Nanofibrous Scaffold Containing Osteoblast-Derived Extracellular Matrix for the Proliferation of Bone Marrow Mesenchymal Stem Cells

Extracellular matrix( ECM) plays a prominent role in establishing and maintaining an appropriate microenvironment for tissue regeneration. The aims of this study were to construct a tissue engineered scaffold by reconstituting osteoblast cell-derived ECM( O-ECM) on the electrospun nanofibrous scaffold,and further to evaluate its subsequent application for promoting the proliferation of bone marrow mesenchymal stem cells( BMSCs). To engineer a biomimetic scaffold, calvarial osteoblasts and electrospun poly-llactic acid( PLLA) nanofibers were prepared and subjected to decellularize for O-ECM deposition. To evaluate and characterize the O-ECM/PLLA scaffold, the morphology was examined and several specific mark proteins of osteoblasts matrix were evaluated.Furthermore,the cell counting kit-8( CCK-8) assay was used to detect the proliferation of the BMSCs cultivated on the O-ECM/PLLA scaffold. The results indicated O-ECM/PLLA scaffold was loaded with Collagen I, Fibronectin, and Laminin, as the composition of the marrow ECM. After decellularization,O-ECM deposition was observed in O-ECM/PLLA scaffold. Moreover,the O-ECM/PLLA scaffold could significantly enhance the proliferation of BMSCs,suggesting better cytocompatibility compared to the other groups tested. Taken together,a biomimetic scaffold based on the joint use of O-ECM and PLLA biomaterials,which represents a promising approach to bone tissue engineering, facilitates the expansion of BMSCs in vitro.     

仿生增强制备聚乳酸基骨组织工程复合材料

依据仿生原理制备了纳米羟基磷灰石聚乳酸(nHA-PLA)复合的骨框架材料.此复合材料中的主要成分是纳米羟基磷灰石,纳米相的羟基磷灰石就是天然骨中主要的无机相.在保持高孔隙率(90%)的同时,复合材料的抗压性能达到2.07 MPa,高于单纯的聚乳酸框架材料(为0.89 MPa).分离成骨细胞并在三维框架材料上培养,用扫描电镜进行观察,复合材料具有很好的细胞贴附性能.仿生制备的三维纳米羟基磷灰石聚乳酸复合骨框架材料,无论从结构还是性能上,都是骨组织工程中的优选材料之一.     

磷酸钙与胶原双相多级仿生骨组织工程支架研究

磷酸钙与胶原是天然骨组织的重要组成成分,介绍了一种仿生设计磷酸钙与胶原双相复合的多级仿生骨组织支架.采用双氧水发泡技术精确定制磷酸钙支架孔结构,结合真空灌注胶原以及仿生矿化技术,构建磷酸钙,胶原双相多级仿生骨组织支架,材料的孔结构及化学组分可实现定制设计.通过对支架材料测试表征,结果显示,这种无机/有机/无机多级仿生支架材有良好的力学性能.材料的体外细胞实验结果证实,这种多级仿生支架材料具有良好的生物相容性.     

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

Objective: Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques. The aim of this study is to investigate the effect of connective tissue growth factor (CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs). Methods: A CTGF-expressing plasmid (pCTGF) was constructed and transfected into MSCs. Then expressions of bone morphogenesis-related genes, proliferation rate, alkaline phosphatase activity, and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs. Results: Overexpression of CTGF was confirmed in pCTGF-MSCs. pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs (P<0.05). CTGF induced a 7.5-fold increase in cell migration over control (P<0.05). pCTGF transfection enhanced the expression of bone matrix proteins, such as bone sialoprotein, osteocalcin, and collagen type I in MSCs. The levels of alkaline phosphatase (ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0- and 3.0-fold higher than those of MSCs cultured in OS-medium, significantly higher than those of mock-MSCs and normal control MSCs (P<0.05). Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules. Conclusion: Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs, and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.     

