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.     

Immobilization of biomacromolecules on poly-L-lactide surface via a layer-by-layer method for the improving of its cytocompatibility to bone marrow stromal cells

Hyaluronic acid （HA） and chitosan （CS） were immobilized on the surface of poly-L-lactide （PLLA） by the following procedure： Firstly, PLLA was aminolyzed with 1, 6-hexanediamine, and part of the PLLA surface ester groups were converted to free amino groups. Then negatively charged hyaluronic acid and positively charged chitosan were deposited onto the surface of aminolyzed PLLA film in a layer-by-layer assembly manner. The effect of the layer-by- layer deposition was evaluated by ATRoFTIR spectroscopy, Raman spectroscopy and static contact angle measurements. The cytocompatibility of PLLA sample to bone marrow stromal cells （BMSCs） was improved after modification with chitosan and HA. The cell attachment, activity, and proliferation on CS/HA modified PLLA films were enhanced comparing with the control. The cells cultured on the modified PLLA samples excreted abundant cytoplasm and can differentiate to vascular smooth muscle （SM）-like （SM-like） cells. A macroporous three-dimensional PLLA scaffold was prepared by integrating both the technique of freeze-drying and particle leaching. Layer-by-layer modification by HA/CS and cell culture was also applied on this scaffold. The scaffold cultured with BMSCs for 2 weeks has been tested successfully in vivo as a patch for repairing the artificial incision on canine pulmonary artery.     

Hippocampal Neuron-Derived Extracellular Matrix Coated Nanofibrous Scaffold for Neural Tissue Engineering

The aim of this study is to prepare poly-L-lactide( PLLA) electrospun nanofibrous scaffolds coated with hippocampal neuron-derived extracellular matrix( N-ECM)and construct a novel neural tissue engineering scaffold.Neonatal rat hippocampal neurons were seeded on PLLA nanofibers,and then decellularized to derive a cell-free extracellular matrix loaded N-ECM/PLLA modified scaffolds. The morphology and ingredients of N-ECM/PLLA were observed by scanning electron microscopy( SEM) and immunofluorescence staining respectively, and the cytocompatibility of the composite scaffolds was characterized by cell count kit-8( CCK-8) assay. The N-ECM was clearly identified loading on scaffolds when being imaged via SEM and immunofluorescence staining results showed that the N-ECM was made up of fibronectin and laminin. Most importantly, compared with tissue culture polystyrene and pure scaffolds, N-ECM/PLLA scaffolds could effectively facilitate the proliferation of rat adrenal neuroma cells( PC12 cells),indicating their better cell compatibilities. Based on the combination of N-ECM and PLLA biomaterials,the present study has fabricated a unique and versatile neural tissue engineering scaffold,offering a new thought for future neural tissue engineering.     

Electrospun Nanofibers of Hydroxyapatite/Collagen/Chitosan Promote Osteogenic Differentiation of BMSCs

Bone tissue engineering, aiming at developing bone substitutes for repair and regeneration of bone defects instead of using autologous bone grafts, has attracted wide attention in the field of tissue engineering and regenerative medicine. Developing biomimetic biomaterial scaffolds able to regulate osteogenic differentiation of stem cells could be a promising strategy to improve the therapeutic efficacy. In this study, clectrospun composite nanofibers of hydroxyapatite/collagen/chitosan （ HAp/Col/CTS ） resembling the fibrous nanostructure and constituents of the hierarchically organized natural bone, were prepared to investigate their capacity for promoting bone mesenchymal stem cells （BMSCs） to differentiate into the osteogenic lineage in the absence and presence of the osteogenlc supplementation, respectively. Call morphology, proliferation and quantified specific osteogenic protein expression on the electrospun HAp/Coi/CTS scaffolds were evaluated in comparison with different controls including dectrospun nanofibrous CTS, HAp/CTS and tissue culture plate. Our remits showed that the nanofibrous HAp/Col/CTS scaffolds supported better spreading and proliferation of the BMSCs than other substrates （ P 〈 0.01 ）. Expressions of osteogenesis protein markers, alkaline phosphatase （ALP） and Col, were significantly upregulated on the HAp/Col/CTS than those on the CTS （P 〈0.01） and HAp/ CTS （P 〈 0. 05 ） scaffolds in the absence of the osteogeulc supplementation. Moreover, presence of osteogeulc supplementation also proved to enhance osteogeule differentiation of BMSCs on HAp/ Col/CTS scaffolds, indicative of a synergistic effect. This study highlights the potential of BMSCs/HAp/Col/CTS cell-scaffold system for functional bone repair and regeneration applications.     

Haemonectin, a bone marrow adhesion protein specific for cells of granulocyte lineage

There is substantial evidence that the haematopoietic microenvironment is crucial to the growth and differentiation of haematopoietic cells. This microenvironment is composed of stromal cells, soluble factors and extracellular matrix (ECM). We have shown that a complex extract of bone marrow ECM can stimulate the growth and differentiation of haematopoietic cells in vitro. Furthermore, the use of inhibitors or stimulators of ECM synthesis in long-term marrow culture affects cell proliferation. On a molecular level, however, the interactions between ECM and haematopoietic cells are not well understood. We have investigated the adhesion between specific bone marrow ECM components and haematopoietic cells, and found a protein, 'haemonectin', of relative molecular mass 60,000 in bone marrow ECM which is a lineage- and organ-specific attachment molecule for cells of granulocyte lineage. This specificity distinguishes haemonectin from previously described adhesion proteins which have a wider tissue distribution and cell type specificity.     

