组织工程软骨生物反应器的设计

目的 根据流体力学原理优化反应器内腔结构,并设计一套组织工程软骨生物反应器.方法 采用计算机仿真的方法对反应器内腔流场进行仿真计算,通过流场分析确定内腔结构,最终构建一套完整的生物反应器系统.结果 确定了生物反应器的内腔结构,生物反应器由控制系统和细胞培养室两部分构成,能置于培养箱中对软骨细胞材料复合物进行动态培养.结论 反应器内腔的结构是合理的.整个生物反应器系统运行可靠.     

组织工程软骨生物反应器的设计

目的 根据流体力学原理优化反应器内腔结构,并设计一套组织工程软骨生物反应器.方法 采用计算机仿真的方法对反应器内腔流场进行仿真计算,通过流场分析确定内腔结构,最终构建一套完整的生物反应器系统.结果 确定了生物反应器的内腔结构,生物反应器由控制系统和细胞培养室两部分构成,能置于培养箱中对软骨细胞材料复合物进行动态培养.结论 反应器内腔的结构是合理的.整个生物反应器系统运行可靠.     

软骨细胞与静电纺丝聚已内酯支架灌流培养构建组织工程软骨的实验研究

目的 采用静电纺丝聚已内酯(polycaprolactone,PCL)支架与软骨细胞复合培养,比较静态和灌流生物反应器培养条件下对细胞增殖及基质分泌的影响.方法 构建PCL支架,自制灌流生物反应器,分离兔软骨细胞,培养后接种于PCL支架,分为灌流培养组和静态培养组.在培养第3、7、14天对支架-细胞复合体行扫描电镜观察,DNA、糖胺聚糖和总胶原定量检测;在培养第14天分析软骨特异性基因表达并观察软骨基质分泌情况.结果 电镜观察PCL支架纤维直径(1.67±0.76) μm,孔径(17.65土7.11)μm,可见支架中软骨细胞黏附生长良好,灌流培养条件下细胞增殖快,且较好地保持了软骨细胞特征形态.在培养第7天,灌流培养组DNA定量高于静态培养组;在培养第3、7和14天,灌流培养组糖胺聚糖定量均高于静态培养组,灌流培养组糖胺聚糖/DNA比值均高于静态培养组.在培养第14天,灌流培养组Ⅱ型胶原、蛋白聚糖基因表达增加;软骨分化指数高于静态培养组.在培养第14天,组织学染色可见灌流培养促进细胞的增殖和渗透生长,提高了软骨基质的分泌,并见软骨陷窝样结构.结论 在灌流生物反应器培养条件下,静电纺丝PCL支架与软骨细胞复合培养可促进软骨细胞的增殖和基质的分泌,提高了组织工程软骨的质量.     

生物反应器系统在软骨组织工程中的应用

关节软骨的退行性变是当今社会主要的健康问题之一。据文献报道关节软骨的再生能力有限,到现在为止还没有一种十持久而且有效的治疗方法来替代关节软骨。组织工程是一个非常有潜力的领域,虽然要建立一套常规治疗软骨缺损的组织工程理论还有很多障碍,但是为治疗软骨损伤为目的的组织工程已经能够快速可靠的培养出适合的软骨组织。生物反应器和一些相关的设备为组织工程提供了一个快速且有效的方法。事实上,在体内生理刺激影响着软骨的功能,所以我们要设计一些特别的生物反应器来对在体外培养的关节软骨施加模拟应力的传导,像流体剪切力,流体静压力,周期压力,和一些混合力。本篇综述总结了一些软骨细胞反应器系统在细胞刺激和培养中的应用。     

转壁生物反应器中动态分化组织工程软骨的实验研究

目的 :在转壁生物反应器动态环境中分化骨髓间充质干细胞 (bMSC) ,制备立体组织工程软骨。方法 :穿刺吸取成年兔骨髓 ,分离扩增bMSC ,复合于纤维蛋白凝胶制成圆柱状三维组织 (细胞密度2 0× 10 7/ml) ,置于转壁生物反应器中进行分化培养 ,同时设单纯静止培养组。 5周后观测大体形态和组织学形态、Ⅰ和Ⅱ型胶原免疫组织化学表达 ,测量分化组织的细胞活力。结果 :动态培养可以有效保持材料大体形态 ,甲苯胺兰染色阳性 ,无明显Ⅰ型胶原表达 ,大量表达Ⅱ型胶原 ,人工组织的细胞活力显著高于单纯静止培养方法。结论 :转壁生物反应器培养明显改善组织工程软骨的活力 ,有利于进行三维材料体外软骨分化。     

软骨组织工程生物反应器研究进展

软骨组织工程技术的发展已进入体外构建的关键阶段.生物反应器能在体外动态模拟体内微环境中物质转运和流体应力刺激反应,从而实现体外经生物力学途径调控软骨细胞的基因表达和增殖.传统的二维或静态压力型生物反应器难以构建功能接近于正常的软骨,各种可即时智能调控的微重力、灌流-液压、微栽体等新型生物反应嚣使得培养基环境的调控更加高...     

