3D-printed composite scaffold with anti-infection and osteogenesis potential against infected bone defects

In the field of orthopedics, an infected bone defect is a refractory disease accompanied by bone infection and defects as well as aggravated circulation. There are currently no personalized scaffolds that can treat bone infections using local stable and sustained-release antibiotics while providing mechanical support and bone induction to promote bone repair in the process of absorption in vivo. In our previous study, rifampicin/moxifloxacin-poly lactic-co-glycolic acid (PLGA) microspheres were prepared and tested for sustained release and antibacterial activity. The composite scaffold of poly-l-lactic acid (PLLA)/Pearl had a positive effect on mechanics supports and promoted osteogenesis. Therefore, in this study, the personalized scaffolds of PLLA/Pearl were first prepared by 3D printing. Then, rifampicin/moxifloxacin-PLGA (RM-P) microspheres were loaded into the scaffold pores to prepare the PLLA/Pearl/RM-P scaffolds. In this in vitro study, we investigated the structural characteristics and cytocompatibility of 3D-printed composite scaffolds, which indicates the integrity of the components in the scaffolds. The PLLA/Pearl and PLLA/Pearl/RM-P composite scaffolds can promote adhesion, proliferation, and differentiation of human bone marrow mesenchymal stem cells. Moreover, a rabbit model of infected bone defects of the radius was established. PLLA, PLLA/Pearl, and PLLA/Pearl/RM-P scaffolds were implanted into the bone nidus. The therapeutic effect of the three scaffolds on the infected bone defects was evaluated through imaging and microbiological and histological analysis after surgery. Among the three scaffolds, only the PLLA/Pearl/RM-P scaffold had anti-infection and bone defect repair in vivo. 3D printing provides support for personalized scaffold structures, and composite materials ensure that the scaffolds exert anti-infection and bone repair effects. Our study suggests that the PLLA/Pearl/RM-P scaffold is a promising new material in the clinical treatment of infected bone defects.

Indication the mechanism of dual-functional scaffold in the treatment of infected bone defects.     

LAPONITE® nanorods regulating degradability,acidic-alkaline microenvironment,apatite mineralization and MC3T3-E1 cells responses to poly(butylene succinate) based bio-nanocomposite scaffolds

Novel bio-nanocomposite scaffolds for bone tissue engineering were prepared by incorporation of LAPONITE® (LAP) nanorods into poly(butylene succinate) (PBSu). The results showed that the scaffolds had well interconnected macroporous structures with macropore size in the range of 200–400 μm and porosity of around 70%. In addition, the water absorption, degradability and apatite mineralization ability of the scaffolds were clearly enhanced with the increase of LAP content. Moreover, the degradation of LAP produced alkaline products, which neutralized the acidic degradable products of PBSu, and formed a weak alkaline microenvironment similar to a biological environment. Furthermore, the adhesion, proliferation and differentiation of MC3T3-E1 cells onto the scaffolds were significantly promoted with the increase of LAP content, in which the scaffold with 30 wt% LAP (sPL30) exhibited the best stimulation effect on the cells responses. The results suggested that the promotion of cells responses could be ascribed to the improvements of surface characteristics (including roughness, hydrophilicity, ions release and apatite formation, etc.) of the scaffolds. The sPL30 scaffold with excellent biocompatibility, bioactivity and degradability had great potential for applications in bone tissue engineering.

PBSu/LAP bio-nanocomposite scaffolds were prepared, and the sPL30 scaffolds significantly stimulated cell adhesion and proliferation.     

MgCHA particles dispersion in porous PCL scaffolds: in vitro mineralization and in vivo bone formation

In this work, we focus on the in vitro and in vivo response of composite scaffolds obtained by incorporating Mg,CO₃‐doped hydroxyapatite (HA) particles in poly(ε‐caprolactone) (PCL) porous matrices. After a complete analysis of chemical and physical properties of synthesized particles (i.e. SEM/EDS, DSC, XRD and FTIR), we demonstrate that the Mg,CO₃ doping influences the surface wettability with implications upon cell–material interaction and new bone formation mechanisms. In particular, ion substitution in apatite crystals positively influences the early in vitro cellular response of human mesenchymal stem cells (hMSCs), i.e. adhesion and proliferation, and promotes an extensive mineralization of the scaffold in osteogenic medium, thus conforming to a more faithful reproduction of the native bone environment than undoped HA particles, used as control in PCL matrices. Furthermore, we demonstrate that Mg,CO₃‐doped HA in PCL scaffolds support the in vivo cellular response by inducing neo‐bone formation as early as 2 months post‐implantation, and abundant mature bone tissue at the sixth month, with a lamellar structure and completely formed bone marrow. Together, these results indicate that Mg²⁺ and CO₃^2– ion substitution in HA particles enhances the scaffold properties, providing the right chemical signals to combine with morphological requirements (i.e. pore size, shape and interconnectivity) to drive osteogenic response in scaffold‐aided bone regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

