离心粘结法制备孔隙连通的三维细胞支架

目的:改进多孔支架制备技术,使多孔支架具有孔隙结构均匀、孔隙连通性良好的特性。方法:间歇离心技术与湿度粘结方法结合,改善致孔剂粘结的均匀性;溶液浇注/颗粒沥析技术制备三维多孔细胞支架;扫描电镜观察支架的孔隙结构,原子吸收光谱检测致孔剂残余,力学实验仪与重量法表征支架的其它物理性能与制备条件的关系。结果:三维多孔支架的孔隙呈球形、分布均匀、孔隙相互连通、通道呈规则的圆形;支架中无残余致孔剂。以聚乳酸为原料制备的支架,其孔隙率、压模量、吸水率分别高达94.7±0.5%、509±6kPa、208.2±20.3%。结论:间歇离心粘结--溶剂浇注/颗粒沥析技术,能够制备出孔隙结构均匀、孔隙相互完全连通的三维细胞支架,支架的孔隙大小和通道尺寸人为可控,支架的孔隙率和强度高,孔隙结构符合组织工程的要求,是一种比较理想的三维细胞支架制备方法。     

溶剂浇铸/颗粒沥滤技术制备组织工程支架材料

生物可降解多孔三维细胞支架是组织工程化组织构建的基础。溶剂浇铸粒子沥滤技术是最简便、也是研究最广泛的一种多孔三维细胞支架制备技术,随着各种改进方法的出现,溶剂浇铸粒子沥滤已成为组织工程用多孔三维细胞支架的理想制备技术 。     

蛙心包淋巴孔的发现

首次报道蛙心包淋巴孔 ,揭示心包腔淋巴转归途径。本实验应用扫描电镜和透射电镜观察心包淋巴孔的超微结构 ,并使用计算机图象处理技术对淋巴孔作定量分析。结果发现 ,正常蛙心包腔面有一些散在分布的心包淋巴孔和少量淋巴窦。构成淋巴孔的间皮细胞常出现粗大的胞质突起 ,伸入淋巴孔 ,形成瓣膜状结构。淋巴孔的平均直径为 0 72±0 33μm ,平均分布密度是 3 57± 2 0 7个 /0 0 1mm2 ;心包间皮淋巴窦的面积是 995 0 8±2 2 1 74μm2 /0 0 1mm2 。蛙心肌无血管 ,其血供仅由心腔内血液直接进入心肌的小梁间隙。心包脏层未发现有淋巴孔。结果表明 :间皮淋巴窦是心包膜正常“漏出”的形态依据。心包淋巴孔的发现 ,证明心包腔淋巴引流途径的存在。淋巴引流对于心肌组织间液的平衡 ,清除组织间液蛋白质 ,防止心肌间质水肿 ,有重要意义     

家兔肋胸膜淋巴孔的发现

为了研究成年家兔肋胸膜淋巴孔的超微结构与三维构形 ,作者应用扫描电镜和透射电镜对成年家兔肋胸膜淋巴孔进行观察 ,用计算机图像处理系统对胸膜淋巴孔作图像数据化处理 ;NaOH溶液消化间皮细胞 ,裸露间皮下结缔组织和筛斑 (maculacribriform)。发现肋胸膜立方形间皮细胞 (cuboidalmesothelialcell)之间有圆形或椭圆形的胸膜淋巴孔 (pleurallymphaticstomata) ,其平均面积和平均密度分别为 :7 2 0± 3 6 9μm2 和 1 2 1±0 72个 / 0 0 1mm2 。扁平形间皮细胞表面未见淋巴孔。胸膜淋巴孔籍胸膜下小管与毛细淋巴管相通。仅在立方形间皮下结缔组织中发现有筛斑。肋胸膜上还可见闭合淋巴孔 (closedlymphaticstomata)和由巨噬细胞组成的乳斑 (milkyspot)。覆盖在肋骨上的胸膜无淋巴孔分布。家兔胸膜淋巴孔通过胸膜下小管与淋巴管直接相连 ,形成从胸膜腔至脉管系的惟一直接通路。     

