新型聚己内酯/磷酸钙支架的制备及生物相容性研究

探讨新型聚己内酯(PCL)/磷酸钙(CPC)复合材料支架的制备方法及对骨髓基质细胞(BMSCs)的生物相容性。采用溶液共混法,利用可溶盐晶体做造孔剂,制备PCL/CPC复合材料支架,以单纯PCL和CPC支架为对照组,Q800型动态力学分析仪进行动态力学性能试验(DMA),采用排水法测量孔隙率;灭菌后通过与犬BMSCs体外共同培养后细胞形态、生长曲线、碱性磷酸酶(ALP)染色和半定量及骨钙素(OC)半定量等方法检测细胞在支架材料上的黏附、增殖及成骨分化情况,动物体内异位成骨检测其成骨情况。结果显示,复合材料的储能模量在PCL/CPC比例为7:3时达到最大,制得的材料孔径为250～350μm,多孔支架的孔隙率为70%～80%;BMSCs在新型PCL/CPC组、CPC组支架表面分布均匀,生长增殖明显较PCL组活跃(P<0.05);PCL/CPC组、CPC组BMSCs成骨行为与PCL组之间有显著差异(P<0.05)。动物体内异位成骨检测提示,4周时PCL/CPC组为13.78%±1.60%、CPC组BMSCs为15.29%±1.20%,成骨显著强于PCL组BMSCs的7.56%±2.20%(P<0.05),表明PCL和CPC的复合明显改善了两种材料的缺陷,获得的PCL/CPC支架具有良好的生物相容性,可与BMSCs共同构建具有成骨能力的三维立体组织工程化骨。     

脱细胞真皮基质/生物矿化胶原一体化骨软骨支架的制备及其生物相容性的研究

目的成功制备脱细胞真皮基质/生物矿化胶原一体化骨软骨支架,并进行理化性质、生物力学与生物相容性评估,为骨软骨缺损修复提供理论依据。方法以物理和化学方法制备牛源脱细胞真皮基质,利用磷酸三钙和胶原,按照不同比例混合,利用自组装和冻干技术,制备以脱细胞真皮基质支架为软骨层,生物矿化胶原为骨层的骨软骨一体化双相支架。通过大体观察、扫描电镜观察、生物力学等检测,对骨软骨一体化支架进行理化性质和力学性能评价。原代培养小鼠软骨细胞和成骨细胞,并分别种植在脱细胞真皮基质和生物矿化胶原双相支架上,利用细胞毒、死/活细胞染色和增殖实验等观察细胞生长情况,测定骨软骨一体化支架的生物相容性。结果大体观察表明支架各层间结合紧密,未出现明显不连续和相互分离。扫描电镜各层内的孔隙结构相互连通且均具有立体多维性,其中脱细胞真皮基质、生物矿化胶原和双相支架的孔径分别为(121.6±8.65)、(98.40±5.56)和(103.2±3.94)μm。同时三组支架的压缩模量分别为(41.05±11.69)、(108.0±12.71)和(84.98±8.51)k Pa,弹性模量分别为(17.24±3.93)、(28.98±3.31)和(21.74±2.92)k Pa。MTT法表明骨软骨一体化支架无细胞毒性。荧光显微镜观察显示,纵切的支架薄片上细胞生长分布均匀,表明支架生物相容性较好,CCK8实验表明支架可以维持良好的细胞活性。结论脱细胞真皮基质/生物矿化胶原骨双相支架中上下层间的理化性能展示了骨软骨组织结构和力学方面的双重仿生,为动物体内实验奠定了基础,有望成为治疗骨软骨缺损修复的一种新手段。     