一种新型血管生物反应器的研究与开发

目的 根据流体力学的分析改进支撑PGA-细胞材料复合物的硅胶管,设计一种应用于体外培养组织工程血管的生物反应器.方法 利用计算机仿真分析的方法对硅胶管内腔的压力场进行仿真计算,通过计算分析确定硅胶管的结构,进而开发一种新型血管生物反应器.结果 确定了硅胶管的尺寸结构以及培养室内相应的辅助结构,使整个反应器系统能够对PGA-细胞材料复合物进行动态培养.结论 改进后的硅胶管结构合理,整个血管生物反应器系统运行稳定.     

三种不同方式构建组织工程软骨修复犬膝关节骨软骨缺损对比的实验研究

[目的]通过对比三种不同方式构建的骨软骨复合体修复比格犬膝关节骨软骨缺损的修复情况,探讨构建组织工程化软骨的最佳可行性方案.[方法]本研究分别采用体外单体培养、分体培养、生物反应器内分体培养体内构建三种方式构建骨软骨复合体,模仿马赛克移植术植入比格犬膝关节骨软骨缺损处,通过大体观察、组织学分析观察其修复情况.[结果]术后3个月,3种方式构建的骨软骨复合体均不同程度修复了犬关节软骨缺损,采用生物反应器内分体培养体内构建方式修复骨软骨缺损效果优于前两种方式.[结论]灌注型生物反应器使软骨和成骨细胞在三维载体内存活并增殖,提高细胞在载体内的复合效率,软骨修复情况优于前两种方法.     

利用脂肪干细胞构建组织工程软骨修复兔膝关节软骨缺损

目的探讨以脂肪于细胞（ADSCs）复合脱细胞软骨基质支架构建组织工程软骨修复兔膝关节软骨缺损的效果。方法以人关节软骨脱细胞基质为支架，复合经诱导的兔ADSCs，体外分别经静态培养和生物反应器培养，构建组织工程软骨。对膝全厚关节缺损进行修复，并与单支架组、空白对照组比较，其中空白对照组12个关节，脱细胞软骨支架组16个关节，静态培养细胞支架组24个关节，生物反应器培养细胞支架组8个关节。分别于术后3、6个月对修复关节进行大体、组织学及免疫组化观察。结果实际完成观察的关节数为44个，其中空白对照组9个，脱细胞软骨支架组11个，静态培养细胞支架组18个，生物反应器培养细胞支架组6个。空白对照组全为纤维组织或纤维软骨样修复；单支架组5个关节为未成熟透明软骨，无成熟透明软骨形成；静态培养细胞支架组83．3％为透明软骨，其中3个关节为成熟透明软骨，12个关节为未成熟透明软骨；生物反应器培养细胞支架组100％为透明软骨，其中2个为成熟透明软骨，4个为未成熟透明软骨。Wakitani评分各组差异有统计学意义（P〈0．05）。结论ADSCs复合脱细胞软骨基质支架能良好地修复兔膝关节全厚软骨缺损，应用生物反应器技术有助于构建组织工程软骨，促进软骨缺损的修复。     

组织工程化肌腱体外构建的环境优化及系统设计

目的 设计一套构建具有一定功能和形态的肌腱组织的体外培养方法。方法 根据肌腱细胞体内的生物力学环境，在细胞的培养过程中，设计一套能够提供张力和参数测量系统的生物反应器，在肌腱的培养过程中对肌腱施加不同形式的张应力作用，以期在体外获得力学性能优化的自体肌腱。结果 细胞培养和张应力控制系统是由完全透明的有机材料制成的，整个系统由细胞培养室、测量系统和排换液系统等部分构成，在长期的肌腱细胞培养中，可提供肌腱一材料支架动态的培养方式。细胞培养室能很方便与气动肌腱连接，产生持续的脉冲张力作用。结论 用肌腱生物反应器可以使肌腱组织复合物在形成过程中始终处于力学环境中，促进肌腱组织中胶原纤维的平行排列，提高肌腱的力学性能，为组织工程化肌腱的产业化提供一种新的方法。     