妊娠和激素干预对豚鼠卵巢囊淋巴孔的影响

本实验以台盼蓝为示踪剂,观察成年雌豚鼠在妊娠、注射促排卵激素和雄激素时,卵巢囊淋巴孔对示踪剂的吸收情况,并结合扫描电镜和计算机图像处理,对豚鼠卵巢囊内壁的淋巴孔进行观察和统计,以研究妊娠和激素干预对豚鼠卵巢囊淋巴孔开放和淋巴吸收功能的影响。结果表明,妊娠组卵巢囊吸收示踪剂的量最多,促排卵组次之,雄激素组最少,且组间两两比较均有显著性意义(P<0.05)。在每1000μm~2的视野中,妊娠组卵巢囊淋巴孔的开放面积为189.9±48.7μm~2,促排卵组为104.4±31.2μm~2,雄激素组为40.5±18.7μm~2,组间两两比较均有显著性意义(P<0.05)。妊娠组卵巢囊内层纤毛柱状上皮的分泌颗粒数量最少,而促排卵组的分泌颗粒较多,且纤毛最活跃。本实验研究结果表明,妊娠和激素干预能影响豚鼠卵巢囊淋巴孔的开放和吸收,囊内卵巢的功能状态与豚鼠卵巢囊淋巴孔的调控密切相关。     

收缩活动促进新生大鼠培养心室肌细胞的~3H-亮氨酸掺入和细胞生长

本工作应用培养的新生大鼠心室肌细胞,以~3H-亮氨酸掺入、细胞直径和细胞体积为指标,观察了收缩活动对心肌蛋白质合成速率及细胞生长的影响。在含10％血清的培养介质中,常钾搏动组的心肌细胞(CMC)的~3H-亮氨酸掺入显著高于高钾停搏组的心肌细胞(QMC),24h的掺入量分别为1229±29与1076±60cpm/10~5细胞(每组n=5,P<0.01),CMC掺入为QMC的114.2％。QMC组细胞直径和体积分别为15.14±0.42μm和1842±123μm~3,而CMC组为16.82±0.64μm和2495±210μm~3,分别比前者高11.1％和35.5％(P<0.01,每组n=6)。这些变化随培养时间的延长更趋明显,但两组间的细胞数目并无显著差异(P>0.05)。上述结果表明:收缩活动本身能促进心肌细胞蛋白质合成和细胞生长。     

纤维蛋白和高分子混合支架的研究及其对大鼠骨髓间充质干细胞增长的影响

通过混合纤维蛋白原和凝血酶溶液与不同量(0、1、2、4 mg)的PLGA无纺丝制得力学强度提高的生物混合支架,检测各组支架对大鼠骨髓间充质干细胞(rMSC)增长的影响.用扫描电镜观察各组支架都具有多孔且孔间相通的特性.根据收缩溶胀的测量,混入PLGA的纤维蛋白支架收缩率明显小于未混合PLGA的支架.检测各组的压缩模量,混合PLGA的支架压缩模量大于未混合的支架,其差异均具有统计学意义.选择具有多向分化潜能的rMSC在混合支架上的生长,进行DNA荧光检测法测得细胞增长值,在混合PLGA无纺丝1 mg的支架上rMSC增长效果最好.实验证明纤维蛋白混合三维支架维持原纤维蛋白支架内部多孔隙三维结构,而且增大了支架的力学强度,在一定程度上提高了骨髓间充质干细胞在支架上的增长,在组织工程中是具有潜力的细胞生长三维支架.     