新型大孔隙磷酸钙骨水泥作为骨组织工程支架的相关研究

目的 探讨新型大孔隙磷酸钙骨水泥(CPC)材料支架的细胞毒性和对细胞黏附、生长和增殖的影响.方法 通过添加甘露醇制孔剂和应用磷酸钠溶液作为CPC固化液的方法合成新型CPC材料.通过CCK8法检测细胞在新型CPC材料浸提液中的生长增殖情况;通过电子扫描电镜测试材料孔径和细胞在材料表面上黏附生长情况;应用力学三点弯曲实验测试新型CPC的生物力学性能.结果 新型CPC材料的孔径值达到(267.43±118.01)μm,孔隙率为(66.15±6.91)％.新型CPC材料的最大负荷、抗弯强度和坚韧度较传统CPC均增加了约1倍(P＜0.05).新型CPC材料浸提液与细胞共培养2、4、6、8d后CCK8法测试吸光度(OD)值与阴性对照组比较其差异无统计学意义(P＞0.05).结论 新型CPC材料具有强大的生物力学性能、大孔隙、高孔隙率和良好的生物相容性,有望成为理想的骨组织工程支架.     

基于3D打印的Ⅰ型胶原涂覆β-TCP骨组织工程支架研究

根据骨缺损形态构建个性化的组织工程支架在骨组织工程应用中有巨大需求。基于3D打印技术制备个性化的I型胶原涂覆的β-磷酸三钙(β-TCP)骨支架。通过比较0/90°、0/60°、0/45°的填充角度,0.10、0.25、0.50 mg/m L涂覆胶原的浓度对β-TCP支架孔径、孔隙率、力学性能的影响,选定最优填充角度为0/90°及最佳涂覆胶原的浓度为0.50 mg/m L的β-TCP/胶原支架。所得支架能准确地再现设计的三维模型,具有多级孔结构,大孔平均直径为315μm,微孔直径为3~5μm,孔隙率为84%。β-TCP/胶原支架的抗压能力为(12.29±0.88)MPa,压缩弹性模量为(116.74±27.75)MPa,与成人松质骨相似。体外大鼠骨髓间充质干细胞(m BMSCs)支架培养实验结果显示,涂覆胶原的支架具有更好的生物相容性,能有效促进m BMSCs的粘附增殖,β-TCP/胶原支架上细胞具有更高的碱性磷酸酶(ALP)活性和Collagen-I、BSP相关成骨基因的表达。研究结果显示,3D打印制备的I型胶原涂覆的β-TCP支架具有匹配的外形,良好可控的孔隙率,对m BMSCs有良好成骨活性,为骨组织支架在临床上应用提供新的技术。     

三维打印双相磁性纳米复合支架材料的性能测定

目的研究制备出一种新型双相磁性纳米复合支架材料(PLGA/Col-I-PLGA/n-HA/Fe_2O_3),通过各项生物学性能检测,评价并探讨其作为骨组织工程支架的可行性。方法通过低温快速成型方法制备双相磁性纳米复合支架材料(PLGA/Col-I-PLGA/n-HA/Fe_2O_3),采用电子试验机检测支架材料的抗弯,抗压,弹性模量评价其力学性能,通过电镜观察支架材料超微结构;以介质(乙醇)浸泡法测定支架材料的孔隙率,将支架材料与骨髓间充质干细胞复合共培养,检测其生物相容性。结果双相磁性纳米复合支架材料力学检测结果显示其具有良好的力学性能,电镜观察结果显示上下两层孔径均匀分布,上层软骨相孔径较小,中间连续相良好融合,孔径及孔隙率检测结果显示软骨层支架的孔径为189um,孔隙率86.5%。骨层支架的孔径为364um,孔隙率77.1%,符合双层支架材料的设计要求。双相磁性纳米复合支架材料与骨髓间充质干细胞共培养,结果显示骨髓间充质干细胞的增殖效果很好,能更好的促进分化为目的细胞,说明双相磁性纳米复合支架材料具有良好的生物相容性。结论双相磁性纳米复合支架材料(PLGA/Col-I-PLGA/n-HA/Fe_2O_3)有很好的力学性能和生物相容性,孔径及孔隙率达到细胞粘附生长的要求,与正常的关节软骨及软骨下骨生理结构更加接近,有望可以更好的修复骨关节炎或者外伤等疾病带来的软骨和软骨下骨损伤。     

