Characterization of osteoblasts derived from bone marrow stromal cells in a modified cell culture system

Bone marrow is a complex tissue composed of hematopoietic and stromal stem cells with the potential to differentiate into adipogenic, fibroblastic, reticular, osteogenic and chondrogenic lineages. Identification of differentiation markers during transformation of stromal cells into osteoblasts in a time-dependent manner may be informative for cell-based tissue engineering. Therefore, we investigated the effects of osteogenic medium (OM) on the proliferation and differentiation of rat bone marrow stromal cells (BMSCs). BMSCs from adult male rat tibia and femur were collected and cultured in alpha-MEM medium with 10% fetal bovine serum, penicillin, streptomycin and gentamycin. After three days of culture, the medium covering the adherent cells in culture was changed to OM containing dexamethasone, Na-beta-glycerophosphate and ascorbic acid. As a control, cell culture was also continued in the original medium for the same time period. Differentiated osteoblast cells were collected after 7, 10, 14, 21 and 30 days of culture, fixed with 4% paraformaldehyde and their immunolabelling for osteoblast markers osteonectin (ON) and osteocalcin (OC) was assessed using an indirect immunoperoxidase technique. Immunolabelling of ON and OC was detectable from day 10 of culture, began to increase on day 14, and increased steadily through to day 21. Labelling was highest on day 30 and was more intense in cells cultured with OM compared to the culture without OM. The control cells cultured in the absence of OM produced negligible levels of both markers. In conclusion, our culture system facilitated differentiation of BMSCs into osteoblasts featuring osteoblast markers, and these cells may be useful in autologous bone implant for the treatment of bone wound healing.     

In vitro culture and characterization of alveolar bone osteoblasts isolated from type 2 diabetics

In order to understand the mechanisms of poor osseointegration following dental implants in type 2 diabetics, it is important to study the biological properties of alveolar bone osteoblasts isolated from these patients. We collected alveolar bone chips under aseptic conditions and cultured them in vitro using the tissue explants adherent method. The biological properties of these cells were characterized using the following methods: alkaline phosphatase (ALP) chemical staining for cell viability, Alizarin red staining for osteogenic characteristics, MTT test for cell proliferation, enzyme dynamics for ALP contents, radio-immunoassay for bone gla protein (BGP) concentration, and ELISA for the concentration of type I collagen (COL-I) in the supernatant. Furthermore, we detected the adhesion ability of two types of cells from titanium slices using non-specific immunofluorescence staining and cell count. The two cell forms showed no significant difference in morphology under the same culture conditions. However, the alveolar bone osteoblasts received from type 2 diabetic patients had slower growth, lower cell activity and calcium nodule formation than the normal ones. The concentration of ALP, BGP and COL-I was lower in the supernatant of alveolar bone osteoblasts received from type 2 diabetic patients than in that received from normal subjects (P < 0.05). The alveolar bone osteoblasts obtained from type 2 diabetic patients can be successfully cultured in vitro with the same morphology and biological characteristics as those from normal patients, but with slower growth and lower concentration of specific secretion and lower combining ability with titanium than normal ones.     

大鼠脊髓星形胶质细胞培养方法的改良

目的:改进大鼠脊髓星形胶质细胞的体外培养方法,为研究脊髓星形胶质细胞的生物学作用及脊髓相关疾病提供实验模型.方法:新生1～2d的SD乳鼠,无菌操作下游离整段脊柱,将脊髓从椎管中冲出,机械吹打制成单细胞悬液,差速黏附处理以去除成纤维细胞成分.利用GFAP免疫荧光显色对培养细胞进行鉴定.结果:经原代和传代培养,获得的星形胶质细胞纯化率高达99%以上.结论:采用本实验室改进的方法,操作简便快捷,培养的大鼠脊髓星形胶质细胞生长良好,纯度高,为后续实验对脊髓星形胶质细胞的生物学作用研究奠定实验基础.     

A three-dimensional tissue culture model of bone formation utilizing rotational co-culture of human adult osteoblasts and osteoclasts

Living bone is a complex, three-dimensional composite material consisting of numerous cell types spatially organized within a mineralized extracellular matrix. To date, mechanistic investigation of the complex cellular level cross-talk between the major bone-forming cells involved in the response of bone to mechanical and biochemical stimuli has been hindered by the lack of a suitable in vitro model that captures the “coupled” nature of this response. Using a novel rotational co-culture approach, we have generated large (>4 mm diameter), three-dimensional mineralized tissue constructs from a mixture of normal human primary osteoblast and osteoclast precursor cells without the need for any exogenous osteoconductive scaffolding material that might interfere with such cell–cell interactions. Mature, differentiated bone constructs consist of an outer region inhabited by osteoclasts and osteoblasts and a central region containing osteocytes encased in a self-assembled, porous mineralized extracellular matrix. Bone constructs exhibit morphological, mineral and biochemical features similar to remodeling human trabecular bone, including the expression of mRNA for SOST, BGLAP, ACP5, BMP-2, BMP-4 and BMP-7 within the construct and the secretion of BMP-2 protein into the medium. This “coupled” model of bone formation will allow the future investigation of various stimuli on the process of normal bone formation/remodeling as it relates to the cellular function of osteoblasts, osteoclasts and osteocytes in the generation of human mineralized tissue.     