羚牛股骨密质骨力学性能的实验研究

为了对国家一级保护动物——羚牛的骨生物力学和临床医学提供必要的骨力学参数,对羚牛新鲜股骨密质骨的拉伸与压缩力学性能进行了实验研究,用光学显微镜和扫描电镜观测了其微观结构,并与同属牛科的家牛进行了比较。实验得到了羚牛新鲜股骨密质骨沿轴向压缩和拉伸弹性模量分别为5.41GPa和0.98GPa,密质骨的力学性能在股骨的不同位置差异不显著。扫描电镜的观察表明:羚牛股骨密质骨在微观结构上是由丛状骨和哈佛氏系统组成的,其中哈佛氏系统沿径向的分布比较平均。     

Preparation and evaluation of biomimetric nano-hydroxyapatite-based composite scaffolds for bone-tissue engineering

In the present study,novel biomimetic composite scaffolds with a composition similar to that of natural bone were prepared,using nano-hydroxyapatite,collagen,and phosphatidylserine.The scaffolds possess an interconnected porous structure with a porosity of 84%.The pore size ranges from several micrometers up to about 400 m.In-vitro studies in simulated body fluids showed that the morphologies of the products derived from mineralization can be regulated by the extracellular matrix components of the scaffolds;this in turn leads to creation of a large number of hydroxyapatite crystals on the scaffold surface.The regulatory properties of collagen and phosphatidylserine also influenced the cell response to the composite scaffolds.MC3T3-E1 cells attached and spread on the surfaces of the materials and interacted with the substrates;this may be the result of charged groups on the composite materials.Radiological analysis suggested that calluses and bone bridges formed in defects within 12 weeks.These composite scaffolds may therefore be a suitable replacement in bone-tissue engineering.     

Dexamethasone-Loaded PLGA Microspheres Incorporated PLLA/PLGA/PCL Composite Scaffold for Bone Tissue Engineering

The combination of micro-carriers and polymer scaffolds as promising bone grafts have attracted considerable interest in recent decades.The poly(L-lactic acid)/poly(lactic-co-glycolic acid)/polycaprolactone(PLLA/PLGA/PCL)composite scaffold with porous structure was fabricated by thermally induced phase separation(TIPS).Dexamethasone(DEX)was incorporated into PLGA microspheres and then loaded on the PLLA/PLGA/PCL scaffoldtopreparethedesiredcompositescaffold.The physicochemical properties of the prepared composite scaffold were characterized.The morphology of rat bone marrow mesenchymal stem cells(BMSCs)grown on scaffolds was observed using scanning electron microscope(SEM)and fluorescence microscope.The resultsshowedthatthePLLA/PLGA/PCLscaffoldhad interconnected macropores and biomimetic nanofibrous structure.In addition,DEX can be released from scaffold in a sustained manner.More importantly,DEX loaded composite scaffold can effectively support the proliferation of BMSCs as indicated by fluorescence observation and cell proliferation assay.The results suggested that the prepared PLLA/PLGA/PCL composite scaffold incorporating drug-loaded PLGA microspheres could hold great potential for bone tissue engineering applications.     

羟基磷灰石基生物材料培养破骨细胞研究现状

骨重建是新骨组织替换旧骨或受损骨的生理过程，在无瘢痕骨愈合和受损骨再生中起着必要的作用。骨重建主要是骨形成细胞如成骨细胞（osteoblast，OB），骨吸收细胞如破骨细胞（osteoclast，OC），和巨噬细胞（macrophage，MP）等细胞之间的相互协调活动。OC作为生物体内唯一多核细胞，负责生物体内骨质的脱钙与骨基质的吸收。自然骨具有一定硬度、韧性，良好的生物活性，保证骨重建中细胞的增殖、分化、骨形成或骨再吸收等活性。以羟基磷灰石（hydroxyapatite，HA）为主的磷酸钙（HA-CaPs）是自然骨中矿物质的主要成分，具有优异的生物性能而被广泛应用于骨组织修复领域。总结了HA-CaPs物相成分、表面形貌等对OC等骨吸收细胞增殖、分化和骨吸收活性的影响，并通过OC对于骨修复材料体内吸收机制及细胞活性调控机制等探讨HA-CaPs与细胞间相互关系，以期为磷酸钙人工骨替代材料更加广泛的生物应用作理论参考。     

Fabrication and biological evaluation of polyether ether ketone(PEEK)/bioceramic composites