应用纳米晶胶原基骨体外构建组织工程骨

目的:应用组织工程技术,建立体外诱导人骨髓间充质干细胞,与纳米晶羟基磷灰石/胶原骨共培养的人工骨模型,探求细胞与纳米晶胶原基骨结合的最佳模式。方法:实验于2005-09/2006-09在江苏大学医学技术学院中心实验室完成。①实验材料:纳米晶胶原基骨材料由清华大学材料系崔福斋等人研制。骨髓取自骨科取髂骨患者(男性,30岁,患者知情同意)。②实验干预:全骨髓法体外培养骨髓间充质干细胞并扩增,应用成骨诱导剂诱导向成骨细胞表型转化。取纳米晶胶原基骨材料经60Co照射灭菌,无水乙醇疏水化,三蒸水洗去乙醇等处理制备支架材料。细胞与支架材料复合培养。③实验分组:骨髓间充质干细胞培养至第3代后分为两组:复合材料后加成骨诱导剂组:骨髓间充质干细胞先与纳米晶胶原基骨复合培养3d后再加入成骨诱导剂;成骨细胞复合材料组:骨髓间充质干细胞在诱导成骨细胞后与纳米晶胶原基骨复合培养。④实验评估:记录第3代骨髓间充质干细胞生长曲线。将诱导后细胞分别进行碱性磷酸酶染色、Von Kossa染色。扫描电镜比较两组复合物经体外孵育2周后,细胞在其中生长情况。结果:①细胞生长情况:倒置显微镜下观察原代培养的骨髓细胞增殖迅速,基本都呈形态均一成纤维样细胞,10～12d左右即可长满,并可稳定传代,传代细胞7～9d即可传代。诱导培养后的细胞呈现典型的成骨细胞形态和生物学特征。构建出骨髓间充质干细胞与骨组织共培养的模型。②体外复合后细胞生长及基质分泌情况:细胞可在纳米晶胶原基骨内表面良好贴壁,复合材料后加成骨诱导剂组,细胞贴附,但成骨细胞少见;成骨细胞复合材料组,细胞数量明显较前者多。复合培养8d,分布于支架材料上的细胞大量增殖、分泌细胞外基质。第14天,大量细胞在材料表面和孔隙中生长。细胞之间广泛存在突起连接,局部有胶原分泌,且可见成骨细胞形成。结论:证实纳米晶胶原基骨适合种子细胞的贴附、生长和增殖,经成骨诱导后的细胞较骨髓间充质干细胞更易与材料贴附、增殖。     

