成骨细胞与多孔生物材料粘附的一种简便检测方法

成骨细胞与生物材料的相互作用是骨组织工程研究的主要领域，其中成骨细胞与生物材料的粘附(Cell adhesion)是基础［１］，成骨细胞必须与材料发生适当的粘附，才能进行迁移、分化和增殖［２］无论在体外，还是体内，直接也是最先与成骨细胞相接触并发生作用的是材料表面，因此成骨细胞与材料表面的粘附相当重要，而且粘附特性的差异还将影响成骨细胞的增殖、分化等一系列反应学者们渐渐认识到成骨细胞与材料的粘附是骨组织工程必须研究的基本问题［３］它不仅可以为骨组织工程研究筛选出适于细胞发挥生理功能的细胞外基质材料，而且…     

nHA/PCL复合材料对成骨细胞增殖的影响

研究了纳米羟基磷灰石/聚己内酯(nHA/PCL)复合材料对成骨细胞的增殖的影响.制备了3种不同的nHA/PCL材料浸提液(3种材料中nHA/PCL质量比分别为0∶100,20∶80和40∶60).采用四唑盐比色法(MTT)检测不同的nHA/PCL材料浸提液对成骨细胞增殖率的影响.应用流式细胞术检测复合材料对成骨细胞周期的影响.结果表明:与对照组相比,实验组细胞增殖率和细胞各时期细胞数量均无显著性差异(p>0.05);研究表明:nHA/PCL浸提液对成骨细胞的增殖无不良影响,具有良好的细胞的相容性,其中40%nHA/PCL具有促进成骨细胞增殖指数的作用.     

脱钙人牙基质复合胶原骨修复材料的制备与性能研究

本研究探讨脱钙人牙基质(Decalcification Tooth Matrices DTM)复合胶原材料作为骨修复材料的可行性.采用冻干后高温交联的方法制备脱钙人牙基质复合胶原材料;利用扫描电镜、X射线衍射、红外(IR)分析及电子能谱(EDS)分析材料表征;通过成骨细胞MCT3T-E1与材料混合培养分析材料的生物相容性及诱导骨细胞生长活性.结果显示材料分布均匀,形成多孔疏松的支架,其主要成分为羟基磷灰石晶体,材料不影响成骨细胞的分裂,且对细胞增殖有明显促进作用.研究表明脱钙人牙基质复合胶原材料具有骨修复材料的结构特点,且具有良好的细胞相容性及诱导成骨细胞增殖活性.     

纳米相陶瓷支架与人成骨细胞生物相容性的体外实验研究

研究了2种新型多孔纳米陶瓷支架材料(hydroxyapatite,HA和tricalcium phosphate,TCP)的生物相容性.从人4月龄胚胎骨膜组织中分离培养出骨膜来源的成骨细胞(periosteal-derived osteoblast,POB),采用人胚骨膜成骨细胞与2种陶瓷支架的体外复合培养,观察细胞在材料上的生长及生理功能表达情况.发现人POB在HA和TCP表面生长繁殖良好,并发挥成骨功能.材料对细胞的增殖还有一定的促进作用(p<0.05).实验表明: 2种新型多孔陶瓷支架材料均有良好的生物相容性.     

电刺激促进成骨细胞粘附特性的实验研究

针对骨组织工程中成骨细胞与基质材料间促粘附这一重要问题,研究微量直流电刺激对成骨细胞粘持性的影响。方法取兔骨髓基质干细胞来源的成骨细胞体培养,于接种即时行微量直流电刺激,刺激电流密度为１００μＡ／ｃｍ＾２,观察电刺激后１、２、６、１２ｈ及２４ｈ各时间点接种成骨细胞粘附情况、体视学计量粘附细胞数量。结果１００μＡ／ｃｍ＾２微量直流电刺激下,阳极区细胞粘附数量与阴极区相比,各时间点均寺加;无直流电刺激     

