心肌组织工程生物反应器研究进展

组织工程的研究主要围绕种子细胞、生物材料和组织构建这三个基本要素而展开。组织构建技术是组织工程研究的核心。组织工程生物反应器是一种体外构建人体组织的系统装置。心肌组织工程在替代和维持梗塞的心肌组织功能,并进而治愈疾病以最大限度地挽救病人生命方面可能发挥巨大作用。主要介绍了国内外工程化心肌组织体外构建技术,特别是用于构建工程化心肌组织的心肌组织工程生物反应器研究方面的进展。     

生物反应器在组织工程研究中的应用

生物反应器在组织工程研究中的应用非常广泛,从最初的种子细胞增殖、分化,到关键的组织体外构建,都可以利用生物反应器来模拟细胞和组织在体内的生长环境,提高工程化组织构建的效率。本文结合本期发表的6篇相关文献,介绍了组织工程中研究中生物反应器的作用,建议通过包括生物力学在内的多学科的研究手段,获得调控不同类型细胞生长和组织构建的关键参数,实现功能化组织工程的研究目标。     

自制双腔生物反应器构建组织工程骨软骨复合体的体外性能

背景：关节软骨损伤往往并发软骨下骨损伤形成骨软骨复合缺损,其治疗仍为骨科急待解决的问题,利用组织工程学构建骨软骨复合体为治疗该类疾患提供了新思路。 目的：探讨利用自行设计制造的双腔搅拌式生物反应器构建一体化组织工程骨软骨复合体的可行性。 方法：在双腔搅拌式生物反应器内对复合于β-磷酸三钙支架材料的羊骨髓间充质干细胞同时进行成骨和成软骨诱导,并根据施加剪切应力分为动态培养组和静态培养组。利用MTT试验、RT-PCR和扫描电镜检测骨髓间充质干细胞体外增殖和诱导分化情况。 结果与结论：MTT试验和扫描电镜结果显示,骨髓间充质干细胞增殖良好。成骨和成软骨相关基因RT-PCR检测结果表明,骨髓间充质干细胞诱导分化良好,动态培养组要优于静态培养组。提示利用自行设计制作的双腔搅拌式生物反应器进行骨软骨复合体的体外构建是可行的,力学刺激环境下的构建效果要优于静态环境。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

软骨组织工程中力学因素的影响及应用

力学因素是软骨组织工程中的重要影响因素之一。近年来的研究表明,力学作用可以刺激细胞因子及激素的分泌,改变三维支架上培养的软骨细胞的新陈代谢,从而促进软骨组织的生长与重建。目前已经有诸多关于体外构建软骨组织的报道,但对于其中的力学因素的影响(包括力学因素对软骨细胞增殖的促进及力学刺激的传导机制等)还没有完全认识。就以上几方面做一综述,并简单介绍生物反应器在软骨组织工程中的应用。     

软骨组织工程中力学因素的影响及应用

力学因素是软骨组织工程中的重要影响因素之一。近年来的研究表明，力学作用可以刺激细胞因子及激素的分泌，改变三维支架上培养的软骨细胞的新陈代谢，从而促进软骨组织的生长与重建。目前已经有诸多关于体外构建软骨组织的报道，但对于其中的力学因素的影响（包括力学因素对软骨细胞增殖的促进及力学刺激的传导机制等）还没有完全认识。就以上几方面做一综述，并简单介绍生物反应器在软骨组织工程中的应用。     