构建壳聚糖-磷酸钙/重组人骨形态发生蛋白2复合材料及在体内的异位成骨

背景：研究表明,重组人骨形态发生蛋白2/壳聚糖-磷酸钙支架复合体具有良好的细胞相容性,且材料内部的多孔结构和孔径也能满足于细胞长入和骨的再生,但要应用于临床还需证明能否体内成骨。目的：构建壳聚糖-磷酸钙与重组人骨形态发生蛋白2的复合材料,并观察其植入兔肌袋模型后的异位成骨能力。设计、时间及地点：观察性实验,于2007-06/08在暨南大学附属第一医院中心实验室、外科实验室及暨南大学理工学院材料科学与工程系实验室完成。材料：将固相的磷酸三钙粉末及液相的壳聚糖溶液在室温下混合,制备壳聚糖-磷酸钙支架。再将壳聚糖-磷酸钙支架放入重组人骨形态发生蛋白2溶液中浸泡,真空抽吸后冷冻干燥,制备壳聚糖-磷酸钙材料/重组人骨形态发生蛋白2复合材料。方法：测量改良后支架的孔隙率及抗压强度,扫描电镜观察其超微结构;20只新西兰大白兔局部麻醉后在左后肢大腿外侧切口,暴露大腿肌肉,分离形成肌袋。随机分为3组,空白对照组5只不植入材料,单纯壳聚糖-磷酸钙材料组5只、壳聚糖-磷酸钙/重组人骨形态发生蛋白2复合材料组10只分别植入壳聚糖-磷酸钙材料、壳聚糖-磷酸钙/重组人骨形态发生蛋白2复合材料。主要观察指标：在植入后4周,通过大体观察、X射线摄片、组织学检测其体内异位成骨能力及生物反应性。结果：制备的复合重组人骨形态发生蛋白2后的壳聚糖-磷酸钙支架的孔隙率为87%,压缩弹性模量为20MPa。扫描电镜结果显示支架材料内部为多孔结构,孔径超过100μm。空白对照组苏木精-伊红染色可见肌肉纤维间少量淋巴细胞浸润。单纯壳聚糖-磷酸钙材料组大体观察可见材料周围有薄层的结缔样组织包裹,材料与周围肌肉连接松散,苏木精-伊红染色可见材料植入区为圆形空腔。壳聚糖-磷酸钙/重组人骨形态发生蛋白2复合材料组大体观察可见材料周围有薄层的结缔样组织包裹,材料中间可见空洞,有降解现象及少量淡红色纤维样结构长入,X射线示左大腿内材料高密度影无明显改变,苏木精-伊红染色可见少量血管样组织长入,Masson三色染色可见编制骨岛中有胶原纤维形成。结论：壳聚糖-磷酸钙/重组人骨形态发生蛋白2复合材料具有良好的孔隙率、抗压强度及超微三维结构,复合材料在兔体内具有异位成骨能力。     

血管化人工骨的体外构建和体内异位成骨

背景:血管化在骨形成和改建中起重要作用,目前大的组织块难以获得充足的氧气和营养供应,因此组织工程中就出现了需要适当血管化的问题.目的:建立体外血管化人工骨模型并且体内异位成骨的实验体系.方法:分离培养鼠骨髓间充质干细胞和肾血管内皮细胞,体外构建鼠骨髓间充质干细胞和鼠肾血管内皮细胞直接接触培养血管化人工骨组,以间接接触三维培养体系和两种细胞单独培养体系作为对照.体外通过测定蛋白质含量,碱性磷酸酶活性和骨钙素,分析骨髓间充质干细胞在不同混合培养模型中的成骨能力.建立鼠骨髓间充质干细胞和肾血管内皮细胞的三维培养体系,再将两种细胞直接接触组材料和骨髓间充质干细胞单独培养组的材料分别植入鼠左右腿肌肉内,通过软X射线摄影和苏木精-伊红染色检测分析不同植入方法的血管形成和成骨能力.结果与结论:当两种细胞混合培养时,具有很好的细胞兼容性.两种细胞单独培养碱性磷酸酶活性和骨钙素较间接接触混合培养降低,而两种细胞混合培养时,体系中碱性磷酸酶活性和骨钙素水平增加.动物实验结果显示在混合培养体系中的骨髓间充质干细胞的成骨能力高于单独培养(P < 0.05).体内实验表明在血管化人工骨中的软X射线骨密度,血管数量和新形成的骨量均高于对照组(P < 0.05).提示在两种细胞混合培养时骨髓间充质干细胞的成骨能力可以被细胞因子和细胞膜蛋白质调节,和传统的人工骨比较,血管化人工骨有加速骨髓间充质干细胞分化,增加了局部的血管微循环生存率,以及加速成骨和更好的抗感染能力.     