成骨细胞诱导骨髓基质细胞体外成骨的初步研究

目的:探讨在不使用细胞因子或化学药物的情况下,成骨细胞(Osteoblast,OB)与骨髓基质细胞(Bone Marrow Stromal Cells,BMSCs)混合培养时,成骨细胞提供的成骨微环境能否在体外诱导BMSCs向成骨细胞分化,并复合支架形成成熟的骨组织.研究成骨细胞诱导BMSCs有效成骨的最小比值(指成骨细胞与骨髓基质干细胞数量的比值).方法:SY别培养SD乳鼠的成骨细胞与SD大鼠的BMSCs,将成骨细胞和BMSCs以1:9、2:8、3:7、1:0的不同比例进行混合培养,通过测定第3、6、9天培养液上清中的碱性磷酸酶(ALP)的含量,研究成骨细胞促BMSCs有效成骨的最小比值.将两种细胞以该最小浓度比混匀接种于涂附Ⅰ型胶原壳聚糖材料支架上(直径9 mm,高3mm)作为混合培养组,相同终浓度的单纯成骨细胞和单纯BMSCs分别接种于相同支架作为阳性对照及阴性对照.另设置低比值成骨细胞对照组(仅含有共培养组中相同的成骨细胞数,但不含有共培养组中的BMSCs).全部标本均于体外培养8周后取材,通过大体观察、组织学及免疫组织化学等相关检测对新生骨进行评价.结果:成骨细胞和BMSCs以3:7的比例进行混合培养时已可实现有效成骨.3:7比例的混合培养组及阳性对照组(成骨细胞组)体外培养8周后大体观察和苏木素-伊红染色(HE)、ALP染色基本相同,均表达骨特异性细胞外基质Ⅰ型胶原,形成了较成熟的骨组织.阴性对照组(单纯BMSCs组)和低比值成骨细胞组,原细胞支架复合物变小、变形.低比值成骨细胞组在局部形成了少量的骨组织,阴性对照组(单纯BMSCs组)未能发现骨样组织形成.结论:在不使用细胞因子或化学药物的情况下,成骨细胞提供的成骨微环境能够在体外诱导BMSCs向成骨细胞分化并形成成熟的骨组织.混合细胞中成骨细胞与BMSCs的比例为3:7时是有效成骨的最小比值.     

PLGA多孔支架降解与成骨细胞相互作用的影响

研究聚丙交酯-乙交酯(PLGA)多孔支架降解和大鼠股骨成骨细胞生理活性之间的相互影响.将大鼠股骨成骨细胞接种于PLGA多孔支架上,观察4周降解时间内成骨细胞增殖活性、碱性磷酸酶(ALP)活性和钙浓度的变化以及PLGA相对分子质量损失、拉伸性能和压缩性能的变化.实验结果表明,支架降解初期,成骨细胞增殖活性较高,ALP活性和分泌钙的浓度较低;支架降解中后期,细胞增殖速度明显减慢,ALP活性和分泌钙的浓度明显下降.接种有细胞的支架降解过程中相对分子质量减小的速度快于对照组,力学拉伸强度和压缩杨氏模量低于对照组.支架材料中后期降解对细胞产生的影响大于降解初期,细胞附着于支架的生理活动加快了支架降解速度.     

Ti基六钛酸钾涂层复合材料生物相容性的实验研究

应用小鼠成骨细胞系OCT-1细胞对两种植入性材料(纯钛及钛基六钛酸钾涂层复合材料)进行体外细胞培养,比较二者的生物相容性.结果显示钛基六钛酸钾涂层复合材料的生物相容性和纯钛材料相当;连续培养可提高材料的生物相容性,材料表面的平整程度对其表面细胞生长状况起着重要的作用.     

兔成骨细胞的体外培养及生物学行为观察

以一月龄幼兔的颅顶骨为材料 ,采用组织块法 ,进行细胞的原代培养并传代之 .结果显示 :(1)原代细胞具有成骨细胞的形态特征 ,经 6次传代后细胞形态变化不明显 ;(2 )细胞具有较强的碱性磷酸酶 (AL P)活性且稳中有升 ;(3)细胞培养液和细胞内均含有骨钙素 ;(4)连续培养 1个月后 ,成骨细胞有矿化结节形成 ;(5 )骨片经 4次贴壁仍有细胞爬出且细胞形态相同 .该结果表明所培养的细胞具有成骨细胞的生物学行为 ,为骨细胞生物学和骨组织工程的研究提供了一种客观而有效的手段     

MACF1对成骨细胞微丝骨架和细胞力学性能的调节作用

为探索细胞骨架关键交联分子微管微丝交联因子1(microtubule actin crosslinking factor 1,MACF1)对成骨细胞微丝骨架和细胞力学性能的调节作用,以MACF1低表达的小鼠成骨细胞及其对照细胞为研究对象,通过细胞免疫荧光染色和激光扫描共聚焦显微镜,观察成骨细胞微丝骨架;运用微丝图像分析系统对微丝特性进行定量分析;采用原子力显微镜检测细胞弹性模量。结果发现,与对照组相比,MACF1低表达显著改变了小鼠成骨细胞的微丝分布角度,增加了成骨细胞的微丝长度与数量,显著减小了成骨细胞的刚度。研究结果为深入认识MACF1在成骨细胞中的功能奠定了实验基础,并为由成骨细胞功能改变引起的骨质疏松等骨骼疾病防治研究提供了新靶标。     

