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1.
目的:三维骨微管结构支架构造方法研究以及成骨细胞复合后的体外培养,观察这种能够为细胞生长提供三维骨微管结构的支架对细胞贴附、生长、增殖以及分化的影响。方法:应用快速成形技术制造支架负型模具,在模具中填充CPC材料,待其固化后,去除模具,形成具有内部相互连通微管的三维支架。复合成骨细胞,进行体外培养。分别于第4d和14d取出样本,用扫描电镜观察细胞生长情况。结果:利用光固化快速成形技术间接构造所得三维支架,具有很好的三维立体结构。扫描电镜下观察,成骨细胞在三维支架表面和微管内贴附生长状况良好,并分泌大量基质。结论:三维骨微管结构支架的快速成形间接构造方法应用于骨组织工程中支架的构造是可行的,所构造的CPC支架结构能够使细胞在其表面和微管内生长、增殖和分化。  相似文献   

2.
构建一种旋转灌注式生物反应器系统,并设计制造具有相互连通微管道结构的大段支架。生物反应器在旋转的同时实现对大段支架的灌注,系统内外气体的实时交换通过安装在反应器两端的半透膜和可透气的蠕动泵软管实现。容器内的氧气和营养物质在得到充分混合之后,连续不断地输送到支架微管道内。使黏附在微管道内的细胞获得充足营养的同时受到一定流体剪应力的刺激,调节细胞功能的发挥。该系统克服了静态培养中存在的各种缺点,改善了培养环境,增加了培养过程的可控性,有助于促进黏附在微管道内部的细胞增殖、分化和产生大量基质。将成骨细胞/支架结构体在该生物反应器系统中培养14d后,用扫描电镜观察细胞在大段支架微管道内的生长情况,结果表明,支架微管道内有大量细胞长入。  相似文献   

3.
背景:微载体培养技术作为一项体外高浓度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。目的:对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。方法:以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组;无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数,对实验组进行形态学观察,包括倒置相差生物显微镜观察和扫描电子显微镜观察。结果与结论:两组培养的细胞数量均呈现前3 d增长,在培养第3天细胞数量达到最高值;实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P0.05),实验组各样本之间细胞数差异无显著性意义(P0.05)。倒置相差生物显微镜下动态观察,可见前3 d微载体表面黏附生长的肝细胞则逐渐增多,培养第3天可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜观察,微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。结果说明,以自制的壳聚糖球形多孔微载体作为一种支架,在体外三维环境下可以进行高浓度细胞培养。  相似文献   

4.
Atelocollagen胶原支架在三维培养大鼠心肌中的生物相容性   总被引:1,自引:1,他引:0  
目的探讨atelocollagen胶原支架在三维培养心肌细胞中的生物相容性,寻找三维培养心肌组织的条件和理想的支架材料。方法原代培养的大鼠心肌细胞经纯化后,将细胞悬液接种到胶原纤维支架上,动态观察第8d、16d、20d在atelocollagen胶原纤维支架上生长的细胞的变化,并进行光镜、扫描电镜和透射电镜观察。结果接种后第1d形成细胞-胶原支架搏动复合体,细胞与支架一起有节律的跳动;心肌细胞填充于支架网孔周围,并与支架融合;在透射电镜下,3个时间段均发现细胞与支架紧密相贴,融合在一起;在扫描电镜下发现,细胞在胶原纤维支架上贴附并充分伸展,相互融合成片。结论atelocollagen胶原支架的生物相容性较好,可作为三维培养细胞和组织的天然材料,适于心肌组织的立体培养。  相似文献   

5.
目的三维立体空间动态诱导骨髓间充质干细胞软骨向分化,分析培养状态对软骨向分化的影响。方法分离培养骨髓间充质干细胞(MSC),以海藻酸钠凝胶载体诱导软骨向分化,于转壁反应器制造周期应力性三维立体环境,比较凝胶立体结构、细胞聚集状态、细胞构型等因素对软骨向分化的影响,分别以凝胶微球悬浮高密度细胞、凝胶包埋离心细胞团、凝胶覆盖细胞及普通细胞培养等方式.进行空间动态诱导培养:同时各培养方式分别设立相应静止培养方式作为对照。10d后观察大体和组织学形态.测量生物化学指标并筛选。结果三维诱导方式比平面诱导有效,动态诱导环境优于静止诱导环境.结合三维立体空间培养和动态环境可进一步提高软骨诱导效率。其中.凝胶微球悬浮高密度细胞的培养方式分化效果最好.优于凝胶包埋细胞团的培养方式。结论海藻酸钠凝胶微球立体动态培养可有效诱导MSC的软骨向分化.为改进软骨组织工程种子细胞的立体空间动态培养提供新思路。  相似文献   

