟基磷灰石/聚酰胺66材料周围细胞形态好于聚酰胺66,且纳米羟基磷灰石/聚酰胺66表面细胞数量多于聚酰胺66,在复合培养的第3天差异尤其显著(P0.01)。②在植入早期,与纳米羟基磷灰石/聚酰胺66相接的骨组织成骨细胞活跃且该组材料周围的骨形成过程较对照组更快。结果说明纳米羟基磷灰石/聚酰胺66复合材料较聚酰胺66有更好的生物相容性。     

胶原复合梯度磷酸三钙负载软骨细胞的体外培养

目的 观察新型三维支架材料胶原复合梯度磷酸三钙在体外与软骨细胞的相容性和黏附性,评价其作为软骨组织工程支架的可行性.方法 取8周龄新两兰大白兔膝关节软骨,以酶消化法获得高纯度软骨细胞,培养3代后与三维支架材料胶原复合梯度磷酸三钙在体外复合培养.用倒置相差显微镜、HE染色、免疫组织化学及扫描电镜观察软骨细胞形态、Ⅱ型胶原表达及成软骨能力,同时观察支架材料与软骨细胞的相容性.结果 扫描电镜观察显示支架材料具有疏松多孔结构,孔隙结构规则,孔径100～150 μm,材料内部孔与孔之间贯通良好.支架亲水性好.软骨细胞吸附于支架表面,增殖并逐渐顺孔隙迁徙至支架内部,在孔壁贴附良好,表型维持稳定,可分泌细胞外基质.结论 胶原复合梯度磷酸三钙三维支架具有良好的细胞相容性.     

Biomineralized porous composite scaffolds prepared by chemical synthesis for bone tissue regeneration

Scaffold design is a key factor in the clinical success of bone tissue engineering grafts. To date, no existing single biomaterial used in bone repair and regeneration fulfils all the requirements for an ideal bone graft. In this study hydroxyapatite/polycaprolactone (HA/PCL) composite scaffolds were prepared by a wet chemical method at room temperature. The physico-chemical properties of the composite materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while scaffold morphology was investigated by scanning electron microscopy (SEM) with energy-dispersive spectroscopy to validate the process used for synthesis. Finally, the response of bone marrow-derived human mesenchymal stem cells (hMSCs) in terms of cell proliferation and differentiation to the osteoblastic phenotype was evaluated using the Alamar blue assay, SEM and alkaline phosphatase activity. Microstructural analysis indicated that the HA particles were distributed homogeneously within the PCL matrix. The biological results revealed that the HA/PCL composite scaffolds are suitable for the proliferation and differentiation of MSCs in vitro, supporting osteogenesis after 15 days. All the results indicate that these scaffolds meet the requirements of materials for bone tissue engineering and could be used for many clinical applications in orthopaedic and maxillofacial surgery.     

Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering

In this study, we prepared nano-hydroxyapatite/polyamide (n-HA/PA) composite scaffolds utilizing thermally induced phase inversion processing technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Mesenchymal stem cells (MSCs) derived from bone marrow of neonatal rabbits were cultured, expanded and seeded on n-HA/PA scaffolds. The MSC/scaffold constructs were cultured for up to 7 days and the adhesion, proliferation and differentiation of MSCs into osteoblastic phenotype were determined using MTT assay, alkaline phosphatase (ALP) activity and collagen type I (COL I) immunohistochemical staining and scanning electronic microscopy (SEM). The results confirm that n-HA/PA scaffolds are biocompatible and have no negative effects on the MSCs in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure n-HA/PA scaffolds and MSC/scaffold constructs were implanted in rabbit mandibles and studied histologically and microradiographically. The results show that n-HA/PA composite scaffolds exhibit good biocompatibility and extensive osteoconductivity with host bone. Moreover, the introduction of MSCs to the scaffolds dramatically enhanced the efficiency of new bone formation, especially at the initial stage after implantation. In long term (more than 12 weeks implantation), however, the pure scaffolds show as good biocompatibility and osteogenesis as the hybrid ones. All these results indicate that the scaffolds fulfill the basic requirements of bone tissue engineering scaffold, and have the potential to be applied in orthopedic, reconstructive and maxillofacial surgery.     