Structural basis of BMP signalling inhibition by the cystine knot protein Noggin

The interplay between bone morphogenetic proteins (BMPs) and their antagonists governs developmental and cellular processes as diverse as establishment of the embryonic dorsal-ventral axis, induction of neural tissue, formation of joints in the skeletal system and neurogenesis in the adult brain. So far, the three-dimensional structures of BMP antagonists and the structural basis for inactivation have remained unknown. Here we report the crystal structure of the antagonist Noggin bound to BMP-7, which shows that Noggin inhibits BMP signalling by blocking the molecular interfaces of the binding epitopes for both type I and type II receptors. The BMP-7-binding affinity of site-specific variants of Noggin is correlated with alterations in bone formation and apoptosis in chick limb development, showing that Noggin functions by sequestering its ligand in an inactive complex. The scaffold of Noggin contains a cystine (the oxidized form of cysteine) knot topology similar to that of BMPs; thus, ligand and antagonist seem to have evolved from a common ancestral gene.     

Identification of the haematopoietic stem cell niche and control of the niche size

Haematopoietic stem cells (HSCs) are a subset of bone marrow cells that are capable of self-renewal and of forming all types of blood cells (multi-potential). However, the HSC 'niche'--the in vivo regulatory microenvironment where HSCs reside--and the mechanisms involved in controlling the number of adult HSCs remain largely unknown. The bone morphogenetic protein (BMP) signal has an essential role in inducing haematopoietic tissue during embryogenesis. We investigated the roles of the BMP signalling pathway in regulating adult HSC development in vivo by analysing mutant mice with conditional inactivation of BMP receptor type IA (BMPRIA). Here we show that an increase in the number of spindle-shaped N-cadherin+CD45- osteoblastic (SNO) cells correlates with an increase in the number of HSCs. The long-term HSCs are found attached to SNO cells. Two adherens junction molecules, N-cadherin and beta-catenin, are asymmetrically localized between the SNO cells and the long-term HSCs. We conclude that SNO cells lining the bone surface function as a key component of the niche to support HSCs, and that BMP signalling through BMPRIA controls the number of HSCs by regulating niche size.     

Haversian bone-mimicking bioceramic scaffolds enhancing MSC-macrophage osteo-imunomodulation

The interaction between immune cells and bone forming cells plays a vital role in maintaining the homeostasis of the skeletal system, and is regulated by the three-dimensional structure of tissues. Whether the construction of biomaterials can activate or reproduce this spatial “cross-talk” between immune cells and bone forming cells in bone natural formation process is a prerequisite for successful fracture healing and bone regeneration. Herein, a bone marrow mesenchymal stem cells (MSCs)/macrophages-laden Haversian bone-mimicking bioceramic scaffold was successfully prepared through the biomimetic design of biomaterials and 3D printing technology. MSCs and macrophages were respectively distributed in the cancellous bone and Haversian canals of the scaffold to simulate the three-dimensional structure regulation of the cell spatial distribution and multiple intercellular interaction in natural bone tissue, and worked in concert to modulate the scaffold material-mediated osteo-immune microenvironment. The in vitro study revealed that the pro-inflammatory response of macrophages was more significantly inhibited when distributed with MSCs in the scaffolds at a cell ratio of 0.5:1 for co-culture, in comparison with multicellular culture at other ratios and unicellular culture. Meanwhile, MSCs exhibited the relatively high osteogenic potential, most likely via the activation of certain key signaling pathways mediated by macrophages-derived paracrine signaling mediators (OSM, BMP-2, and WNT10b). This work not only establishes a bionic platform for the regulation of multicellular osteo-immune response and regeneration but also offers a promising tissue-engineered biomimetic scaffold with improved immunomodulatory function for promoting bone tissue regeneration.     