Defining the extracellular matrix using proteomics

The cell microenvironment has a profound influence on the behaviour, growth and survival of cells. The extracellular matrix (ECM) provides not only mechanical and structural support to cells and tissues but also binds soluble ligands and transmembrane receptors to provide spatial coordination of signalling processes. The ability of cells to sense the chemical, mechanical and topographical features of the ECM enables them to integrate complex, multiparametric information into a coherent response to the surrounding microenvironment. Consequently, dysregulation or mutation of ECM components results in a broad range of pathological conditions. Characterization of the composition of ECM derived from various cells has begun to reveal insights into ECM structure and function, and mechanisms of disease. Proteomic methodologies permit the global analysis of subcellular systems, but extracellular and transmembrane proteins present analytical difficulties to proteomic strategies owing to the particular biochemical properties of these molecules. Here, we review advances in proteomic approaches that have been applied to furthering our understanding of the ECM microenvironment. We survey recent studies that have addressed challenges in the analysis of ECM and discuss major outcomes in the context of health and disease. In addition, we summarize efforts to progress towards a systems‐level understanding of ECM biology.     

神经干细胞克隆在不同细胞外基质上的生长特性

目的：观察不同细胞外基质对培养神经干细胞生长特性的影响。方法：经体外扩增和传代,神经干细胞克隆被接种于多聚赖氨酸、多聚鸟氨酸和鼠尾胶原形成的细胞外基质上,观察其增殖和迁移特性。结果：未经包被的塑料培养板作为基质时,体积较大的神经干细胞克隆才能贴壁,贴壁后的克隆仍呈球形,在其底部有细胞呈放射状迁出,数量逐渐增多,以梭形为主,细胞排列稀疏;多聚鸟氨酸和多聚赖氨酸作为细胞外基质时,迁移细胞排列密集,形态多样;鼠尾胶原作为细胞外基质时,促贴壁迁移的作用最差。当多聚赖氨酸和鼠尾胶原形成界限时可明显观察到鼠尾胶原对细胞迁移的抑制作用。结论：在各种基质上,克隆体积均可继续增长,但在鼠尾胶原上增长较慢。     

A method for measuring steroid adsorption to tissue culture plasticware

Summary Tritiated test steroids were added to culture medium in plastic tissue culture vessels. At different time intervals, levels of radioactivity in the plastic itself and in the culture medium were measured. There was significant adsorption of steroids to the test vessel. We describe here a general procedure to measure the amount of steroid hormone adsorbed from culture media by tissue culture plasticware, and the remaining amount actually available to the cells in the medium. Such measurements may be critical in establishing doses of steroids for in vitro endocrinology experiments.     

三维细胞培养中支架的研究现状

细胞培养技术是生物技术中最核心、最基础的技术,目前常用的二维细胞培养方法,例如,多孔板、烧瓶和培养皿等存在诸多不足和限制,三维细胞培养能够改善细胞局部微环境、促进细胞与细胞、细胞与基质之间的交流,模拟体内生理环境,从而增强细胞功能,在新药研发、肿瘤研究等领域具有广泛应用前景。支架是实现三维细胞培养最常用的手段之一,相当于人工细胞外基质,能够模拟体内组织复杂的三维结构及其主要特征。本文从基于支架的培养体系和无支架的培养体系两方面讨论了三维细胞培养方法,尤其详细介绍了各种支架。     

Initial responses of human osteoblasts to sol-gel modified titanium with hydroxyapatite and titania composition

Sol-gel thin films of hydroxyapatite (HA) and titania (TiO(2)) have received a great deal of attention in the area of bioactive surface modification of titanium (Ti) implants. Sol-gel coatings were developed on Ti substrates of pure HA and TiO(2) and two composite forms, HA+10% TiO(2) and HA+20% TiO(2), and the biological properties of the coatings were evaluated. All the coating layers exhibited thin and homogeneous structures and phase-pure compositions (either HA or TiO(2)). Primary human osteoblast cells showed good attachment, spreading and proliferation on all the sol-gel coated surfaces, with enhanced cell numbers on all the coated surfaces relative to uncoated Ti control at day 1, as observed by MTT assay and scanning electron microscopy. Cell attachment rates were also enhanced on the pure HA coating relative to control Ti. The pure HA and HA+10% TiO(2) composite coating furthermore enhanced proliferation of osteoblasts at 4 days. Moreover, the gene expression level of several osteogenic markers including bone sialoprotein and osteopontin, as measured by RT-PCR at 24h, was shown to vary according to coating composition. These findings suggest that human primary bone cells show marked and rapid early functional changes in response to HA and TiO(2) sol-gel coatings on Ti.     