The repair of vascularized bone defects represents a significantly clinical challenge, and vascular regeneration is one of the necessary factors to promote bone tissue regeneration. To effectively repair large bone defects, new bone tissue must regenerate with a rich vascular network. Therefore, the development of biomaterials that can promote the regeneration of vascularized bone tissue is currently receiving attention from researchers. In this study, Li–Ca–Si bioceramics (LCS) containing Li, Ca, and Si elements was developed, then LCS was compounded with PEEK to prepare PEEK+10% LCS, PEEK+20% LCS, PEEK+30% LCS, and the effect of LCS-PEEK composite biomaterials on the proliferation and angiogenic ability of human umbilical vascular endothelial cells (HUVECs) further explored by Cell Counting Kit-8 (CCK-8), scanning electron microscope (SEM), quantitative real-time PCR (QPCR), Western Blotting and enzyme linked immunosorbent assay (ELISA). The results showed that HUVECs inoculated on 30%LCS ?+ ?PEEK material exhibited the best proliferation ability. And the adhesion ability of endothelial cells on PEEK gradually increased with the increase of LCS contents. Furthermore, the angiogenic ability of HUVECs on LCS-PEEK composites was examined using QPCR and Western blotting, and the results showed that the expression of angiogenic-related genes and proteins of HUVECs on PEEK composites gradually increased with increasing LCS concentration. These results demonstrated that the angiogenic ability of HUVECs was effectively stimulated by LCS-modified PEEK materials. The present results indicate that the PEEK material can be modified with bioceramics to promote angiogenesis, and this study lay the foundation for the subsequent development of scaffolds that promote vascularized bone tissue regeneration.     

纳米仿生骨组织材料的生理响应及生物矿化

利用溶胶－凝胶法合成了两种具有纳米结构的新型高生物活性骨修复及骨组织工程支架材料，并利用体外实验方法（In Vitro)以及X－射线衍射（XRD），扫描电子显微镜（SEM），红外光谱（FTIR），氮气吸附－解吸（BET）和等离子发射光谱（ICP）等技术对材料的显微结构及其在模拟生理溶液（SBF）中的降解过程，表面反应产物及生物矿化机理进行了研究，研究表明：两种溶胶－凝胶材料均具有较高的生物活性；由于化学组成不同，它们在SBF溶液中的离子扩散规律及生物矿化行为有所不同。     

Toll样受体信号通路的3条途径在大鼠肝再生中抑制库普弗细胞免疫反应

为从基因转录水平了解大鼠肝再生中Toll样受体信号通路调节库普弗细胞免疫反应的途径和方式,首先建立大鼠2/3肝切除(partial hepatectomy,PH)模型,从中分离库普弗细胞进行基因微阵列分析.发现Toll样受体信号通路的23个基因及其调节免疫反应的62个基因与大鼠肝再生相关.基因协同作用(Ep(t)值)和同类提取法分析表明,大鼠肝再生进展阶段,Toll样受体信号通路通过TLR/NF-κB,TLR/MAPK,TLR/IRF等3条途径和TLR4,MAL,UNC93B1,AP1S2,IRF1等5个关键基因抑制库普弗细胞免疫反应.     

Comparative study of microstructural remodification to porous β-TCP and HA in rabbits

To assess the remolding ability of repaired bone in hydroxyapatite （HA） and β-calcium phosphate （β-TCP） scaffold, two 75% porosity bioceramics with the same three-dimensional geometry were implanted into femoral condyles of rabbits. Histological and micro-computed tomography （micro-CT） results demonstrated abundant new bone formation in the porous HA scaffold along with indistinctive scaffold degradation. Results also indicated that scaffold resorption in the β-TCP group, which was followed by a replacement with newly formed bone, was significantly higher than that in the HA group. The crosslinking trabeculae remodeled from the mixtures of the newly formed bone and β-TCP scaffold remnants might be helpful to promoting even loading and reducing stress. The bone remodeling pattern resulted from bone formation and scaffold resorption was significantly different for the two bioceramics. The results demonstrated that the 75% porous β-TCP was more suitable for new bone remodification than HA scaffold.     