电刺激提高骨骼肌微纤维致动器致动性能

生物混合致动器是生物混合机器人的重要部分,该研究利用可光交联水凝胶(GelMA)作为小鼠成肌细胞C2C12细胞的细胞间基质,制作了一种直径为500 μm的微纤维致动器,并探究长时间电刺激对微纤维中C2C12细胞分化及致动能力的影响。该研究利用不同电压的方波脉冲信号在细胞分化期间对细胞进行每天15 min的电刺激,同时利用刚性支架固定微纤维以保持其方向与电场方向一致,并对微纤维施加30%机械拉伸。为表征电刺激对细胞分化的影响,利用ImageJ软件统计肌管长度、宽度与取向角,利用肌球蛋白重链(MHC)荧光染色表征其肌球蛋白重链表达,利用ANSYS有限元仿真计算致动器主动张力。结果表明,该微纤维致动器适合C2C12细胞生长并使其分化形成可收缩的骨骼肌,通过分化期间的长时间电刺激可有效促进C2C12细胞的分化、增大骨骼肌中肌管尺寸并提高微纤维致动器致动性能。其中18 V的电刺激效果最为显著,能使肌管长度有效提高30%,肌管宽度提高24%,肌管主动张力提高198%,使其达到0.21 μN。含有性能增强的骨骼肌的微纤维致动器可进一步应用于生物混合机器人组装领域,在药物筛选和骨骼肌再生等基础研究领域...     

四种显微技术观测大鼠颌下腺细胞与丝素-壳聚糖体外共培养的形态学特点

目的采用倒置显微镜、扫描电镜（scanning electron microscopy,SEM）、荧光显微镜和激光共聚焦显微镜（（laser scanning confocal microscopy,LSCM））技术对大鼠颌下腺细胞（rat submandibular gland cells,RSMGs）与丝素-壳聚糖（silk fibroin-chitosan,SFCs）的体外复合培养进行形态学观察。为观测、评估种子细胞在三维支架的内部生长情况提供技术支持。方法取0～8 d龄SD大鼠的颌下腺,对大鼠颌下腺细胞进行原代培养、分离纯化并传代;用抗细胞角蛋白单克隆抗体（CK8）及淀粉酶抗体的免疫细胞化学染色鉴定细胞来源。选取传至第二代的对数生长期的RSMGs作为种子细胞,选取SFCs共混膜（5×5×2）mm作为支架材料构建组织工程化涎腺样结构。将种子细胞与支架材料复合培养并分别于倒置显微镜、SEM、荧光显微镜和LSCM下观察二者复合生长情况。结果倒置显微镜可以直接观察活细胞与支架复合生长情况,方法简单易行。SEM可以较精确的展示细胞支架复合生长的表面超微结构。经过荧光染料的着色,荧光显微镜和LSCM都可以观察到支架上锚定的种子细胞。荧光显微镜可见细胞核的荧光信号均匀的分布在支架孔隙内。LSCM通过层扫描及三维重建技术对较厚的标本获取图像;并可以通过旋转图像,从不同角度观察细胞支架复合物的三维剖面或整体结构,得到更为准确的定位信息。结论四种显微技术均可应用于RSMGs与SFCs体外共培养的形态学观测。LSCM的三维重建技术结合荧光染料标记可以较好地获得RSMGs与SFCs复合生长的情况,有着较广泛的应用价值。     

雪旺氏细胞与同种异体骨支架的体外共培养研究

目的:探讨雪旺细胞(Schwann’s cells,SCs)在同种异体骨支架上的生物相容性,体外构建组织工程骨神经化模型。方法:利用新鲜人体骨骼制备同种异体骨支架材料,检测其物理性能;采用优化方法提取新生SD大鼠坐骨、臂丛神经培养SCs,实验分为三维培养实验组(SCs+同种异体骨)、二维培养对照组(SCs+胶原玻片),S-100抗体免疫荧光染色鉴定SCs纯度;细胞计数法检测两组细胞增殖特点;细胞接种后第3、7天取样,扫描电镜观察。结果:同种异体骨支架具有良好的三维孔隙结构,适宜细胞贴附生长;S-100免疫荧光染色证实SCs纯度95%;扫描电镜检测显示两组SCs均可正常粘附增殖,细胞间排布规律相似,培养早期实验组SCs胞体更加细长,伪足更加明显,随着培养时间的延长表现出较强的迁移能力;细胞增殖检测:两组SCs生长曲线特征基本一致,支架材料对SCs无毒性作用。结论:同种异体骨支架SCs具有良好的生物相容性,其三维立体多孔结构有利于SCs的粘附与迁移,初步构建了体外组织工程骨神经化模型。     