Spatial bistability of Dpp-receptor interactions during Drosophila dorsal-ventral patterning

In many developmental contexts, a locally produced morphogen specifies positional information by forming a concentration gradient over a field of cells. However, during embryonic dorsal-ventral patterning in Drosophila, two members of the bone morphogenetic protein (BMP) family, Decapentaplegic (Dpp) and Screw (Scw), are broadly transcribed but promote receptor-mediated signalling in a restricted subset of expressing cells. Here we use a novel immunostaining protocol to visualize receptor-bound BMPs and show that both proteins become localized to a sharp stripe of dorsal cells. We demonstrate that proper BMP localization involves two distinct processes. First, Dpp undergoes directed, long-range extracellular transport. Scw also undergoes long-range movement, but can do so independently of Dpp transport. Second, an intracellular positive feedback circuit promotes future ligand binding as a function of previous signalling strength. These data elicit a model in which extracellular Dpp transport initially creates a shallow gradient of BMP binding that is acted on by positive intracellular feedback to produce two stable states of BMP-receptor interactions, a spatial bistability in which BMP binding and signalling capabilities are high in dorsal-most cells and low in lateral cells.     

The status of Wnt signalling regulates neural and epidermal fates in the chick embryo

The acquisition of neural fate by embryonic ectodermal cells is a fundamental step in the formation of the vertebrate nervous system. Neural induction seems to involve signalling by fibroblast growth factors (FGFs) and attenuation of the activity of bone morphogenetic protein (BMP). But FGFs, either alone or in combination with BMP antagonists, are not sufficient to induce neural fate in prospective epidermal ectoderm of amniote embryos. These findings suggest that additional signals are involved in the specification of neural fate. Here we show that the state of Wnt signalling is a critical determinant of neural and epidermal fates in the chick embryo. Continual Wnt signalling blocks the response of epiblast cells to FGF signals, permitting the expression and signalling of BMP to direct an epidermal fate. Conversely, a lack of exposure of epiblast cells to Wnt signals permits FGFs to induce a neural fate.     

Scaling of the BMP activation gradient in Xenopus embryos

In groundbreaking experiments, Hans Spemann demonstrated that the dorsal part of the amphibian embryo can generate a well-proportioned tadpole, and that a small group of dorsal cells, the 'organizer', can induce a complete and well-proportioned twinned axis when transplanted into a host embryo. Key to organizer function is the localized secretion of inhibitors of bone morphogenetic protein (BMP), which defines a graded BMP activation profile. Although the central proteins involved in shaping this gradient are well characterized, their integrated function, and in particular how pattern scales with size, is not understood. Here we present evidence that in Xenopus, the BMP activity gradient is defined by a 'shuttling-based' mechanism, whereby the BMP ligands are translocated ventrally through their association with the BMP inhibitor Chordin. This shuttling, with feedback repression of the BMP ligand Admp, offers a quantitative explanation to Spemann's observations, and accounts naturally for the scaling of embryo pattern with its size.     

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.     

Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells

Transformed, oncogenic precursors, possessing both defining neural-stem-cell properties and the ability to initiate intracerebral tumours, have been identified in human brain cancers. Here we report that bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, trigger a significant reduction in the stem-like, tumour-initiating precursors of human glioblastomas (GBMs). Transient in vitro exposure to BMP4 abolishes the capacity of transplanted GBM cells to establish intracerebral GBMs. Most importantly, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality that occur in 100% of mice after intracerebral grafting of human GBM cells. We demonstrate that BMPs activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human glioblastomas (GBMs). This is followed by a reduction in proliferation, and increased expression of markers of neural differentiation, with no effect on cell viability. The concomitant reduction in clonogenic ability, in the size of the CD133+ population and in the growth kinetics of GBM cells indicates that BMP4 reduces the tumour-initiating cell pool of GBMs. These findings show that the BMP-BMPR signalling system--which controls the activity of normal brain stem cells--may also act as a key inhibitory regulator of tumour-initiating, stem-like cells from GBMs and the results also identify BMP4 as a novel, non-cytotoxic therapeutic effector, which may be used to prevent growth and recurrence of GBMs in humans.     

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.     

利用快速成型技术制造人工生物活性骨

以快速成型、三维重构和生物学技术为基础，建立了新型人工生物活性骨工程化制造系统。该系统制作的人工骨外形与被替代骨一样，内部都具有模拟真实骨组织微管系统的三维网架结构，可通过骨生长因子和活体细胞的复合，使其具有生物活性，因此弥补了传统生物填充材料由于缺乏活性布无法实现骨诱导的缺陷。动物实验的结果表明，所制造的内部仿真结构，为人工骨的快速活化和新生骨细胞的三维并行生长提供了理想的空间条件，其中骨引导和骨诱导的双重机制可有效地加速骨组织的生长，从而证明了人工骨具有良好的生物学性能。