基于3D打印羟基磷灰石支架的填充结构与力学性能研究

3D打印骨组织工程支架是近来的研究热点,而制备同时具有高孔隙率和足够力学性能的骨组织工程支架是研究的难点之一。在孔隙率相同条件下,探究不同填充角度结构对3D打印支架力学性能影响。首先用SolidWorks软件设计孔隙率相同的3种不同填充角度(45°、60°、90°)支架结构,以交点处结构作为支架的最小支撑单元,并用ABAQUS软件对其进行力学性能仿真,对仿真所得单元结构压缩模量进行累加,探究填充角度对支架力学性能的影响;进而通过3D打印制备3种填充结构的羟基磷灰石支架,测试支架的孔隙率和力学性能,对仿真结果进行验证。结果表明,仿真所得3种填充结构的压缩模量比为E_s(90°)∶E_s(60°)∶E_s(45°)=12.3∶10.9∶10.0。打印得到3种不同填充角度(90°,60°,45°)的羟基磷灰石支架孔隙率无显著性差异,其压缩模量比为E_s(90°)∶E_s(60°)∶E_s(45°)=15.4∶13.1∶10.0,与仿真结果趋势一致,90°填充的支架具有最高的抗压强度((7.36±0.63) MPa)和压缩模量((33.55 ± 2.49) MPa),与力学性能最低的45°填充支架相比,抗压强提高74.8%,压缩模量提高55.18%。在孔隙率相同的条件下,单个孔型面积越小,其压缩模量和抗压强度越高。该研究为制备最优填充结构的3D打印生物支架提供分析方法和理论依据。     

PCL/ZrO_2骨组织工程支架3D打印制备方法及其性能研究

目的为制备出满足骨缺损修复需要的具有一定力学强度和生物活性的骨组织工程支架,本文选取聚己内酯(polycaprolactone, PCL)和纳米氧化锆(ZrO_2)粉末制备出三维多孔复合材料支架。方法采用高温熔融挤出3D打印方式制备PCL/ZrO_2复合材料支架,为获取支架的几何形态、力学性能和生物学性能,利用扫描电子显微镜(scanning electron microscope, SEM)和万能试验机(material testsystem,MTS)分别分析了支架的形貌和压缩性能,并通过体外细胞培养的方式测试复合材料支架的生物相容性。结果制备完成的复合材料支架具有良好的三维孔隙结构,孔径≥400μm,孔隙率≥40%。对比纯PCL支架,PCL/ZrO_2复合材料支架的力学性能显著提高,杨氏模量提高0.4倍左右,抗压强度提高0.5倍左右。在体外实验中,细胞培养7 d后PCL/ZrO_2复合材料支架上的细胞增殖对比纯PCL支架有显著提高。结论基于该结果,本文制备出的PCL/ZrO_2生物活性骨组织支架在骨组织工程方面有一定的应用前景。     

蚕丝蛋白/PCL共混取向纤维的力学性质与细胞响应

蚕丝蛋白因其良好的机械强度、生物相容性和耐灭菌性,是目前骨修复领域不可多得的天然蛋白支架材料。为提高蚕丝蛋白支架对细胞定向移动的引导能力和力学强度,采用静电纺丝方式制备较高取向度的PCL/蚕丝蛋白共混纤维支架,并与非取向型对比。通过扫描电镜图像分析技术表征静电纺丝纤维表面的取向度,并比较不同纤维排列结构下的力学性质。间充质干细胞在骨修复中应用广泛。人源骨髓间充质干细胞来自健康献髓者,在体外培养条件下,测试不同结构材料对间充质干细胞增殖的影响,并使用活细胞工作站实时追踪细胞在不同材料表面的运动功能。结果表明,在取向静电纺丝纤维支架中,91%的纤维分布于"10°范围以内,且该支架具有力学的各向异性,具有较大的轴向拉伸模量。在间充质干细胞增殖96 min后,中取向静电纺丝纤维支架组的细胞数量产生显著差异。在取向纤维材料表面生长的间充质干细胞展现出最小的细胞分布夹角,仅为6.57°±4.45°,同时,取向纤维表面的细胞长度有显著提高(236.46±82.00)μm。在进一步的实时细胞追踪中,在细胞的主要移动方向上,取向纤维表面的细胞分解移动速度达6.66μm/h,远远高于其他表面。因而,取向静电纺丝纤维支架可能在体内应用中支持成体干细胞增殖,并加快早期细胞定向迁移。     