Photoinitiated cross-linking of the biodegradable polyester poly(propylene fumarate). Part II. In vitro degradation

This study investigated the in vitro degradation of both solid PPF networks and porous PPF scaffolds formed by photoinitiated cross-linking of PPF polymer chains. Three formulations of scaffolds of differing porosity and pore size were constructed by varying porogen size and content. The effects of pore size and pore volume on scaffold mass, geometry, porosity, mechanical properties, and water absorption were then examined. Throughout the study, the solid networks and porous scaffolds exhibited continual mass loss and slight change in length. Porogen content appeared to have the greatest effect upon physical degradation. For example, scaffolds initially fabricated with 80 wt % porogen content lost approximately 30% of their initial PPF content after 32 weeks of degradation, whereas scaffolds fabricated with 70 wt % porogen content lost approximately 18% after 32 weeks of degradation. For all scaffold formulations, water absorption capacity, porosity, and compressive modulus were maintained at constant values following porogen leaching. These results indicate the potential of photo-cross-linked PPF scaffolds in tissue engineering applications which require maintenance of scaffold structure, strength, and porosity during the initial stages of degradation.     

Development of biodegradable scaffolds based on magnetically guided assembly of magnetic sugar particles

Biodegradable scaffolds with controlled pore layout and porosity have great significance in tissue engineering for cell penetration, tissue ingrowth, vascularization, and nutrient delivery. Porogen leaching has been commonly used to control pore size, pore structure and porosity in the scaffold. In this paper we focus on the use/development of two magnetically guided porogen assembly methods using magnetic sugar particles (MSPs) for scaffold fabrication. First, a patterning device is utilized to align MSPs following designed templates. Then a magnetic sheet film is fabricated by mixing poly(vinyl alcohol, PVA) and NdFeB powder for steering the MSPs. After poly(l-lactide-co-?-caprolactone) (PLCL) casting and removal of the sugar template, a scaffold with spherical pores is obtained. The surface and the inner structure of the scaffolds are evaluated using light and electron micrographs showing their interconnection of pores, pore wall morphology and porosity. Single layer scaffolds with the size of 8mm in width and 10mm in length were constructed with controllable pore diameters in the ranges of 105-150 μm, 250-300 μm and 425-500 μm.     

Porogen Effect on Structural and Physical Properties of β-TCP Scaffolds for Bone Tissue Regeneration

Scaffolds for bone tissue applications have been an outstanding alternative to repair and regenerate bone tissue defects caused by traumas or illness. There are many methods available to fabricate porous scaffold such as solvent casting, gas bubble, phase separation, electrospinning, particle-leaching, among others. The particle-leaching technique has shown advantages in bone tissue regeneration applications, the main benefit of this technique is related to the porogen particle size and the porogen content in the manufacture of scaffolds. Tricalcium phosphate is one calcium phosphate that presented appropriated characteristic to be used for bone tissue engineering due to the chemical properties similar to the human bones. Scaffolds of tricalcium phosphate β phase were made using sugar particles. The porogen was varied in amounts of 50, 60 and 70 wt.% of two commercial sugars with the remainder of the composition made up of tricalcium phosphate powders. The pore sizes in all the scaffolds were in the range of 90 to 600 μm with an irregular pore morphology and the porosity was in the range of 63 to 77%.     