6.
骨组织微结构观察分析及仿生支架立体光固化间接制造   总被引:5,自引:0,他引:5  
通过对骨组织显微结构观察分析,指导人工骨支架内部微管道结构设计,结合CAD、反求工程和快速成形技术制造仿生结构生物活性人工骨支架.观察骨组织切片,获取骨组织微观结构数据,进行三维重构和辅助设计,应用快速成形技术制造相应的支架模具.在模具中填充磷酸钙骨水泥,烧结后得到仿生结构生物活性人工骨支架.光学显微镜和扫描电镜观察和测量支架微结构,X射线衍射分析磷酸钙骨水泥在热分解前后的主要成分,结果表明,所得支架与设计相符,热分解前的磷酸钙骨水泥主要成分为低结晶度的羟基磷灰石,而热分解之后为结晶度更高的羟基磷灰石.体外培养试验表明支架无细胞毒性,且黏附在支架上的细胞保持着良好的形态和功能发挥.  相似文献   

7.
目的初步探讨模拟微重力对许旺细胞三维培养的影响.方法分别把粘附在聚羟基乙酸支架上生长3、7、10及14天的许旺细胞和新鲜分离的原代许旺细胞悬液连同聚羟基乙酸支架送入转壁式生物反应器(RWVB)中旋转培养5天,观察细胞在支架上的生长情况及超微结构的改变.结果粘附在支架上的许旺细胞进入模拟微重力环境后细胞变圆变小,核染色质聚集,细胞器水肿扩张,核/浆比例明显增大,微丝微管松散紊乱,细胞在3天内快速凋亡或碎裂溶解;尚未贴壁的原代许旺细胞进入模拟微重力环境后在支架表面均匀贴壁生长、增殖,蛋白合成活跃,个别细胞内可见髓鞘样结构,5天内未见细胞死亡现象.结论贴壁生长3~14天的许旺细胞进入模拟微重力环境后表现为快速凋亡、溶解,而未贴壁的原代许旺细胞进入模拟微重力环境后则功能活跃表达及快速增殖,可用于构建比较理想的许旺细胞三维培养体系.  相似文献   

8.
背景:与传统二维培养相比,三维培养软骨微组织具有更大的优势,但仍需进一步探索更有利的三维培养方式。目的:评价2种三维培养方式下微组织的细胞行为及促软骨形成能力。方法:通过化学脱细胞方法和组织粉碎方法制备软骨源性微载体,采用DNA定量和核染色验证脱细胞是否成功,通过组织学染色观察脱细胞前后基质保留情况,采用扫描电子显微镜和CCK-8方法对微载体进行表征;通过三维静态培养法和三维动态培养法将软骨源性微载体与人脂肪间充质干细胞结合构建软骨源性微组织,利用扫描电子显微镜、活死染色、RT-q PCR等手段检测两组微组织的细胞活力及成软骨能力。结果与结论:(1)成功制备软骨源性微载体,与脱细胞前相比,脱细胞后DNA含量显著降低(P <0.001);扫描电子显微镜观察微载体表面有胶原包绕,保持天然软骨细胞外基质特征;CCK-8法检测表明微载体无细胞毒性且能够促进细胞增殖;(2)扫描电子显微镜及活死染色结果显示,相比三维静态组,三维动态组微组织细胞具有更舒展的形态,细胞与细胞间、细胞与基质间、基质与基质间形成广泛的连接;(3)RT-qPCR结果表明两组微组织SOX9、蛋白聚糖、Ⅱ型胶原表达在培养...  相似文献   