Incorporation of Sphingosine 1-Phosphate Loaded Mesoporous Silica Nanoparticles into Poly( L-lactic acid ) Nanofibrous Scaffolds for Bone Tissue Engineering

Controlled release of the functional factors is the key to improve clinical therapeutic efficacy during the tissue repair and regeneration.The three-dimensional(3D)scaffold can provide not only physical properties such as high strength and porosity but also an optimal environment to enhance tissue regeneration.Sphingosine1-phosphate(S1P),an angiogenic factor,was loaded into mesoporous silica nanoparticles(MSNs)and then incorporated into poly(L-lactic acid)(PLLA)nanofibrous scaffold,which was fabricated by thermally induced phase separation(TIPS)method.The prepared scaffolds were examined by attenuated total reflection Fourier transform infrared spectroscopy(ATR-FTIR),scanning electron microscopy(SEM),and transmission electron microscopy(TEM)and compressive mechanical test.The ATR-FTIR result demonstrated the existence of MSNs in the PLLA nanofibrous scaffold.The SEM images showed that PLLA scaffold had regular pore channel,interconnected pores and nanofibrous structure.The addition of MSNs at appropriate content had no visible effect on the structure of scaffold.The compressive modulus of scaffold containing MSNs was higher than that of the scaffold without MSNs.Furthermore,fluorescein isothiocyanate(FITC)was used as model molecule to investigate the release behavior of S1P from MSNsincorporated PLLA(MSNs/PLLA)nanofibrous scaffold.The result showed that the composite scaffold largely reduced the initial burst release and exhibited prolonged release of FITC than MSNs.Thus,these results indicated that S1P-loaded composite nanofibrous scaffold has potential applications for bone tissue engineering.     

Biological and mechanical investigation of novel flax/silk protein-based nanofibrous scaffold for bone regeneration

The process of bone tissue regeneration involves an intricate network of a biological macromolecules that includes proteins and peptides to support stimulation and healing response. Here, we have fabricated a novel flaxseed/silk fibroin based (dual protein) composite nanofibers using β-TCP to improve the biological properties and promote cartilage tissue regeneration along with a controllable rate of biodegradation. The physiochemical properties of PVA/β-TCP/FP:SF were characterized by FTIR, XRD, SEM, porosity, contact angle measurements and mechanical evaluation etc. This study demonstrates the biological performance of the developed nanofibers by using hemocompatibility, biodegradation ability, and apatite deposition in stimulated body fluid. Moreover, the cell viability assay was performed on MG-63 osteoblast cells and long-term antibacterial rate against E. coli and S. aureus was analysed. Docking results of flax protein and silk fibroin protein further confirmed the stable modes of intramolecular bonding and the most stable electrostatic values using theoretical calculations. Taken together, the experimental results proved that the dual protein-based fibrous scaffold was found to be up-regulated in terms of biological activity with enhanced cell adhesion and proliferation.     

面向组织工程的多细胞结构三维自组装成形方法的研究进展

多细胞结构是由细胞群体构成的有机体,其体外构建对于组织工程和再生医学的发展具有重要的基础意义。利用细胞自身的自组织特性构建三维(3D)多细胞结构正成为生物制造和组织再生的一种重要途径,并受到越来越多的关注。对三维多细胞结构的自组装式构建与调控的相关基础研究及关键技术进行了综述及分析,主要涉及凝胶内3D细胞培养、多细胞结构可控形成,及其与图灵反应-扩散机制的联系等方面的研究工作。为进一步研究多细胞3D自组装机理,使得该自组装过程可控,且满足同时调控外部施加和细胞自身分泌的作用因子的浓度梯度分布的需求,提出对内部结构特征梯度化的3D凝胶体内细胞3D自组装模型进行研究,以推进三维多细胞结构及组织前体形成的理性调控技术。     