血管化人工骨的体外构建和体内异位成骨

背景：血管化在骨形成和改建中起重要作用,目前大的组织块难以获得充足的氧气和营养供应,因此组织工程中就出现了需要适当血管化的问题。目的：建立体外血管化人工骨模型并且体内异位成骨的实验体系。方法：分离培养鼠骨髓间充质干细胞和肾血管内皮细胞,体外构建鼠骨髓间充质干细胞和鼠肾血管内皮细胞直接接触培养血管化人工骨组,以间接接触三维培养体系和两种细胞单独培养体系作为对照。体外通过测定蛋白质含量,碱性磷酸酶活性和骨钙素,分析骨髓间充质干细胞在不同混合培养模型中的成骨能力。建立鼠骨髓间充质干细胞和肾血管内皮细胞的三维培养体系,再将两种细胞直接接触组材料和骨髓间充质干细胞单独培养组的材料分别植入鼠左右腿肌肉内,通过软 X 射线摄影和苏木精-伊红染色检测分析不同植入方法的血管形成和成骨能力。结果与结论：当两种细胞混合培养时,具有很好的细胞兼容性。两种细胞单独培养碱性磷酸酶活性和骨钙素较间接接触混合培养降低,而两种细胞混合培养时,体系中碱性磷酸酶活性和骨钙素水平增加。动物实验结果显示在混合培养体系中的骨髓间充质干细胞的成骨能力高于单独培养（P〈0.05）。体内实验表明在血管化人工骨中的软 X 射线骨密度,血管数量和新形成的骨量均高于对照组（P〈0.05）。提示在两种细胞混合培养时骨髓间充质干细胞的成骨能力可以被细胞因子和细胞膜蛋白质调节,和传统的人工骨比较,血管化人工骨有加速骨髓间充质干细胞分化,增加了局部的血管微循环生存率,以及加速成骨和更好的抗感染能力。     

生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸仿生复合支架的成骨及矿化效应

背景:研制具有结构与功能化仿生作用的骨修复替代材料,应在模拟体内细胞生长环境中进行.目的:观察生物活性玻璃,胶原蛋白/透明质酸/磷酸丝氨酸仿生复合支架材料植入体内后诱导成骨和促进矿化的能力.设计、时间及地点:随机对照动物实验,于2005-0612006-02在南方医科大学珠江医院血液科实验室完成.材料:生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸支架、生物活性玻璃,胶原蛋白支架和58S生物玻璃支架为自制.40只健康成年日本大耳白兔制造两侧桡骨10 mm骨缺损模型.干预:将40只模型兔随机分成4组,生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸组12只、生物活性玻璃,胶原蛋白组12只、58S生物玻璃组12只分别植入相应支架材料,空白对照组4只不植入任何物质.主要观察指标:检测植入材料2,4,8,12周后缺损部位X射线、硬组织切片、骨形成率和矿化沉积率.结果:40只模型兔80条桡骨全部进入结果分析.①术后所有动物伤口愈合良好,未发生骨折.②生物活性玻璃/胶原蛋白/透明质酸,磷酸丝氨酸组术后4周硬组织切片可见大量玫瑰红色新骨和绿色骨小梁形成,术后12周支架已基本由新生骨组织替代,哈弗系统形成:术后8周X射线显示骨皮质连接完整,12周缺损完全修复,髓腔基本再通.③生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸组术后4周的矿化沉积率和新骨形成速率比58S生物玻璃组高出了2.85倍和3.16倍,且明显优于生物活性玻璃/胶原蛋白组(P＜0.001).结论:生物活性玻璃/胶原蛋白/透明质酸/磷酸丝氨酸仿生复合支架在诱导成骨和促进生物矿化方面性能优越,其矿化机制有待进一步观察探讨.     

骨髓基质细胞与快速成形仿生人工骨支架载体的体外附着实验

目的：探讨具有良好生物相容性的聚乳酸-羟基乙酸与磷酸三钙人工合成仿生复合材料骨移植支架材料的可行性.方法：实验于2004-11在温州医学院附属第二医院完成.新西兰兔,雌性,体质量2.5～3.0 kg.仿生人工骨材料为清华大学机械学院快速成形实验室提供,将其剪成2 mm&;#215;4 mm&;#215;1 mm大小,750 mL/L乙醇浸泡消毒30 min,紫外灯照射2 h灭菌,烘干备用.在新西兰兔股骨上端,抽取约1.5 mL骨髓,进行骨髓基质细胞的分离和培养.将第3代骨髓基质细胞以4&;#215;1010L-1细胞密度接种于仿生人工骨材料中,第7天取材,透射显微镜及其扫描电镜下观察细胞粘附情况.结果：仿生人工骨复合物表面及其孔隙内长满细胞,透射显微光镜下可见细胞覆盖的光照影.扫描电镜下观察：3 d时,仿生人工骨复合物载体表面细胞生长密集,胞体呈不规则的球形、梭形或多角形,从胞体生出的细胞突长短不一,有的与其他细胞相连.第7天时细胞在孔洞中长满,有大量的胶原纤维分泌.结论：快速成形仿生人工骨复合材料与骨髓基质细胞有较好的生物相容性,具备作为细胞支架骨移植材料的性质.     