多孔聚乙烯醇/明胶软骨组织工程支架复合材料的制备及其生物相容性

背景：以明胶为基体制备的组织工程支架材料具有良好的生物相容性和生物降解性能,但存在力学性能低,降解速率难以控制的缺陷。 目的：制备一种软骨组织工程支架材料多孔聚乙烯醇/明胶复合物,并检测其理化性能和生物相容性。 方法：采用乳化发泡法制备聚乙烯醇/明胶多孔支架,并通过电镜分析、力学测试、皮下植入实验,检测材料孔径和孔隙率、IR光谱、力学性能和生物相容性。 结果与结论：多孔材料内部呈三维网状多孔结构,孔径均匀,有相似的孔隙率61.8%,含水率44.6%,抗拉强度为(5.01±0.03) MPa,抗压强度为(1.47±0.36) MPa,有较好的力学性能,IR光谱分析表明材料内部结构均匀。皮下植入后,炎症反应逐渐减轻,囊壁逐渐变薄,并趋于稳定,提示多孔聚乙烯醇/明胶支架材料具有较好的生物相容性和力学性能。     

基于高温挤出打印的PLGA/β-TCP人工骨支架制备及其性能研究

目的制备具有较优力学性能和细胞增殖特性的聚乳酸-羟基乙酸共聚物/β-磷酸三钙[poly(lactic-co-glycolic acid)/β-tricalcium phosphate,PLGA/β-TCP]人工骨支架,为骨组织工程支架材料和结构的选择提供实验依据。方法以PLGA和纳米β-TCP为原材料,先将PLGA在加热筒中高温熔融,再将β-TCP混合搅拌均匀进行打印。采用高温挤出打印方法制备人工骨支架,研究不同材料配比(PLGA与β-TCP配比为4∶1、3∶1、2∶1)、不同孔径(200μm、300μm和400μm)、不同层高(4.2 mm、5.4 mm和6.6 mm)以及不同孔形(方形、45°倾斜和60°倾斜)对支架力学性能和细胞增殖性能的影响。打印9组支架,利用扫描电镜分析支架的形貌特征,用万能材料试验机测试支架的抗压强度、弹性模量,用CCK-8试剂盒进行细胞增殖性能测试。最后对实验数据进行极差分析,得出最优组合,并制作相应的支架,对其进行力学性能和生物性能的测试。结果在力学性能实验中,S5组支架显示出最好的抗压强度(7.23 MPa),S9组支架显示出最好的杨氏模量(356.1 MPa),与骨小梁相当;在细胞增殖实验中,S1组支架显示出最好的增殖特性。通过正交实验法,分析并实验验证,得出综合性能最优的支架参数为材料配比为4∶1、层高为6.6 mm、孔型为45°倾斜以及孔径为200μm。结论材料配比为4∶1、层高为6.6mm、孔型为45°倾斜以及孔径为200μm的支架基本能够满足某些松质骨(如骨小梁)的力学性能和细胞增殖性能,并可为后续相关研究提供实验和理论基础。     