Fabrication and characterization of a PAM modified PHBV/BG scaffold

In order to improve the bioactivity and mechanical strength of the scaffold used in bone repair simultaneously, a novel porous PAM-poly （β-hydroxybutyrate-co-β-hydroxyvalerate） （PHBV）/bioactive glass （BG） scaffold was prepared by photo-initiated polymerization. PAM was used to improve the hydrophilicity of PHBV matrix while the BG particles were added to increase the bioactivity and strength of the matrix synchronously. The grafted amide group and Si-O moieties from acrylamide and the added BG were confirmed by Fourier Transform Infrared Spectrometry （FTIR）. The micromorphology of the scaffolds before and after grafting was observed by scanning electron microscopy （SEM）. The resulting images demonstrate that the PAM-PHBV/BG scaffold has a well connected pore structure and appropriate pore size which may be convenient for cells to grow and discharge metabolites. The specific gravity method was used to evaluate the pore property of the scaffold and the result shows that the scaffold has an average porosity up to 82.0%. Mercury intrusion porosimetry （MIP） indicated that the pores of PAM-PHBV/BG scaffold were mainly distributed between 75 and 150 μm. The compressive strength test was adopted to evaluate the mechanical property of the scaffold. The result shows that the PAM-PHBV/BG scaffold has a relatively high compressive strength （0.91 MPa） when compared with the pure PHBV scaffold. Besides, the properties of the pure PHBV scaffold, PHBV/BG scaffold were also evaluated. The newly prepared PAM-PHBV/BG scaffold may be worthy of further studying as a bone repair material.     

生物材料的3D打印研究进展

在生物医药领域,通过对生物材料或活细胞进行3D打印,可构建复杂生物三维结构如个性化植入体、可再生人工骨、体外细胞三维结构体、人工器官等,因而基于生物3D打印在个性化定制及复杂结构调控制造上的独特优势,综述了生物3D打印技术的基本工艺、应用领域与研究进展.重点针对3D打印生物材料这一研究热点,全面讨论了喷墨打印和注射挤出打印两种路径,分析总结了3D打印相关生物材料并应用于体外模型、医疗器械和植入体的制造以及可降解组织支架、细胞三维结构体的构建,最后对该技术未来发展趋势和研究重点提出展望.     

三维微流道支架直接压印成形方法

针对软质生物材料支架内部三维微结构成形难题,提出了结合分层制造、微压印与冷冻干燥技术的三维微流道支架直接压印成形方法.通过开发自动化成形设备,实现了材料溶液填充、微结构压印、结构预冻、层间黏结等工艺过程的可控化,从而解决了传统手工操作所造成的结构重复性差、成形效率低等问题.研究了模具表面等离子处理、亲水物质添加、改变溶液填充方式等工艺对自动化成形过程中微结构复型性能的影响.结果表明,向材料溶液中添加微量亲水物质并辅助溶液填充引导,可实现软质水溶性天然生物材料内部复杂微流道结构的精确三维压印成形.通过工艺装备保证了成形过程的自动化和稳定性,从而实现了支架微结构制造的可重复性与可控性.     

The multiple interactions between growth factors and microenvironment in vivo

The extracellular matrices (ECMs), which form the supportive meshwork around the cells, are largely composed of proteins and polysaccharide secreted by cells, while, growth factors, which almost all exist in ECMs, are polypeptides synthesized and secreted…     

Preparation of fiber-microsphere scaffolds for loading bioactive substances in gradient amounts

Gradient scaffolds are needed for interface tissue regeneration. In this study, a technique combining electrospinning and electrospraying was developed for preparing poly(L-lactide-co-glycolide) (PLGA) fiber-microsphere scaffolds for loading bioactive substances in gradient amounts. The gradient fiber-microsphere scaffolds contain two sheets of electrospun membranes and a sheet of microspheres loaded with bioactive substances in gradient amounts between the electrospun membranes. The morphologies of the gradient scaffolds were characterized and bovine serum albumin (BSA) was loaded as a model bioactive substance. The amount of BSA-loaded microspheres decreased gradually along the length of the gradient scaffold. The addition of poly (ethylene glycol) significantly improved the hydrophilicity of the gradient scaffold and the release behavior of BSA with respect to the gradient became apparent, with differences in the release amounts along the length of the gradient scaffold being observed. The biocompatibility of the gradient scaffold was verified using MC3T3-E1 pre-osteoblastic cells. The study demonstrated that the combination of electrospinning and electrospraying was a feasible method for the preparation of gradient scaffolds for potential applications in interface tissue engineering.