A new generation of sodium chloride porogen for tissue engineering

Porogen leaching is a widely used and simple technique for the creation of porous scaffolds in tissue engineering. Sodium chloride (NaCl) is the most commonly used porogen, but the current grinding and sieving methods generate salt particles with huge size variations and cannot generate porogens in the submicron size range. We have developed a facile method based on the principles of crystallization to precisely control salt crystal sizes down to a few microns within a narrow size distribution. The resulting NaCl crystal size could be controlled through the solution concentration, crystallization temperature, and crystallization time. A reduction in solution temperature, longer crystallization times, and an increase in salt concentration resulted in an increase in NaCl crystal sizes due to the lowered solubility of the salt solution. The nucleation and crystallization technique provides superior control over the resulting NaCl size distribution (13.78 ± 1.18 μm), whereas the traditional grinding and sieving methods produced NaCl porogens 13.89 ± 12.49 μm in size. The resulting NaCl porogens were used to fabricate scaffolds with increased interconnectivity, porous microchanneled scaffolds, and multiphasic vascular grafts. This new generation of salt porogen provides great freedom in designing versatile scaffolds for various tissue-engineering applications.     

Freestanding stacked mesh‐like hydrogel sheets enable the creation of complex macroscale cellular scaffolds

Hydrogel‐based bottom‐up tissue engineering depends on assembly of cell‐laden modules for complex three‐dimensional tissue reconstruction. Though sheet‐like hydrogel modules enable rapid and controllable assembly, they have limitations in generating spatial microenvironments and mass transport. Here, we describe a simple method for forming large‐scale cell‐hydrogel assemblies via stacking cell‐embedded mesh‐like hydrogel sheets to create complex macroscale cellular scaffolds. Freestanding stacked hydrogel sheets were fabricated for long‐term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets (8.0 mm × 8.7 mm) were aligned using a polydimethylsiloxane drainage well. The stacked hydrogel sheets were precisely aligned so that the openings could facilitate mass transport through the stacked sheets. Despite the relatively large height of the stacked structure (400–700 μm), which is larger than the diffusion limit thickness of 150–200 μm, the freestanding cell‐ydrogel assemblies maintained cell viability and exhibited enhanced cellular function compared with single hydrogel sheets. Furthermore, a three‐dimensional co‐culture system was constructed simply by stacking different cell‐containing hydrogel sheets. These results show that stacked hydrogel sheets have significant potential as a macroscale cell‐culture and assay platform with complex microenvironments for biologically relevant in vitro tissue‐level drug assays and physiological studies.     

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug‐induced liver injury (DILI), and has led to a demand for robust, human cell‐based, in vitro assays for drug discovery. Microporous polymer‐based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high‐throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi‐like structures. Poly‐l‐ lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 μm; B: 175 ± 6 μm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost‐effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer‐based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505–514, 2018     

Systematic investigation of porogen size and content on scaffold morphometric parameters and properties

A systematic investigation of tissue engineering scaffolds prepared by salt leaching of a photopolymerized dimethacrylate was performed to determine how the scaffold structure (porosity, pore size, etc.) can be controlled and also to determine how the scaffold structure and the mechanical properties are related. Two series of scaffolds were prepared with (1) the same polymer-to-salt ratio but different salt sizes (ranging from average size of 100 to 390 microm) and (2) the same salt size but different polymer-to-salt ratios (ranging from salt mass of 70 to 90%). These scaffolds were examined to determine how the fabrication parameters affected the scaffold morphometric parameters and corresponding mechanical properties. Combined techniques of X-ray microcomputed tomography (microCT), mercury porosimetry, and gravimetric analysis were used to determine the scaffold parameters, such as porosity, pore size, and strut thickness and their size distributions, and pore interconnectivity. Scaffolds with porosities ranging from 57% to 92% (by volume) with interconnected structures could be fabricated using the current technique. The porosity and strut thickness were subsequently related to the mechanical response of the scaffolds, both of which contribute to the compression modulus of the scaffold. The current study shows that the structure and properties of the scaffold could be tailored by the size and the amount of porogen used in the fabrication of the scaffold.     