Extracellular calcium sensing and extracellular calcium signaling

The cloning of a G protein-coupled extracellular Ca(2+) (Ca(o)(2+))-sensing receptor (CaR) has elucidated the molecular basis for many of the previously recognized effects of Ca(o)(2+) on tissues that maintain systemic Ca(o)(2+) homeostasis, especially parathyroid chief cells and several cells in the kidney. The availability of the cloned CaR enabled the development of DNA and antibody probes for identifying the CaR's mRNA and protein, respectively, within these and other tissues. It also permitted the identification of human diseases resulting from inactivating or activating mutations of the CaR gene and the subsequent generation of mice with targeted disruption of the CaR gene. The characteristic alterations in parathyroid and renal function in these patients and in the mice with "knockout" of the CaR gene have provided valuable information on the CaR's physiological roles in these tissues participating in mineral ion homeostasis. Nevertheless, relatively little is known about how the CaR regulates other tissues involved in systemic Ca(o)(2+) homeostasis, particularly bone and intestine. Moreover, there is evidence that additional Ca(o)(2+) sensors may exist in bone cells that mediate some or even all of the known effects of Ca(o)(2+) on these cells. Even more remains to be learned about the CaR's function in the rapidly growing list of cells that express it but are uninvolved in systemic Ca(o)(2+) metabolism. Available data suggest that the receptor serves numerous roles outside of systemic mineral ion homeostasis, ranging from the regulation of hormonal secretion and the activities of various ion channels to the longer term control of gene expression, programmed cell death (apoptosis), and cellular proliferation. In some cases, the CaR on these "nonhomeostatic" cells responds to local changes in Ca(o)(2+) taking place within compartments of the extracellular fluid (ECF) that communicate with the outside environment (e.g., the gastrointestinal tract). In others, localized changes in Ca(o)(2+) within the ECF can originate from several mechanisms, including fluxes of calcium ions into or out of cellular or extracellular stores or across epithelium that absorb or secrete Ca(2+). In any event, the CaR and other receptors/sensors for Ca(o)(2+) and probably for other extracellular ions represent versatile regulators of numerous cellular functions and may serve as important therapeutic targets.     

Cytoplasmic calcium oscillations and store-operated calcium influx

Intracellular calcium oscillations have fascinated scientists for decades. They provide an important cellular signal which, unlike most signalling mechanisms, is digitally encoded. While it is generally agreed that oscillations most frequently arise from cyclical release and re-uptake of intracellularly stored calcium, it is becoming increasingly clear that influx of calcium across the plasma membrane also plays a critical role in their maintenance and even in delivering their signal to the correct cellular locus. In this review we will discuss the role played by Ca²⁺ entry mechanisms in Ca²⁺ oscillations, and approaches to understanding the molecular nature of this Ca²⁺ entry pathway.     

Effects of calcium chelators on intracellular calcium and excitotoxicity

In an attempt to probe the relationship between excitotoxicity and increases in intracellular calcium ([Ca²⁺]_i), BAPTA-AM and its analogs were applied to cultured hippocampal neurons. Chelation of [Ca²⁺]_i depressed and prolonged transient responses to glutamate and did not effect elevation of [Ca²⁺]_i by prolonged exposure. This explains the inability of the chelators to prevent glutamate-induced toxicity.     

Gastrointestinal absorption of calcium from milk and calcium salts

Whether ingested calcium is absorbed more efficiently from freely water-soluble calcium salts than from poorly soluble salts is unclear. It is also unknown whether calcium is absorbed better from dairy products than from calcium salts. Using a method by which the net absorption of calcium can be accurately measured after a single dose, we studied eight healthy fasting subjects after they took a 500-mg dose of calcium from each of five calcium salts with various degrees of water solubility and from milk. The order of administration of the agents given was randomly determined. The mean (+/- SEM) net calcium absorption, in decreasing order of the solubility of the salts, was 32 +/- 4 percent from calcium acetate, 32 +/- 4 percent from calcium lactate, 27 +/- 3 percent from calcium gluconate, 30 +/- 3 percent from calcium citrate, and 39 +/- 3 percent from calcium carbonate. The differences in absorption were not statistically significant according to analysis of variance. On the basis of in vitro solubility experiments in acid mediums, we hypothesize that acid dissolution in the gastrointestinal tract may be responsible for the similar absorption of calcium from salts with widely different water solubilities. Calcium absorption from whole milk (31 +/- 3 percent) was similar to absorption from calcium salts. We conclude that calcium absorption from carbonate, acetate, lactate, gluconate, and citrate salts of calcium, and from whole milk, is similar in fasting healthy young subjects. Further study will be required to determine whether the results would be different in older subjects, with a higher dose of calcium, or if the calcium was ingested with food.     