骨髓基质细胞与快速成形仿生人工骨支架载体的体外附着实验

目的:探讨具有良好生物相容性的聚乳酸-羟基乙酸与磷酸三钙人工合成仿生复合材料骨移植支架材料的可行性。方法:实验于2004-11在温州医学院附属第二医院完成。新西兰兔,雌性,体质量2.5～3.0kg。仿生人工骨材料为清华大学机械学院快速成形实验室提供,将其剪成2m m×4m m×1mm大小,750mL/L乙醇浸泡消毒30m in,紫外灯照射2h灭菌,烘干备用。在新西兰兔股骨上端,抽取约1.5mL骨髓,进行骨髓基质细胞的分离和培养。将第3代骨髓基质细胞以4×1010L-1细胞密度接种于仿生人工骨材料中,第7天取材,透射显微镜及其扫描电镜下观察细胞粘附情况。结果:仿生人工骨复合物表面及其孔隙内长满细胞,透射显微光镜下可见细胞覆盖的光照影。扫描电镜下观察:3d时,仿生人工骨复合物载体表面细胞生长密集,胞体呈不规则的球形、梭形或多角形,从胞体生出的细胞突长短不一,有的与其他细胞相连。第7天时细胞在孔洞中长满,有大量的胶原纤维分泌。结论:快速成形仿生人工骨复合材料与骨髓基质细胞有较好的生物相容性,具备作为细胞支架骨移植材料的性质。     

Study on antibacterial properties and cytocompatibility of EPL coated 3D printed PCL/HA composite scaffolds

Biomaterial scaffolds play a critical role in bone tissue engineering. Moreover, 3D printing technology has enormous advantage in the manufacture of bioengineering scaffolds for patient-specific bone defect treatments. In order to provide an aseptic environment for bone regeneration, ε-poly-l-lysine (EPL), an antimicrobic cationic polypeptide, was used for surface modification of 3D printed polycaprolactone/hydroxyapatite (PCL/HA) scaffolds which were fabricated by fused deposition modeling (FDM) technology. The scaffold morphology and micro-structure were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and transform infrared spectroscopy (FT-IR). The release profile surface roughness, open porosity, and mechanical properties of the scaffolds were evaluated. Cell adhesion, proliferation, differentiation potential and antibacterial properties were also examined. As a result, 3D printed PCL/HA scaffolds with interconnected pores showed a slightly rough surface and improved mechanical properties due to adding hydroxyapatite (HA) particles. After being modified by EPL, favorable biocompatibility and osteoconductivity of ε-poly-l-lysine/polycaprolactone/hydroxyapatite (EPL/PCL/HA) scaffolds were observed. Moreover, antibacterial activity of the EPL/PCL/HA scaffolds was apparent. As a consequence, the EPL/PCL/HA scaffolds had great potential for bone regeneration and prevention of infections. This would yield a patient-specific bioactive and antibacterial composite scaffold for advanced bone tissue engineering applications.

Biomaterial scaffolds play a critical role in bone tissue engineering.     