Biocompatibility and osteogenesis of biomimetic Bioglass-Collagen-Phosphatidylserine composite scaffolds for bone tissue engineering

A novel biomimetic composite scaffold Bioglass-Collagen-Phosphatidylserine (BG-COL-PS) was fabricated with a freeze-drying technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM) showed that the BG-COL-PS scaffolds exhibited interconnected porous structures with pore sizes of several microns up to about 300 μm. The scaffolds have a porosity of 75.40% and the corresponding compressive strength of 1.5469 Mpa. Rat mesenchymal stem cells (rMSCs) were seeded on BG-COL-PS or BG-COL scaffolds and cultured for 21 days in vitro. Based on the results of SEM, dsDNA content, alkaline phosphatase (ALP) activity, osteogenic gene expression analysis and alizarin red staining, the responses of MSCs to the scaffold exhibited a higher degree of attachment, growth as well as osteogenic differentiation than those on BG-COL scaffolds in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure BG-COL-PS scaffolds and MSC/scaffold constructs were implanted in rat femurs defects for 6 weeks and studied histologically and radiographically. The in vivo results showed that BG-COL-PS composite scaffolds exhibited good biocompatibility and extensive osteoconductivity with host bone. Moreover, the BG-COL-PS/MSC constructs dramatically enhanced the efficiency of new bone formation than pure BG-COL-PS scaffolds or BG-COL/MSC constructs. All these results demonstrate the usefulness of PS composited BG-COL-PS scaffolds for inducing enhanced bone formation. The BG-COL-PS scaffolds fulfill the basic requirements of bone tissue engineering scaffold and have the potential to be applied in orthopedic and reconstructive surgery.     

双基因转染犬骨髓间充质干细胞复合HA/ZrO2生物材料新型组织工程骨的构建及其体外成骨能力的研究

目的 构建骨形态发生蛋白2(BMP-2)、血管内皮细胞生长因子165(VEGF165)双基因修饰的骨髓间充质干细胞(BMSCs)复合羟基磷灰石复合二氧化锆(HA/ZrO₂)生物材料的新型组织工程骨,并观察该组织工程骨在体外的成骨能力。方法 采用有机泡沫作为模版,干铺烧制法制备新型的蜂窝状HA/ZrO₂梯度生物材料,电镜观察新型生物材料的表面特性,生物力学试验机检测其力学性能。采取1岁龄健康beagle犬骨髓分离原代BMSCs进行培养,建立双基因修饰的BMSCs复合蜂窝状HA/ZrO₂梯度生物材料的共培养体,构建新型组织工程骨。实验分为4组:未转染组,只转染BMP-2(BMP-2组)和VEGF165(VEGF165组)单一目的基因的BMSCs,以及转染BMP-2、VEGF165共基因慢病毒的BMSCs组(BMP-2+VEGF165组)。显微镜下观察细胞在支架材料上的生长情况,用碱性磷酸酶染色检测各组细胞成骨分化能力,免疫组织化学染色检测其成骨细胞特异性蛋白骨Ⅰ型胶原及骨钙素的分泌。结果 新型材料电镜下其表面整体呈多孔状,孔径125~550 μm,各孔之间存在缝隙联结;其平均抗弯强度为812.25 MPa,最高可达987.12 MPa;共培养体建立后扫描电镜观察转染后的BMSCs在支架材料上黏附生长状况良好,双基因联合转染组细胞分泌基质旺盛;BMP-2+VEGF165组细胞碱性磷酸酶活性检测明显高于其他各组(F=1 029.398,P<0.01),免疫组织化学染色在不同阶段发现成骨细胞早晚期分泌的骨Ⅰ型胶原及骨钙素特异性蛋白。结论 新型的蜂窝状HA/ZrO₂梯度生物材料是一种合适种子细胞生长的支架材料,并且其力学满足人体四肢承重骨的需要;VEGF165、BMP-2双基因转染BMSCs后具有协同作用,能够促进其在体外的成骨分化。     

Preparation,characterization and in vitro analysis of novel structured nanofibrous scaffolds for bone tissue engineering