Intracellular calcium buffering shapes calcium oscillations in Xenopus melanotropes

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.     

Sodium/calcium exchange regulates cytoplasmic calcium in smooth muscle

The sodium/calcium (Na⁺/Ca²⁺) exchanger is often considered to be a key regulator of the cytoplasmic calcium concentration ([Ca²⁺]) in smooth muscle but neither its precise role in Ca²⁺ homeostasis nor even its existence in smooth muscle are generally agreed upon. Here we directly assessed the role Na⁺/Ca²⁺ exchange plays in regulating [Ca²⁺] in single voltage-clamped smooth muscle cells. Following an elevation of [Ca²⁺], its decline was found to have both voltage-dependent and voltage-independent components. The voltage-dependent component was abolished when Na⁺ was removed from the external bathing solution. During the fall of [Ca²⁺] a small and declining Na⁺-dependent inward current was observed of a magnitude predicted by 31 Na⁺/Ca²⁺ exchange stoichiometry. At [Ca²⁺] above 400 nM the principal efflux of Ca²⁺ above rest was attributed to this Na⁺-dependent removal mechanism. These results establish that a Na⁺/Ca²⁺ exchanger exists in smooth muscle and argue that it can regulate [Ca²⁺] at physiological Ca²⁺ concentrations.     

B-lymphocyte calcium inFlux

Summary:  Dynamic changes in cytoplasmic calcium concentration dictate the immunological fate and functions of lymphocytes. During the past few years, important details have been revealed about the mechanism of store-operated calcium entry in lymphocytes, including the molecular identity of calcium release-activated calcium (CRAC) channels and the endoplasmic reticulum (ER) calcium sensor (STIM1) responsible for CRAC channel activation following calcium depletion of stores. However, details of the potential fine regulation of CRAC channel activation that may be imposed on lymphocytes following physiologic stimulation within an inflammatory environment have not been fully addressed. In this review, we discuss several underexplored aspects of store-operated (CRAC-mediated) and store-independent calcium signaling in B lymphocytes. First, we discuss results suggesting that coupling between stores and CRAC channels may be regulated, allowing for fine tuning of CRAC channel activation following depletion of ER stores. Second, we discuss mechanisms that sustain the duration of calcium entry via CRAC channels. Finally, we discuss distinct calcium permeant non-selective cation channels (NSCCs) that are activated by innate stimuli in B cells, the potential means by which these innate calcium signaling pathways and CRAC channels crossregulate one another, and the mechanistic basis and physiologic consequences of innate calcium signaling.     

Dialysate calcium and plasma calcium fractions during and after haemodialysis

Summary Dialysate Calcium and Plasma Calcium Fractions during and after Haemodialysis:The effect of different dialysate Ca concentrations on the plasma Ca fractions was examined in 28 patients. In 10 patients dialysed with a dialysate Ca concentration of 3.0 mEq/l the Ca fractions were determined at the start and end of dialysis. 8 patients were dialysed with dialysate Ca of 3.5 mEq/l. In this group the Ca fractions were also estimated in the dialysis-free interval. The third group was dialysed with a dialysate Ca of 4.5 mEq/l. Total calcium and protein-bound calcium rose significantly in all groups. Ionised calcium in the first group was significantly reduced, in the second group it remained constant and in the third group it was significantly raised. Since parathyroid function depends on the plasma ionised calcium it is concluded that a dialysate concentration of 3.0 mEq/l is partly responsible for the pathogenesis of secondary hyperparathyroidism and of renal osteodystrophy. In normocalcaemic patients a dialysate Ca concentration of 3.5 to 4.0 mEq/l is optimal. In patients entering long-term haemodialysis treatment with pronounced calcium deficiency symptoms a dialysate Ca of up to 4.5 mEq/l may be indicated for a short period after having normalized the inorganic phosphate levels in order to prevent extraosseous calcification.