纳米羟基磷灰石／壳聚糖支架与诱导骨髓间充质干细胞体外构建软骨支架复合体

背景:国内外许多研究通过不同的构建方法构建组织工程化软骨支架复合体修复骨软骨联合缺损,且取得了一定的进展,但目前各种方法存在的问题突出表现为组织工程化的软骨和骨组织之间的界面、移植体和宿丰骨和软骨之间的界面耦合不够理想.目的:将体外提纯、扩增的骨髓间充质下细胞诱导成软骨细胞.将其接种于穿"靴"的纳米羟基磷灰石/壳聚糖支架的底部上联合培养,探索其用于组织工程软骨复合体的可行性.设计、时间及地点:细胞和材料复合的体外观察实验,于2008-03/07在暨南大学附属第一医院中心实验室和暨南大学理工学院材料系实验室完成.材聿斗:通过原位复合和冷冻干燥结合的方法制各纳米羟基磷灰石/壳聚糖支架.健康新西兰兔10只由广东省医学实验动物中心提供.方法:密度梯度离心法提取分离骨髓间充质干细胞,软骨诱导液诱导骨髓闻充质干细胞2周后甲苯胺蓝染色检测.把诱导得到的软骨细胞接种于穿"靴"的纳米羟基磷灰石/壳聚糖支架的底部,将细胞-支架复合物置入成软骨条件培养液中培养2周.主要观察指标:倒置相差显微镜下观察细胞形态特征,鉴定CD29,CD44,CD34和CD45抗原的表达,观察细胞生长特性,测定细胞活力和生长周期,扫描电镜观察纳米羟摹磷灰石/壳聚糖史架结构和细胞与支架的复合情况.结果:骨髓间充质干细胞可在体外分离扩增,表达CD29和CD44,不表达CD34和CD45,细胞活力为95.27%,G0～G1期细胞占94.68%.经软骨诱导液诱导后骨髓问充质十细胞转化成软骨细胞;制备的纳米羟基磷灰石/壳聚糖多孔支架孔隙率为90%,平均孔径为150μm,与软骨细胞有较好的黏附性.结论:初步证实纳米羟基磷灰石,壳聚糖支架与骨髓间充质干细胞诱导成的软骨细胞复合可以在体外构建组织工程软骨复合体.     

Visible-light photocatalytic performance,recovery and degradation mechanism of ternary magnetic Fe3O4/BiOBr/BiOI composite

The ternary magnetic Fe₃O₄/BiOBr/BiOI (x : 3 : 1) photocatalysts were successfully synthesized by a facile solvothermal method. The samples were characterized by XRD, SEM, EDS, ICP-AES, XPS, UV-vis DRS, PL and VSM. Nitrogen-containing dye RhB was used as a degradation substrate to evaluate the photocatalytic degradation activities of the samples. The photocatalytic performance of Fe₃O₄/BiOBr/BiOI (0.4 : 3 : 1) is superior to other Fe₃O₄/BiOBr/BiOI (x : 3 : 1). Compared with binary magnetic Fe₃O₄/BiOBr (0.5 : 1) prepared in our previous work, the Fe₃O₄/BiOBr/BiOI (0.4 : 3 : 1) has obvious advantages in photocatalytic activity and adsorption capacity. And the specific surface area (48.30 m² g⁻¹) is much larger than that of the previous report (Fe₃O₄/BiOBr/BiOI (0.5 : 2 : 2)) synthesized by a co-precipitation method. Besides, after 25 s of magnetic field, Fe₃O₄/BiOBr/BiOI (0.4 : 3 : 1) can be rapidly separated from water. After eight recycling cycles, the magnetic properties, photocatalytic activity, crystallization and morphology of the Fe₃O₄/BiOBr/BiOI (0.4 : 3 : 1) catalyst remain good. The possible photocatalytic degradation mechanism of RhB under Fe₃O₄/BiOBr/BiOI (0.4 : 3 : 1) photocatalyst was also proposed. The results indicate that the ternary magnetic Fe₃O₄/BiOBr/BiOI (0.4 : 3 : 1) composite with high photocatalytic degradation efficiency, good magnetic separation performance and excellent recyclability and stability has potential application prospect in wastewater.

The ternary magnetic Fe₃O₄/BiOBr/BiOI (x : 3 : 1) photocatalysts were successfully synthesized by a facile solvothermal method.     

蚕丝支架与3T3-L1前脂肪细胞生物相容性的体外实验(英文)

背景:蚕丝是天然制品,其力学性能及生物相容性优于传统人工合成的可降解高分子材料,在医疗领域中已获得了广泛的应用而受到关注.目的:观察蚕丝对3T3-L1前脂肪细胞吸附作用及蚕丝对3T3-L1前脂肪细胞形态和功能的影响.方法:取原料蚕丝和用胰酶消化后的蚕丝任意缠绕成网状立体构型纤维条索,架空固定在自制的不锈钢支架上,支架网孔70～200 μm,厚200-300 μm,孔隙率为20%.消化后的蚕丝三维支架放入24孔培养板中,将浓度为6×10L~(-1)的3T3-L1前脂肪细胞悬液每孔滴入3滴,每孔细胞数量为1×10~7个.悬空孵育4 h,待细胞充分吸附在支架上后加入培养液,令细胞完全浸没,隔两三天换半液,培养1～4周.结果与结论:①倒置显微镜观察3T3-L1前脂肪细胞-蚕丝复合物可见细胞伸出细长的突起沿着蚕丝不断向前迁移延伸,细胞首尾相互融合,渐渐连成一片分布于蚕丝网眼内.②扫描电镜观察3T3-L1前脂肪细胞-蚕丝复合物可见细胞与支架紧密贴附,适度伸展,并有基质分泌.提示蚕丝对3T3-L1前脂肪细胞具有良好的吸附作用,并能维持3T3-L1前脂肪细胞正常形态和功能.     