生物力学作用对骨髓间充质干细胞增殖和成骨分化影响研究进展

骨髓间充质干细胞在体内所处的力学环境比较复杂,为了探索生物力学与骨髓间充质干细胞增殖分化之间的关系,对近年来生物力学微环境对骨髓间充质干细胞增殖分化影响的研究现状和最新进展进行了综述。认为牵张力与流体剪切力对骨髓间充质干细胞产生的刺激大部分会使其向成骨方向分化,而较大的压缩力和静水压力对骨髓间充质干细胞产生的刺激大部分会使其偏向软骨方向分化,小部分向成骨方向分化,每种分化方向都有其最适的分化条件。通过综述生物力学对骨髓间充质干细胞增殖及成骨分化的影响,为骨髓间充质干细胞的骨组织工程与再生医学研究及更好地应用于临床治疗提供思路和参考依据。     

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.     

小直径血管支架的仿生构建及其种子细胞

静电纺丝仿生天然细胞外基质(ECM)结构,所制备的高度多孔、高比表面积的纳米级(50～500nm)纤维赋予了丰富的分子架构和生化信号,为种子细胞提供理想的生长微环境.不同组分、纤维尺寸及取向和种子细胞类型,可以裁剪获得不同生化和力学性能的电纺支架细胞复合物.总结了血管组织工程仿生ECM的设计与构建,并强调与支架复合的种子细胞在血管组织工程的作用.     

Green Electrospun Silk Fibroin/Galactose Chitosan Composite Nanofibrous Scaffolds for Hepatic Tissue Engineering

The electrospun nanofibrous scaffolds made of proteins and polysaccharides were thought to be able to simulate the structure of natural extracellular matrix well.Silk fibroin(SF)and chitosan(CS)are probably the most widely used natural materials in biomedical fields including liver tissue engineering for their good properties and wide variety of sources.The asialoglycoprotein receptors of hepatocyte were reported to specifically recognize and interact with galactose.In this work,a green electrospun SF/galactosylated chitosan(GC)composite nanofibrous scaffold was fabricated and characterized.The data indicated that the addition of GC greatly influenced the spinning effect of SF aqueous solution,and the average diameter of the composite nanofibers was about 520nm.Moreover,the green electrospun SF/GC nanofibrous scaffolds were demonstrated significantly enhancing the adhesion and proliferation of hepatocyte(RH35)according to our data.The present study did a useful exploration on constructing scaffolds for liver regeneration by green electrospinning,and also laid a good foundation for the further applicative research of this green electrospun scaffolds in liver tissue engineering.     

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.     

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.     

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.     

兔骨髓间充质干细胞复合脱细胞牛松质骨体外相容性研究

探讨脱细胞牛松质骨（Acellular Bovince Cancellous Bone，ABcB）生物衍生材料与骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）的生物相容性，为组织工程方法修复骨缺损提供依据。将兔骨髓间充质干细胞与ABCB复合并体外培养，然后进行形态学、细胞增殖、蛋白质含量测定。骨髓间充质干细胞能够贴附在ABCB孔隙内生长，细胞生长及蛋白合成功能不受ABCB的影响。ABCB生物相容性良好，可作为组织工程的支架材料应用于骨缺损的修复；BMSCs可以作为骨组织工程种子细胞。     

红景天苷对BMSC生物学特性影响

目的：研究红景天苷对骨髓间充质干细胞（Bonemesenchymalstemcells,BMSCs）生物学活性影响。方法：采用贴壁法分离培养BMSCs,检测细胞表面分子表达对所获干细胞进行鉴定。以终浓度为10¨g／mL红景天苷与BMSCs共培养,通过形态学观察和MTT法分析研究其对BMSCs生物学特性影响。结果：成功培养出BMSCs,免疫荧光染色显示,培养的第3代BMSCs表达CD29、CD44干细胞标志。10μg／mL红景天苷与BMSCs共培养后,较对照组细胞生长更为旺盛,更为规整,形成典型的“鱼群状”。生长曲线显示,实验组细胞较对照组生长稳定,增殖能力活跃。结论：10μg／mL红景天苷可促进BMSCs增殖分化,又不影响细胞增殖分化等生物学特性。     