In a previous study, a three-dimensional nanofibrous spiral scaffold for bone tissue engineering was developed, which showed enhanced human osteoblast cell attachment, proliferation and differentiation compared with traditional cylinder scaffolds, owing to the incorporation of spiral structures and nanofiber. However, the application of these scaffolds to bone tissue engineering was limited by their weak mechanical strength. This limitation triggered the design for novel structured scaffolds with reinforced physical characteristics. In this study, spiral polycaprolactone (PCL) nanofibrous scaffolds were inserted into poly(lactide-co-glycolide) (PLGA) microsphere sintered tubular scaffolds to form integrated scaffolds to provide mechanical properties and bioactivity appropriate for bone tissue engineering. Four experiment groups were designed: PLGA cylinder scaffold; PLGA tubular scaffold; PLGA tubular scaffold with PCL spiral structured inner core; PLGA tubular scaffold with PCL nanofiber containing spiral structured inner core. The morphology, porosity and mechanical properties of the scaffolds were characterized. Furthermore, human osteoblastic cells were seeded on these scaffolds, and the cell attachment, proliferation, differentiation and mineralized matrix deposition on the scaffolds were evaluated. The integrated scaffolds had Young’s modulus 250–300 MPa, and compressive strength 8–11 MPa under uniaxial compression. With the addition of an inner highly porous insert to the tubular shell, human osteoblast cells seeded on the integrated scaffolds showed slightly higher cell proliferation, 20–25% more alkaline phosphatase expression and twofold higher calcium deposition than those on the cylinder and tubular scaffolds. Furthermore, compared with sintered PLGA cylinder scaffolds, the integrated scaffolds allowed better cellular infiltration Therefore, this design demonstrates great potential for integrated scaffolds in bone tissue engineering applications.     

Fabrication and characterization of hydrophilic poly(lactic-co-glycolic acid)/poly(vinyl alcohol) blend cell scaffolds by melt-molding particulate-leaching method

Porous PLGA/PVA scaffolds were fabricated by blending poly(lactic-co-glycolic acid) (PLGA) with polyvinyl alcohol (PVA) to improve the hydrophilicity and cell compatibility of the scaffolds for tissue engineering applications. PLGA/PVA blend scaffolds with different PVA compositions up to 20wt% were fabricated by a melt-molding particulate-leaching method (non-solvent method). The prepared scaffolds were investigated by scanning electron microscopy (SEM), mercury intrusion porosimetry, the measurements of water contact angles and bi-axial tensile strengths, etc. for their surface and bulk characterizations. The scaffolds exhibited highly porous and open-cellular pore structures with almost same surface and interior porosities (pore size, 200-300 microm; porosity, about 90%). The PLGA/PVA blend scaffolds with PVA compositions more than 5% were easily wetted in cell culture medium without any prewetting treatments, which is highly desirable for tissue engineering applications. In vitro cell compatibility of the control hydrophobic PLGA and hydrophilized PLGA/PVA (5wt%) blend scaffolds was compared by the culture of human chondrocytes in the scaffolds and the following analyses by MTT assay and SEM observation. It was observed that the PLGA/PVA blend scaffold had better cell adhesion and growth than the control PLGA scaffold. For in vivo evaluation of tissue compatibility, the scaffolds were implanted into the skull defects of rabbits. The results were evaluated by histology examinations. The PLGA/PVA (5wt%) blend scaffold showed better bone ingrowth into the scaffold and new bone formation inside the scaffold than the PLGA scaffold. It seems that 5% addition of PVA to PLGA to fabricate PLGA/PVA blend scaffolds is enough for improving the hydrophilicity and cell compatibility of the scaffolds.     

骨髓间充质干细胞与纳米羟基磷灰石支架材料的体外相容性研究

目的　探讨兔骨髓间充质干细胞(rBMSCs)与纳米羟基磷灰石(nano-HA)支架材料的相容性,进一步验证nano-HA材料作为骨组织工程支架材料的可行性。 方法　将rBMSCs与nano-HA支架材料在体外复合培养,通过倒置显微镜、扫描电镜观察细胞与材料的复合情况,用MTT法、碱性磷酸酶活性检测法检测材料对细胞增殖、分化的影响。 结果　rBMSCs可以在nano-HA支架材料表面及孔隙中良好的黏附、迁移、增殖和分化,复合培养5 d后nano-HA支架材料对rBMSCs的增殖分化表现出一定的促进作用。 结论　rBMSCs与nano-HA支架材料具有良好的生物相容性, nano-HA支架材料可以作为rBMSCs良好的载体。