Cardiovascular tissue engineering I. Perfusion bioreactors: a review

Tissue engineering is a fast-evolving field of biomedical science and technology with future promise to manufacture living tissues and organs for replacement, repair, and regeneration of diseased organs. Owing to the specific role of hemodynamics in the development, maintenance, and functioning of the cardiovascular system, bioreactors are a fundamental of cardiovascular tissue engineering. The development of perfusion bioreactor technology for cardiovascular tissue engineering is a direct sequence of previous historic successes in extracorporeal circulation techniques. Bioreactors provide a fluidic environment for tissue engineered tissue and organs, and guarantee their viability, maturation, biomonitoring, testing, storage, and transportation. There are different types of bioreactors and they vary greatly in their size, complexity, and functional capabilities. Although progress in design and functional properties of perfusion bioreactors for tissue engineered blood vessels, heart valves, and myocardial patches is obvious, there are some challenges and insufficiently addressed issues, and room for bioreactor design improvement and performance optimization. These challenges include creating a triple perfusion bioreactor for vascularized tubular tissue engineered cardiac construct; designing and manufacturing fluidics-based perfused minibioreactors; incorporation of systematic mathematical modeling and computer simulation based on computational fluid dynamics into the bioreactor designing process; and development of automatic systems of hydrodynamic regime control. Designing and engineering of built-in noninvasive biomonitoring systems is another important challenge. The optimal and most efficient perfusion and conditioning regime, which accelerates tissue maturation of tissue-engineered constructs also remains to be determined. This is a first article in a series of reviews on critical elements of cardiovascular tissue engineering technology describing the current status, unsolved problems, and challenges of bioreactor technology in cardiovascular tissue engineering and outlining future trends and developments.     

A perfusion bioreactor for intestinal tissue engineering

BACKGROUND: Short gut syndrome is a devastating clinical problem with limited long-term treatment options. A unique characteristic of the normal intestinal epithelium is its capacity for regeneration and adaptation. Despite this tremendous capacity in vivo, one of the major limitations in advancing the understanding of intestinal epithelial differentiation and proliferation has been the difficulty in maintaining primary cultures of normal gut epithelium in vitro. A perfusion bioreactor system has been shown to be beneficial in long-term culture and bioengineering of a variety of tissues. The purpose of this study is to design and fabricate a perfusion bioreactor for intestinal tissue engineering. MATERIALS AND METHODS: A perfusion bioreactor is fabricated using specific parameters. Intestinal epithelial organoid units harvested from neonatal rats are seeded onto biodegradable polymer scaffolds and cultured for 2 d in the bioreactor. Cell attachment, viability, and survival are assessed using MTT assay, scanning electron micrograph, and histology. RESULTS: A functional perfusion bioreactor was successfully designed and manufactured. MTT assay and scanning electron micrograph demonstrated successful attachment of viable cells onto the polymer scaffolds. Histology confirmed the survival of intestinal epithelial cells seeded on the scaffolds and cultured in the perfusion bioreactor for 2 days. CONCLUSIONS: A functional perfusion bioreactor can be successfully fabricated for the in-vitro cultivation of intestinal epithelial cells. With further optimization, the perfusion bioreactor may be a useful in in-vitro system for engineering new intestinal tissue.     

利用灌注式生物反应器体外构建大段组织工程化骨的实验研究

目的 研究体外利用灌注式生物反应器构建大段组织工程化骨的可行性. 方法把在体外培养扩增的第三代人骨髓基质干细胞与大段多孔β-磷酸三钙(β-TCP)支架复合.将细胞/支架复合体放入灌注式生物反应器中,进行连续灌注培养.28 d后,检测细胞的增殖及碱性磷酸酶(ALP)活性,同时对培养后的细胞/支架复合体进行组织学检测及形态学计量,用以评价体外组织工程化骨的构建.以静态培养作为对照组. 结果培养28 d后,灌注培养组的细胞活性明显高于静态培养组.灌注培养组细胞的ALP活性显著高于静态培养组.静态培养组细胞仅在多孔β-TCP支架周缘增殖,形成的新骨量较少.灌注培养组细胞在整个β-TCP支架内增殖,形成的新骨量较多. 结论利用灌注式生物反应器的灌注培养,可以使人骨髓基质干细胞在大段β-TCP载体内增殖并形成新骨,使体外大段组织工程化骨的构建成为可能.     