Effect of some factors on fabrication of poly(L-lactic acid) microporous foams by thermally induced phase separation using N,N-dimethylacetamide as solvent

Biocompatible, highly interconnected microporous poly(L-lactic acid) (PLLA) foams or scaffolds with nano-fibrous structure, containing pores with diameters of 0.1-3.5 μm and fibers with diameters of 300-700 nm scale, were prepared through the thermally induced liquid-liquid phase separation (TIPS) method using N,N'-dimethyl acetamide (DMAc) as solvent. Various foam morphologies were obtained by changing parameters involved in the TIPS process, such as polymer concentration, solvent composition, and quenching temperatures. The morphology of different foams was examined by scanning electron microscopy, whereas the pore size and the pore size distribution were calculated. The results showed that most porous foams presented nano-fibrous structure with interconnected open pores. In the case of using DMAc as solvent, with increasing polymer concentration, either the average pore diameter or the pore size distribution exhibited a maximum value at 0.05 g/mL polymer concentration and quenching temperature of -30°C. It was found that all the pore size distribution fit the F-distribution equation. With increasing the quenching temperature from -30°C to -10°C, the maximum average pore diameter of the foams decreased and the pore size distribution became narrower, whereas the polymer concentration exhibiting the maximum pore size and widest pore size distribution increased from 0.05 g/mL to 0.07 g/mL. In the case of using the mixed solvent of DMAc/DOX (1,4-dioxane) from 9/1 to 7/3 (v/v) there appeared a maximum value of average pore diameter and a widest pore size distribution all at 0.05 g/mL PLLA concentration and quenching temperature of -30°C. The maximum pore size tends to increase with increasing DOX content.     

Fabrication of highly porous scaffolds for tissue engineering based on star-shaped functional poly(ε-caprolactone)

The potential of novel functional star‐shaped poly(ε‐caprolactone)s of controlled molecular weight and low molecular weight distribution bearing acrylate end groups as material for biomedical applications was demonstrated in this study. The polymers were functionalized via Michael‐type addition of amino acid esters containing amino or thiol groups showing the potential for immobilization of biomolecules. Furthermore, scaffolds of different geometries were prepared by uniaxial freezing of polymer solutions followed by freeze drying. Different solvents and polymer concentrations were investigated, resulting in scaffolds with porosities between 76 and 96%. Mechanical properties of the scaffolds were investigated and the morphology was determined via scanning electron microscopy. Scaffolds with interconnected channels were prepared using benzene, 1,2‐dichloroethane or dioxane as solvent. The tubular longitudinal pores in honeycomb arrangement extend throughout the full extent of the scaffolds (typical pore sizes: 20–100 µm). Biotechnol. Bioeng. 2011; 108:694–703. © 2010 Wiley Periodicals, Inc.     

Tissue engineering scaffolds based on photocured dimethacrylate polymers for in vitro optical imaging

Model tissue engineering scaffolds based on photocurable resin mixtures with sodium chloride have been prepared for optical imaging studies of cell attachment. A photoactivated ethoxylated bisphenol A dimethacrylate was mixed with sieved sodium chloride (NaCl) crystals and photocured to form a cross-linked composite. Upon soaking in water, the NaCl dissolved to leave a porous scaffold with desirable optical properties, mechanical integrity, and controlled porosity. Scaffolds were prepared with salt crystals that had been sieved to average diameters of 390, 300, 200, and 100 microm, yielding porosities of approximately 75 vol %. Scanning electron microscopy and X-ray microcomputed tomography confirmed that the pore size distribution of the scaffolds could be controlled using this photocuring technique. Compression tests showed that for scaffolds with 84% (by mass fraction) salt, the larger pore size scaffolds were more rigid, while the smaller pore size scaffolds were softer and more readily compressible. The prepared scaffolds were seeded with osteoblasts, cultured between 3 and 18 d, and examined using confocal microscopy. Because the cross-linked polymer in the scaffolds is an amorphous glass, it was possible to optically image cells that were over 400 microm beneath the surface of the sample.