人脐带Wharton胶中间充质干细胞体外无支架组织工程软骨的构建

背景:构建组织工程软骨的方法多为自体种子细胞复合天然或合成物支架,目前多存在种子细胞来源有限、支架安全性和生物相容性、以及细胞在支架中分布不均的问题。目的:探讨人脐带Wharton胶中间充质干细胞向软骨诱导分化并体外构建无支架组织工程软骨的可行性。方法:分离培养人脐带Wharton胶中的间充质干细胞,进行流式细胞学鉴定。对软骨诱导前后的细胞进行组织学和免疫组织化学染色,对其表达的葡萄糖胺聚糖和Ⅱ型胶原进行定量研究,并应用RT-PCR检测软骨诱导前后Ⅱ型胶原和Sox-9mRNA的表达。采用密集诱导培养→离心管培养→生物反应器培养,进行体外构建无支架软骨组织。结果与结论:人脐带Wharton胶富含干细胞,流式细胞仪检测结果显示这些细胞不表达造血干细胞标志,表达CD44,CD105、CD271等间充质干细胞表面标志;HLA-ABC阳性表达,HLA-DPDQDR阴性表达。未进行软骨诱导的细胞弱表达软骨细胞标志,诱导后葡萄糖胺聚糖和Ⅱ型胶原显著增高。RT-PCR结果显示人脐带Wharton胶间充质干细胞诱导前后均表达Sox-9、Ⅱ型胶原mRNA。说明人脐带Wharton胶间充质干细胞具有前软骨细胞的特性。采用密集诱导培养结合生物反应器培养,不用支架,体外可以构建成大块组织工程软骨。表明人脐带Wharton胶间充质干细胞是一种良好的构建组织工程软骨的种子细胞。     

软骨组织工程研究的新进展

软骨组织的再生能力有限 ,组织工程软骨的构建对修复软骨缺损意义重大。本文从四方面介绍了软骨组织工程的研究的新进展 ,包括软骨种子细胞的研究、软骨细胞与支架的体外培养、细胞支架复合物植入体内的研究及软骨细胞移植的临床应用。     

组织工程软骨研究进展

组织工程学是 2 0世纪 80年代末期发展起来的一门新兴边缘学科。它是综合应用工程学和生命科学的基本原理、理论和技术 ,在体外预先构建有生命的种植体 ,然后植入体内修复组织缺损 ,替代组织器官的一部分或全部功能。软骨为单一细胞 (软骨细胞 )构成组织 ,透明软骨、弹性软骨和纤维软骨组成人体重要结构 ,具有十分重要的生理功能。组织工程软骨是第一个组织工程化组织 ,在矫形外科和整形外科领域 ,组织工程软骨研究受到高度重视 ,发展迅速。本文介绍矫形外科领域中组织工程软骨研究发展。1　种子细胞1 1　软骨细胞多数研究以软骨细胞为种子…     

软骨组织工程研究的新进展

软骨组织的再生能力有限，组织工程软骨的构建对修复软骨缺损意义理大。本从四方面介绍了软骨组织工程的研究的新进展，包括软骨种子细胞的研究，软骨细胞与支架的体外培养，细胞支架复合物植入体内的研究及软骨细胞移植的临床应用。     

旋转生物反应器用于提高组织工程气管软骨力学强度的实验研究

针对体外静态培养方法的缺点,采用旋转生物反应器培养组织工程气管软骨,探讨工程化软骨合适的体外培养条件.选用大鼠剑突软骨细胞种植到DegraPol管状支架上,然后分别于传统静态培养和生物反应器内培养.于体外培养3周和6周后,取出软骨细胞-DegraPol支架复合物,以噻唑蓝(MTT)法测定细胞增殖活性,GAG浓度测定细胞外基质分泌情况,应力-应变机械力学方法测定最大应变和应力的变化,并制备扫描电镜标本观察软骨细胞在DegraP01支架中培养后的超微结构.体外培养3周应用MTT法测定A值分别为:静态培养的细胞-支架复合物组0.12±0.01,生物反应器组0.17±0.05(每组n=6).GAG浓度测定静态培养组为(0.14±0.03) μg/mg,生物反应器组为(0.22±O.03) μg/mg(每组n=6).应力-应变机械力学测定结果为,体外培养3周应变值:生物反应器组为(3.53±0.91),静态培养组为(1.71±0.13).应力值生物反应器组为(0.33±0.04) MPa,静态培养组为(0.26±0.01) MPa.体外培养6周应变值:生物反应器组为(0.57±0.10),静态培养组为(0.48±0.07),应力值生物反应器组为(0.16±0.02) MPa,静态培养组为(0.09±0.02) MPa(每组n=4).扫描电镜观察显示生物反应器组获得更好的软骨样结构和更多的细胞外基质.应用旋转生物反应器能够提供适宜的机械应力刺激,可作为体外构建组织工程化气管软骨的可行的培养方法.     