9.
体外LAK细胞杀伤人直肠腺癌细胞时的细胞骨架变化   总被引:1,自引:0,他引:1  
应用免疫荧光细胞化学和透射电镜观察体外LAK细胞杀伤人直肠腺癌细胞时效靶细胞内细胞骨架的变化。结果表明:两种细胞相互识别后均出现趋向运动,趋向运动的细胞突起内含有丰富的微丝,微丝的排列与细胞运动的方向一致。效靶细胞相互接触后,两种细胞浆内的微管均出现重排现象。两种细胞质膜接触处可见成束的微丝及微管,微丝及微管集中处含有丰富的溶酶体颗粒。微丝及微管抑制试验均不出现上述变化。结果提示:微丝可能在效靶细  相似文献   

10.
本研究目的是观测硫酸肝素-胶原蛋白支架材料对Schwann细胞的相容性,采用: (1)冷冻干燥法制备硫酸肝素-胶原蛋白支架材料,培养、纯化并鉴定Schwann细胞后,将Hoechst荧光标记的Schwann细胞复合于硫酸肝素-胶原支架材料中,荧光显微镜、扫描电镜及光镜下观察其在材料内部的空间排列形式; (2)以台盼蓝染色、MTT法检测Schwann细胞与硫酸肝素-胶原蛋白共培养时的生物活性。结果显示:作者制备的支架材料具有纵行的、平行排列的微管结构,Schwann细胞在支架材料微管内成线性平行排列,类似于神经基底膜与Schwann细胞形成的Büngner带;台盼蓝染色Schwann细胞死亡率仅6. 47%,MTT法证明Schwann细胞与硫酸肝素-胶原蛋白材料共培养时保有高度的生物活性。本研究结果表明:以硫酸肝素复合胶原蛋白结合特定条件下冷冻干燥技术可以制备在组分和内部空间结构上高度仿生神经的新型神经组织工程支架材料,与Schwann细胞有良好的细胞相容性。  相似文献   

11.
Current methods for formation of microvascular channel scaffolds are limited with non-circular channel cross-sections, complicated fabrication, and less flexibility in microchannel network design. To address current limitations in the creation of engineered microvascular channels with complex three-dimensional (3-D) geometries in the shape of microvessels, we have developed a reproducible, cost-effective, and flexible micromanufacturing process combined with photolithographic reflowable photoresist and soft lithography techniques to fabricate cylindrical microchannel and networks. A positive reflowable photoresist AZ P4620 was used to fabricate a master microchannel mold with semi-circular cross-sections. By the alignment and bonding of two polydimethylsiloxane (PDMS) microchannels replicated from the master mold together, a cylindrical microchannel or microchannel network was created. Further examination of the channel dimensions and surface profiles at different branching levels showed that the shape of the microfluidic channel was well approximated by a semi-circular surface, and a multi-level, multi-depth channel network was created. In addition, a computational fluidic dynamics (CFD) model was used to simulate shear flows and corresponding pressure distributions inside of the microchannel and channel network based on the dimensions of the fabricated channels. The fabricated multi-depth cylindrical microchannel network can provide platforms for the investigation of microvascular cells growing inside of cylindrical channels under shear flows and lumen pressures, and work as scaffolds for the investigation of morphogenesis and tubulogenesis.  相似文献   

12.
Engineered smooth muscle tissue requires ordered configurations of cells to reproduce native function, and microtechnology offers possibilities for physically and chemically controlling cell organization with high spatial resolution. In this work, poly(dimethylsiloxane) microchannel scaffolds, modified by layer-by-layer self-assembly of polyelectrolytes to promote cell adhesion, were evaluated for use as substrates for the culture of aligned smooth muscle cells. The hypothesis that narrower channels would result in better alignment was tested using channel width dimensions of 20, 30, 40, 50, and 60 microm, in addition to flat (control) surfaces. Alignment of cells was assessed by two different methods, each sensitive to a different aspect of cell alignment from fluorescence micrographs. Two-dimensional fast Fourier transform analysis was performed to analyze the orientation distribution of actin filaments in cells. This was complemented by connectivity analysis of stained nuclei to obtain nuclear orientation distributions. Both methods produced consistent data that support the hypothesis that narrow microchannels promote a highly aligned culture of smooth muscle cells, and the degree of alignment is dependent on the microchannel width. Precise replication of in vivo cell alignment in engineered tissue, with the ability to tailor specific surface chemistries of the scaffold to the desired application, will potentially allow the production of artificial tissue that more closely duplicates the structure and function of native tissue.  相似文献   