Development of glass-ceramic scaffolds for bone tissue engineering: characterisation, proliferation of human osteoblasts and nodule formation

Glass-ceramic macroporous scaffolds for tissue engineering have been developed using a polyurethane sponge template and bioactive glass powders. The starting glass (CEL2) belongs to the system SiO(2)-P(2)O(5)-CaO-MgO-Na(2)O-K(2)O and has been synthesised by a conventional melting-quenching route. A slurry of CEL2 powder, polyvinyl alcohol and water has been prepared in order to coat, by impregnation, the polymeric template. An optimised thermal treatment was then use to remove the sponge and to sinter the glass powders, leading to a glass-ceramic replica of the template. Morphological observations, image analyses, mechanical tests and in vitro tests showed that the obtained devices are good candidates as scaffolds for bone-tissue engineering, in terms of pore-size distribution, pore interconnection, surface roughness, and both bioactivity and biocompatibility. In particular, a human osteoblast cell line (MG-63) seeded onto the scaffold after a standardised preconditioning route in simulated body fluid showed a high degree of cell proliferation and a good ability to produce calcium nodules. The obtained results were enhanced by the addition of bone morphogenetic proteins after cell seeding.     

使用碱性蛋白酶去除人体骨骼标本软组织的技术研究

<正>未防腐的人体骨骼标本的常用制备方法包括自然腐蚀法、水煮法和药液浸泡法[1]。1989年开始有动物解剖学者尝试使用蛋白酶分解蛋白的方式来去除(仓鼠和家兔)肌肉组织来获取骨骼标本,他们使用蛋白酶的浓度为0.3%～0.5%[2～4]。然而这一方法是否适     

A micro cell culture method utilizing modified microscope slides for use in fluorescent antibody and enzyme immunoassays

Summary A technique for culturing small quantities of mammalian cells on modified microscope slides is described. The modified microscope slides were Bellco Glass, Inc., toxoplasmosis slides and the cell cultures used were early passage bovine embryonic lung cells and continuous cell lines of porcine and canine origins. The slide cell cultures were either uninfected or infected with selected viruses or the obligate intracellular protozoanEncephalitozoon caniculi for utilization in direct and indirect fluorescent antibody testing or in peroxidase antiperoxidase immunosorbant assays.     

A simple electronic device for gassing tissue culture vessels

Summary A device is described which will (a) mix 100% CO₂ with compressed air in any desired proportion, (b) deliver the mixed CO₂-air at any desired flow rate, and (c) permit automatic control of the time period for which gas flows. This device employs two solenoid valves controlling gas flow, a commercially available electronic timer, and two gas regulators.     

改良组织块培养法体外培养人角膜上皮干细胞

文题释义：角膜上皮干细胞：属于单能干细胞,具有细胞周期长、低分化状态、增殖潜力大、不对称分裂等特点,定位于角膜缘基底细胞层,又称之为角膜缘干细胞,对角膜上皮细胞更新及维持角膜透明起着重要作用。角膜缘干细胞的体外培养方法：主要包括酶消化培养法和组织块培养法。酶消化培养法是利用DispaseⅡ酶破坏角膜缘上皮细胞与基底膜之间的半桥粒连接,然后剥取角膜缘上皮层,再使用胰酶将其消化为单个细胞进行培养。组织块培养法没有经过酶的双重消化,将剖取的角膜缘组织块进行贴壁,细胞游离出组织块进行贴壁生长,需要一个漫长的过程。  摘要背景：角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手。目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向。目的：探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性。方法：人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于 37 ℃、体积分数为5%CO₂细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养。组织块周边有细胞游离出贴壁生长记作“培养第1天”,每日相差显微镜下观察细胞生长变化。利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达。结果与结论：①改良组织块培养组出膜时间明显短于常规组织块培养组(P < 0.05),出膜率明显高于常规组织块培养组(P < 0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞。ORCID: 0000-0001-8370-174X(许中中) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Cell-matrix biology in vascular tissue engineering

We are developing biocompatible small-calibre vascular substitutes based on polymeric scaffolds that incorporate cell-matrix signals to enhance vascular cell attachment and function. Our graft scaffold comprises an outer electrostatically spun porous polyurethane layer seeded with smooth muscle cells, and a luminal polycaprolactone layer for endothelial cell attachment. Vascular cell adhesion properties of three vascular elastic fibre molecules, tropoelastin, fibrillin-1 and fibulin-5, have been defined, and adhesion fragments optimized. These fragments are being used to coat the scaffolds to enhance luminal endothelial cell attachment, and to regulate smooth muscle cell attachment and function. Tropoelastin-based cell seeding materials are also being developed. In this way, vascular cell-matrix biology is enhancing graft design.     

Modified culture method for human corneal epithelial cells

Summary An improved procedure for the primary culture of pure human corneal epithelial cells from corneal explants is described. Confluent monolayers of epithelial cells can be consistently produced from small segments of donor corneas regardless of donor age and without feeder layers. Incubating segments on collagen at the air-liquid interface significantly improves the yield of cells per cornea and shortens cell migration time as compared to culturing on plastic. Fibroblast contamination is eliminated by serum-free medium and confirmed by indirect immunofluorescent staining for keratin.