以海藻酸盐及异种骨复合技术制备组织工程化骨及体内成骨(英文)

背景:海藻酸有相对温和的凝胶条件与良好的生物相容性,已广泛应用于生物组织工程.目的:采用海藻酸钠凝胶复合异种骨的方法,构建骨组织工程载体,观察载体中细胞的生物性能及体内成骨能力.方法:取2只2周龄新西兰兔的骨髓,以1 ×10~(-8)mol/L重组人骨形态发生蛋白2诱导骨髓间充质干细胞.取诱导后第2代骨髓间充质干细胞接种于1%海藻酸钠凝胶中,培养4 d苏木精-伊红染色观察凝胶中细胞形态.将第2代骨髓间充质干细胞分为单纯DMEM凝胶组和含1%海藻酸钠的DMEM凝胶组,分别培养7 d后行骨形态发生蛋白2免疫组织化学染色观察.取24只裸鼠,随机分为2组,于两侧股部肌袋中分别植入骨髓间充质干细胞,海藻酸钠凝胶/牛松质骨复合体作为实验组,骨髓间充质干细胞,牛松质骨复合体作为对照组.术后2,4周后组织学观察复合体成骨情况,图像分析系统分析各组成骨或软骨的面积百分比.结果与结论:海藻酸钠凝胶中骨髓间充质干细胞形态饱满,细胞悬浮于凝胶中,可见细胞分裂和核分裂相.单纯DMEM凝胶组和含1%海藻酸钠的DMEM凝胶组免疫组织化学观察,细胞分裂增殖正常,伸出多种形态的突起,胞核大,核仁清晰.单纯DMEM凝胶组和含1%海藻酸钠的DMEM凝胶组的骨形态发生蛋白2表达阳性率差异无显著性意义(P>0.05).扫描电镜观察海藻酸钠凝胶均匀地复合于牛松质骨微孔中,不同平面均有细胞生长.动物实验显示术后2,4周实验组和对照组的成骨或软骨的面积百分比差异有显著性意义(P<0.05).提示以海藻酸钠凝胶,牛松质骨构建骨组织工程载体,合乎组织工程载体的超结构原理,能最大限度地承载细胞,生物性能好,对骨髓间充质干细胞增殖和成骨表型及相关的生物性能无不良影响,在体内成骨效率较高.     

Self-assembly of renal cells into engineered renal tissues in collagen/Matrigel scaffold in vitro

To tissue engineer a kidney is a formidable task because of the complex cell composition and structures in the kidney. This study reconstructed renal tissues using mixed renal cells in collagen/Matrigel® scaffolds in vitro. Neonatal rat renal cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During in vitro culture, the renal constructs were observed under microscope and analyzed by histological and immunofluorescent examinations. Results showed that the mixed renal cells reconstituted renal tubular and glomeruli‐like structures with different appearances at varying developmental stages. Tubular structures were formed by CK18‐positive cells with similar appearances lining the surrounding hollow centres. The glomeruli‐like structures were tufts of cell aggregates containing Flk‐1‐positive cells. These results show that neonatal rat renal cells self‐assembled into engineered renal tissues containing both tubules and glomeruli‐like structures when cultured in 3D collagen/Matrigel scaffold in vitro. Copyright © 2011 John Wiley & Sons, Ltd.     