13.
For tissue-engineered vascular grafts to reach their full potential, three-dimensional (3D) cellular micro-integration will be necessary. In this study, we utilize femtosecond laser ablation to produce microchannels inside electrospun polycaprolactone (PCL) scaffolds. These microchannels potentially provide spatially controlled cell distributions approaching those observed in vivo. The ability of such laser-ablated microchannels to direct cell seeding was evaluated. The dimensions chosen were 100 μm wide, 100 μm deep and 10 mm long. Femtosecond laser ablation successfully produced these microchannels in the scaffolds without substantially altering the ~900 nm diameter fibers. Flow within these microchannels was studied by injecting fluorescent polystyrene bead solutions. Direct measurement of bead motion yielded an inlet velocity of 2.78 cm s(-1). This was used for modeling two-dimensional (2D) flow using computational fluid dynamics to estimate flow profiles within the microchannel. Successful demonstrations of bead flow were followed by seeding of 500,000 human coronary artery smooth muscle cells (HCASMCs) in proliferative medium at a rate of ~500 μL min(-1). Confocal microscopy and scanning electron microscopy confirmed that the HCASMCs were seeded down the full 10-mm length of the microchannel and stayed within its boundaries. Both nuclei and F-actin were observed within the seeded cells. The presence of F-actin filaments shows that the cells were adhered strongly to the scaffold and remained viable throughout the culture. The concept of "vascular wall engineering" producing intricate cell seeding through microchannels produced via femtosecond laser ablation was validated.  相似文献   

14.
The medial layer of small diameter blood vessels contains circumferentially aligned vascular smooth muscle cells (vSMC) that possess contractile phenotype. In tissue-engineered constructs, these cellular characteristics are usually achieved by seeding planar scaffolds with vSMC, rolling the cell-laden scaffold into a tubular structure, and maturing the construct in a pulsatile bioreactor, a lengthy process that can take up to two months. During the maturation phase, the cells circumferentially orient, their contractile protein expression increases, and they obtain a contractile phenotype. Generating cell culture platforms that enable the rapid production of directionally oriented vSMC with increased contractile protein expression would be a major step forward for blood vessel tissue engineering and would greatly facilitate the in vitro study of vSMC biology. Previously, we developed a micropatterned cell culture surface that promotes orientation and contractile protein expression of vSMC. Herein, we explore two potential applications of this technology. First, we fabricate tubular and biodegradable scaffolds that possess the micropatterning on their exterior surface. When vSMC are seeded on these scaffolds, they initially proliferate in order to fill the microchannels and as confluence is reached the cells align in the direction of the micropatterning resulting in a biodegradable scaffold that is inhabited by circumferentially aligned vSMC within a week. Second, we illustrate that we can generate biostable cell culture surfaces that allow the in vitro study of the cells in a more contractile state. Specifically, we explore contractile protein expression of cells cultured on the micropatterned surfaces with the addition of soluble transforming growth factor beta one (TGFβ1).  相似文献   

15.
Various micro cell culture systems have recently been developed. However, it is extremely difficult to recover cultured cells from a microchannel because the upper and lower substrates of a microchip are permanently combined. Therefore, we developed a cell culture and recovery system that uses a separable microchip with reversible combining that allows separation between closed and open channels. To realize this system, two problems related to microfluidic control-prevention of leakage and non-invasive recovery of cultured cells from the substrate-must be overcome. In the present study, we used surface chemistry modification to solve both problems. First, octadecyltrimethoxysilane (ODTMS) was utilized to control the Laplace pressure at the liquid/vapor phase interface, such that it was directed toward the microchannels, which suppressed leakage from the slight gap between two substrates. Second, a thermoresponsive polymer poly(N-isopropyl acrylamide) (PNIPAAm) was used to coat the surface of the ODTMS-modified microchannel by UV-mediated photopolymerization. PNIPAAm substrates are well known for controlled cell adhesion/detachment by alteration of temperature. Finally, the ODTMS- and PNIPAAm-modified separable microchips were subjected to patterning, and human arterial endothelial cells (HAECs) were cultured in the resulting microchannels with no leakage. After 96 h of the culture, the HAECs were detached from the microchips by decreasing the temperature and were then recovered from the microchannels. This study is the first to demonstrate the recovery of living cells cultured in a microchannel, and may be useful as a fundamental technique for vascular tissue engineering.  相似文献   