β-磷酸三钙复合骨髓间充质干细胞修复山羊骨软骨缺损的实验研究

目的将β-磷酸三钙（β-TCP）与山羊骨髓间充质干细胞（BMSCs）复合后,在生物反应器中分别向成软骨和成骨诱导,并植入骨软骨缺损处,观察软骨修复效果。方法分离、培养山羊BMSCs,在生物反应器中分别向成软骨及成骨诱导2周,植入骨软骨缺损部位。实验组分为A组：旋转力刺激＋成软骨、成骨诱导组（力学刺激组）,B组：单纯成软骨、成骨诱导组（无力学刺激组）,并设空白对照组。术后12周和24周进行大体观察、组织学染色等,并行O＇Driscoll Keeley and Salter评分。结果 A、B组均有新生软骨形成;A组软骨在12周与24周均优于B组（P〈0.05）;术后12、24周A组评分优于B组,差异有统计学意义（P〈0.05）。对照组无新生软骨形成。结论将BMSCs复合于β-TCP,可用于组织工程修复骨软骨缺损;体外培养阶段的旋转力刺激有利于改善组织工程软骨的质量。     

培养软骨移植修复关节软骨缺损的实验研究

目的：为探讨一种新的关节软骨缺损修复方法。方法：将体外培养２周形成软骨样组织,移植修复兔关节软骨全层缺损。于移植术后２、４、８周分别行功能评价、大体形态及组织学检查。结果：全部实验兔于术后２周内恢复正常活动。２周时移植修复组织由非成熟透明软骨组成。４周时部分移植组出现成熟透明软骨。８周时移植组关节软骨缺损全部由成熟透明软骨充填修复,修复组织与邻近关节软骨融合。培养软骨移植修复关节软骨全层缺损明显优于自身修复（Ｐ＜００１）。结论：本实验提示使用具有高有丝分裂率的软骨细胞,经离心管培养形成骺软骨样组织,植入关节软骨全层缺损后,软骨细胞生长良好,逐渐成熟和转化,能发挥良好的修复作用。     

Bone tissue engineering bioreactors: dynamic culture and the influence of shear stress

A bone tissue engineering strategy involving the in vitro expansion of cells on a scaffold before implantation into the body represents a promising alternative to current clinical treatments. To improve in vitro culture, bioreactor systems have been widely researched for bone tissue engineering purposes. Spinner flask, rotating wall bioreactors, and perfusion systems have all been the focus of experiments, and each system has advantages and disadvantages. This review seeks to summarize these efforts and provide the current status of research in this area. Research using spinner flasks and rotating wall bioreactors is discussed, but focus is placed on perfusion bioreactor systems. While spinner flasks and rotating wall bioreactors have been shown to improve in vitro culture conditions by increasing homogeneity of nutrients in the media, perfusion systems expose cells to shear stress and more efficiently enhance nutrient transfer. Enhanced mineralized matrix deposition and enhancement of osteoblastic signal expression in response to culture in these systems have been widely reported. This review provides analysis of the causes of these changes in signal expression as well as reports on bioreactor systems that have been commercialized.     

Rotating three‐dimensional dynamic culture of adult human bone marrow‐derived cells for tissue engineering of hyaline cartilage

The method of constructing cartilage tissue from bone marrow‐derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow‐derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per µg DNA of the tissues were 10.01 ± 3.49 µg/µg DNA in the case of the RWV bioreactor and 6.27 ± 3.41 µg/µg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three‐dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow‐derived cells. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 517–521, 2009     

Long-Term Expression of Highly Differentiated Functions by Isolated Porcine Hepatocytes Perfused in a Radial-Flow Bioreactor

To overcome the limitations of standard hollow-fiber module in ensuring efficient cell perfusion and long-term expression of highly differentiated hepatocyte functions, we developed a novel bioreactor in which a three-dimensional hepatocyte culture system was perfused in radial-flow geometry. Isolated porcine hepatocytes were cultured for 2 weeks in recirculating serum-free tissue culture medium, in which NH4Cl and lidocaine were repeatedly added, and ammonia removal, urea synthesis, monoethylglycinexylide (MEGX) production, albumin secretion, Po2, Pco2, O2 consumption, and pH were measured thereafter. During the whole duration of the study, ammonia removal was paralleled by urea production, while MEGX concentration was constantly increased. Our results indicated that hepatocytes remained differentiated and metabolically active throughout the duration of the study. The radial-flow bioreactor allowed physiological contact between recirculating fluid and cells by equalizing the concentration of the perfusing components, including O2, throughout the module, suggesting a potential use of this configuration for extracorporeal liver support.