Cartilage tissue engineering on the surface of a novel gelatin-calcium-phosphate biphasic scaffold in a double-chamber bioreactor

Tissue engineering is a new approach to articular cartilage repair; however, the integration of the engineered cartilage into the host subchondral bone is a major problem in osteochondral injury. The aim of the present work, therefore, was to make a tissue-engineered osteochondral construct from a novel biphasic scaffold in a newly designed double-chamber bioreactor. This bioreactor was designed to coculture chondrocytes and osteoblasts simultaneously. The aim of this study was to prove that engineered cartilage could be formed with the use of this biphasic scaffold. The scaffold was constructed from gelatin and a calcium-phosphate block made from calcined bovine bone. The cartilage part of the scaffold had a uniform pore size of about 180 microm and approximate porosity of 75%, with the trabecular pattern preserved in the bony part of the scaffold. The biphasic scaffolds were seeded with porcine chondrocytes and cultured in a double-chamber bioreactor for 2 or 4 weeks. The chondrocytes were homogeneously distributed in the gelatin part of the scaffold, and secretion of the extracellular matrix was demonstrated histologically. The chondrocytes retained their phenotype after 4 weeks of culture, as proven immunohistochemically. After 4 weeks of culture, hyaline-like cartilage with lacuna formation could be clearly seen in the gelatin scaffold on the surface of the calcium phosphate. The results show that this biphasic scaffold can support cartilage formation on a calcium-phosphate surface in a double-chamber bioreactor, and it seems reasonable to suggest that there is potential for further application in osteochondral tissue engineering.     

Hydrodynamic parameters modulate biochemical, histological, and mechanical properties of engineered cartilage

Functional engineered cartilage constructs represent a promising therapeutic approach for the replacement of damaged articular cartilage. The in vitro generation of cartilage tissue suitable for repair requires an understanding of the complex interrelationships between environmental cues, such as hydrodynamic forces, and tissue growth and development. In the present study, engineered cartilage constructs were cultivated in four well-defined hydrodynamic environments within a bioreactor, and correlations were established between construct ultrastructural and mechanical properties and key hydrodynamic parameters. Results suggest that even for similar composition, constructs may exhibit different mechanical properties due to differences in their ultrastructure that can be modulated by hydrodynamic parameters. For example, improved mechanical properties were observed in constructs that exhibited a thick fibrous outer capsule as a result of cultivation under increased hydrodynamic shear. In particular, uniformity in the contribution of the fluid velocity vectors (axial, radial, and tangential) to the total fluid velocity and shear stress were the hydrodynamic parameters that most affected the construct properties under investigation. The correlations identified here may be useful in the development of engineered tissue growth models that inform the design of bioreactor cultivation systems toward the production of clinically relevant engineered cartilage.     

A novel customizable modular bioreactor system for whole-heart cultivation under controlled 3D biomechanical stimulation

In the last decade, cardiovascular tissue engineering has made great progress developing new strategies for regenerative medicine applications. However, while tissue engineered heart valves are already entering the clinical routine, tissue engineered myocardial substitutes are still restrained to experimental approaches. In contrast to the heart valves, tissue engineered myocardium cannot be repopulated in vivo because of its biological complexity, requiring elaborate cultivation conditions ex vivo. Although new promising approaches—like the whole-heart decellularization concept—have entered the myocardial tissue engineering field, bioreactor technology needed for the generation of functional myocardial tissue still lags behind in the sense of user-friendly, flexible and low cost systems. Here, we present a novel customizable modular bioreactor system that can be used for whole-heart cultivation. Out of a commercially obtainable original equipment manufacturer platform we constructed a modular bioreactor system specifically aimed at the cultivation of decellularized whole-hearts through perfusion and controlled 3D biomechanical stimulation with a simple but highly flexible operation platform based on LabVIEW^®. The modular setup not only allows a wide range of variance regarding medium conditioning under controlled 3D myocardial stretching but can also easily be upgraded for e.g. electrophysiological monitoring or stimulation, allowing for a tailor-made low-cost myocardial bioreactor system.     