16.
The development of 3D scaffolds consisting of stacked multi-layered porous sheets featuring microchannels is proposed and investigated in this work. In this concept, the inner-porosity of the sheets allows diffusion of nutrients and signalling products between the layers whereas the microchannels facilitate nutrient supply on all layers as they provide space for the culture medium to be perfused throughout the scaffold. Besides the above, these scaffolds have excellent distribution of the cells as seeding and attaching of the cells occurs on individual layers that are subsequently stacked. In addition, these scaffolds enable gaining local data from within the scaffolds as unstacking of the stacked layers allows for determination of various parameters per layer. Here, we show the proof of this concept by culturing C2C12 pre-myoblasts and A4-4 cells on stacked Poly(l-lactic acid) (PLLA) sheets featuring microchannels. The results obtained for culturing under static conditions clearly indicate that despite inhibited cell proliferation due to nutrient limitations, diffusion between the layers takes place and cells on various layers stay viable and also affect each other. Under dynamic conditions, medium flow through the channels improves nutrient availability to the cells on the various layers, drastically increasing cell proliferation on all layers.  相似文献   

17.
Morphogenesis is a fundamental process by which new blood vessels are formed during angiogenesis. The ability to control angiogenesis would lead to improvements in tissue engineering constructions; indeed, the study of angiogenesis has numerous clinical applications, for example, in the investigation of metastatic cancer, peripheral and coronary vascular disease, and wound healing. Conventional in vitro organotypic cell culture approaches to these studies are limited primarily by their reliance on microvascular vessel formation through a random process of morphogenesis that lacks the spatial reproducibility and orientation needed for high-throughput drug testing. We have developed a bioreactor system for scaffold-guided tubulogenesis coupled with 3-D organotypic culture to spatially control vessel formation and its orientation. To create microchannels to guide microvessel formation, we fabricated rigid scaffolds using photolithography and light curing epoxy, and soft scaffolds formed by a polydimethylsiloxane (PDMS) stamp directly into collagen. Scaffolds seeded with dermal microvascular endothelial cells were placed between gelled layers of collagen containing dermal fibroblasts within a Transwell filter system and cultured for up to 2 weeks to allow for vessel maturation. Morphological analysis of thin tissue sections following standard histology and immunohistochemical detection of endothelial cells, fibroblasts, and basement membrane confirmed vessel formation along the microchannel walls with either scaffold. This system may also provide a means to explore revascularization within decellularized extracellular matrices, the culture of microvessel networks with controlled geometries, and possibly the spatial guidance of angiogenesis for interfacing with an external microfluidic supply network. As a new tool for guided angiogenesis, our approach introduces new possibilities for identification of anti-angiogenic therapeutics.  相似文献   

18.
Adipose derived stem cells (ADSCs) were cultured on collagen–silk fibroin films with microchannel and micropillar patterns to investigate the effects of cell morphology changes on osteogenic differentiation. Channel and pillar micropatterned films were prepared from collagen type I and silk fibroin. While higher ADSC proliferation profiles were obtained on micropillar blend film, microchannel blend films, however, caused twice higher aspect ratio and effective orientation of cells. Alkaline phosphatase activity of ADSCs was several times higher on microchannel surface when the measured activities were normalized to cell number. Effective deposition of collagen type I and mineral by the cells were observed for patterned and unpatterned films, and these extracellular matrix components were oriented along the axis of the microchannels. In conclusion, the use of collagen–fibroin blend film with microchannel topography increased the aspect ratio and alignment of cells significantly, and was also effective in the differentiation of ADSCs into osteogenic lineage  相似文献   

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