复合胰岛素样生长因子 1 壳聚糖胶原支架与人牙周膜细胞的增殖简

背景：胰岛素样生长因子1具有促进成纤维细胞有丝分裂的作用,同时具有促进牙周细胞生长、分化及合成细胞外基质的作用。 目的：观察负载胰岛素样生长因子1的壳聚糖胶原支架对于人牙周膜细胞增殖的作用。 方法：将人牙周膜细胞分别接种于负载胰岛素样生长因子1的壳聚糖胶原支架与普通胶原支架上,于接种的1 h、24 h及1周检测重组人转化生长因子β1的释放,于第1,7,28天检测两组细胞的黏附和增殖情况。结果与结论：负载胰岛素样生长因子1的壳聚糖胶原支架组第1,24小时和第1周的重组人转化生长因子β1释放率明显低于普通胶原支架组（P 0.05）,负载胰岛素样生长因子1的壳聚糖胶原支架组接种第7,28天的细胞黏附和增殖情况优于普通胶原支架组（P 〈0.01）。表明负载胰岛素样生长因子1的壳聚糖胶原支架可显著促进人牙周膜细胞的增殖。     

Enhancement of osteogenesis in vitro and in vivo by a novel osteoblast differentiation promoting compound, TAK-778.

TAK-778 [(2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxyamide; mw 505.53], a novel osteoblast differentiation promoting compound, was characterized in vitro and in vivo models. TAK-778 at doses of 10(-6) M and higher promoted potently bone-like nodule formation in the presence of dexamethasone in rat bone marrow stromal cell culture. This was accompanied by increases in cellular alkaline phosphatase activity, soluble collagen release, and osteocalcin secretion. Under the culture conditions, TAK-778 also stimulated the secretion of transforming growth factor-beta and insulin-like growth factor-I, indicating that TAK-778 may exert regulatory effects on osteoblast differentiation via autocrine/paracrine mechanisms. Furthermore, the in vivo osteogenic potential of TAK-778 was studied in bony defect and osteotomy animal models, using sustained release microcapsules consisted of a biodegradable polymer, poly (dl-lactic/glycolic) acid (PLGA). Single local injection of TAK-778/PLGA-microcapsules (PLGA-MC) (0.2-5 mg/site) to rat skull defects resulted in a dose-dependent increase in new bone area within the defects after 4 weeks. When the pellet containing TAK-778/PLGA-MC (4 mg/pellet) was packed into place to fill the tibial segmental defect in rabbit, this pellet induced osseous union within 2 months, whereas the placebo pellet did not. In addition, single local application of TAK-778/PLGA-MC (10 mg/site) to rabbit tibial osteotomy site enhanced callus formation accompanied by an increase in breaking force after 30 days. These results reveal for the first time that a nonendogenous chemical compound promotes potently osteogenesis in vitro and enhances new bone formation during skeletal regeneration and bone repair in vivo and should be useful for the stimulation of fracture healing.     

Composite clinoptilolite/PCL‐PEG‐PCL scaffolds for bone regeneration: In vitro and in vivo evaluation

In this study, clinoptilolite (CLN) was employed as a reinforcement in a polymer‐based composite scaffold in bone tissue engineering and evaluated in vivo for the first time. Highly porous, mechanically stable, and osteogenic CLN/PCL‐PEG‐PCL (CLN/PCEC) scaffolds were fabricated with modified particulate leaching/compression molding technique with varying CLN contents. We hypothesized that CLN reinforcement in a composite scaffold will improve bone regeneration and promote repair. Therefore, the scaffolds were analyzed for compressive strength, biodegradation, biocompatibility, and induction of osteogenic differentiation in vitro. CLN inclusion in PC‐10 (10% w/w) and PC‐20 (20% w/w) scaffolds revealed 54.7% and 53.4% porosity, higher dry (0.62 and 0.76 MPa), and wet (0.37 and 0.45 MPa) compressive strength, greater cellular adhesion, alkaline phosphatase activity (2.20 and 2.82 mg/g_DNA/min), and intracellular calcium concentration (122.44 and 243.24 g Ca/mg_DNA). The scaffolds were evaluated in a unicortical bone defect at anterior aspect of proximal tibia of adult rabbits 4 and 8 weeks postimplantation. Similar to in vitro results, CLN‐containing scaffolds led to efficient regeneration of bone in a dose‐dependent manner. PC‐20 demonstrated highest quality of bone union, cortex development, and bone‐scaffold interaction at the defect site. Therefore, higher CLN content in PC‐20 permitted robust remodeling whereas pure PCEC (PC‐0) scaffolds displayed fibrous tissue formation. Consequently, CLN was proven to be a potent reinforcement in terms of promoting mechanical, physical, and biological properties of polymer‐based scaffolds in a more economical, easy‐to‐handle, and reproducible approach.