新型前交叉韧带支架材料的构建及其生物相容性的实验研究

探讨以聚羟基丁酸己酯/聚左旋乳酸(PHB/PLLA1∶1)胶原杂化支架作为前交叉韧带组织工程载体材料的可行性。制备"三明治"样结构PHB/PLLA共聚物并测量其孔隙率等指标。以I型胶原对制备的PHB/PLLA支架进行杂化,获得PHB/PLLA胶原杂化支架。扫描电镜观察其表面结构。将兔皮肤成纤维细胞(SF)接种于PHB/PLLA胶原杂化支架,共培养5d后,扫描电镜下观察其在材料上生长情况。PHB/PLLA支架杂化后胶原填充于纤维空隙,分布比较均匀。体外培养的皮肤成纤维细胞成功种植在支架材料上,在材料上粘附、生长良好。说明构建的支架材料具有良好的三维构型和生物相容性,有望为前交叉韧带损伤的修复提供了一种新型的支架材料。     

Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment

Haematopoietic progenitor cells proliferate and mature in semisolid media when stimulated by exogenous haematopoietic cell growth factors (HCGFs) such as granulocyte-macrophage colony-stimulating factor (GM-CSF). They also proliferate in association with marrow-derived stromal cells although biologically active amounts of HCGFs cannot be detected in stromal culture supernatants. It is possible that HCGFs are synthesized in small amounts by stromal cells but remain bound to the stromal cells and/or their extracellular matrix (ECM). This interpretation accords with haematopoietic progenitor cell proliferation in close association with stromal layers in long-term cultures. Glycosaminoglycans (GAGs) are found in the ECM produced by stromal cells. They are prime candidates for selectively retaining HCGFs in the stromal layer; they influence embryonic morphogenesis and cyto-differentiation and they may regulate haematopoiesis. We now report that granulocyte-macrophage colony-stimulating activity can be eluted from cultured stromal layers and that exogenous GM-CSF binds to GAGs from bone marrow stromal ECM. Selective compartmentalization of HCGFs in this manner may be an important function of the marrow microenvironment and may be involved in haematopoietic cell regulation.     

不同移植骨材料修复骨缺损的病理学研究

目的研究比较3种不同骨移植材料对修复骨缺损的病理学的影响。方法分别对骨缺损动物模型进行实验性治疗,通过病理形态学观察,比较这些移植骨的成骨能力。结果各组移植物均可形成骨组织,修复骨缺损。POB/TBCc植入4周后,可见大量成骨细胞围绕在TBCc材料周围,并有类骨组织,8周后形成骨小梁,16周形成板层骨,骨细胞成熟并出现髓腔。结论三组材料比较,TBCc/POB组为较好的骨移植材料。     

Covalently immobilized gelatin gradients within three-dimensional porous scaffolds

A stable gelatin gradient providing continuous increment of signaling for cell adhesion and proliferation was fabricated within 3D poly（L-lactic acid） （PLLA） scaffolds. The porous PLLA scaffold fabricated by NaCI particle leaching was vertically fixed on a glass vial. 1,6-Hexanediamine/propanol solution was continuously injected into the vial by a micropump to aminolyze the PLLA scaffold. As a result of reaction time difference, the introduced -NH2 groups increased continuously along with the longitude of the PLLA scaffold in the z-direction. After covalent immobilization of gelatin by glutaraldehyde coupling, the gelatin gradient scaffold was thus obtained. In vitro chondrocyte culture showed that the cells had higher viability and more extending morphology in the gelatin gradient scaffold than that in the uniform gelatin control.     

Effect of Dy3+on osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and adipocytic trans-differentiation of mouse primary osteoblasts

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement, mineralized function, Oil Red O stain and measurement were employed to assess the effect of Dy³⁺ on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (BMSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts (OBs). The results showed that Dy³⁺ had no effect on BMSC proliferation at concentrations of 1×10⁻⁸ and 1×10⁻⁵ mol/L, but inhibited BMSC proliferation at other concentrations. Dy³⁺ had no effect on OB proliferation at concentrations of 1×10⁻¹⁰ and 1×10⁻⁹ mol/L, but inhibited OB proliferation at other concentrations. Dy³⁺ had no effect on the osteogenic differentiation of BMSCs at concentrations of 1×10⁻⁹ and 1×10⁻⁷ mol/L, and promoted osteogenic differentiation of BMSCs at other concentrations at the 7th day. The osteogenic differentiation of BMSCs was inhibited by Dy³⁺ at concentration of 1×10⁻⁵ mol/L at the 14th day, but promoted osteogenic differentiation of BMSCs at concentrations of 1×10⁻⁹, 1×10⁻⁸, 1×10⁻⁷ and 1×10⁻⁶ mol/L with the maximal effect at concentration of 10⁻⁶ mol/L. Dy³⁺ promoted mineralized function of BMSCs at any concentration. Dy³⁺ had no effect on adipogenic differentiation of BMSCs at concentration of 1×10⁻⁷ mol/L, but inhibited adipogenic differentiation of BMSCs at other concentrations. Dy³⁺ inhibited adipocytic trans-differentiation of OBs at any concentration, suggesting that Dy³⁺ had protective effect on bone and the protective effect on bone may be mediated by modulating differentiation of BMSCs away from the adipocyte and inhibiting adipocytic trans-differentiation of OBs which may promote differentiation and mineralization of OBs. These results may be valuable for better understanding the mechanism of the effect of Dy³⁺ on pathogenesis of osteoporosis. Supported by the Foundation for Key Program of Ministry of Education of China (Grant No. 208018)