Wavy-walled bioreactor supports increased cell proliferation and matrix deposition in engineered cartilage constructs

Hydrodynamic forces in bioreactors can decisively influence extracellular matrix deposition in engineered cartilage constructs. In the present study, the reduced fluid shear, high-axial mixing environment provided by a wavy-walled bioreactor was exploited in the cultivation of cartilage constructs using polyglycolic acid scaffolds seeded with bovine articular chondrocytes. Increased growth as defined by weight, cell proliferation and extracellular matrix deposition was observed in cartilage constructs from wavy-walled bioreactors in comparison with those from spinner flasks cultured under the same conditions. The wet weight composition of 4-week constructs from the wavy-walled bioreactor was similar to that of spinner flask constructs, but the former were 60% heavier due to equally higher incorporation of extracellular matrix and 30% higher cell population. It is most likely that increased construct matrix incorporation was a result of increased mitotic activity of chondrocytes cultured in the environment of the wavy-walled bioreactor. A layer of elongated cells embedded in type I collagen formed at the periphery of wavy-walled bioreactor and spinner flask constructs, possibly as a response to local shear forces. On the basis of the robustness and reproducibility of the extracellular matrix composition of cartilage constructs, the wavy-walled bioreactor demonstrated promise as an experimental cartilage tissue-engineering vessel. Increased construct growth in the wavy-walled bioreactor may lead to enhanced mechanical properties and expedited in vitro cultivation.     

旋转生物反应器培养对组织工程气管软骨力学强度的影响

目的研究旋转生物反应器培养对组织工程气管软骨力学强度的影响，探索适宜的组织工程气管软骨培养方法。 方法分离2周龄Lewis大鼠剑突软骨细胞传代培养，收集第3代软骨细胞种植到DegraPol管状支架上，静态培养7d，然后将软骨细胞-支架复合物分别置于旋转生物反应器内培养（生物反应器组）或继续静态培养培养3周（静态培养组）。取出软骨细胞-支架复合物，以噻唑蓝（MTT）法测定软骨细胞增殖活性，结果以吸光度（A）值表示（每组n=6）；以Zwick1445型材料试验机测定软骨细胞-支架复合物的最大应变值和应力值（每组n=4）；并制备扫描电镜标本，观察软骨细胞在DegraPol支架中培养后的超微结构变化。 结果不同条件下培养3周，生物反应器组和静态培养组A值分别0.17±0.05、0.12±0.01，最大应力值分别为（0.33±0.04）和（0.26±0.01）MPa，最大应变值分别为（3.53±0.91）和（1.71±0.13）mm/mm，2组间3项指标的差异均有统计学意义（均P〈0.05）。扫描电镜观察显示生物反应器组获得更好的软骨样结构和更多的细胞外基质。 结论旋转生物反应器能够提供更好的体外培养条件，有利于组织工程气管软骨的形成。     

Squeeze pressure bioreactor: a hydrodynamic bioreactor for noncontact stimulation of cartilage constructs

A novel squeeze pressure bioreactor for noncontact hydrodynamic stimulation of cartilage is described. The bioreactor is based on a small piston that moves up and down, perpendicular to a tissue construct, in a fluid-filled chamber. Fluid displaced by the piston generates a pressure wave and shear stress as it moves across the sample, simulating the dynamic environment of a mobile joint. The fluid dynamics inside the squeeze pressure bioreactor was modeled using analytical and computational methods to simulate the mechanical stimuli imposed on a construct. In particular, the pressure, velocity field, and wall shear stress generated on the surface of the construct were analyzed using the theory of hydrodynamic lubrication, which describes the flow of an incompressible fluid between two surfaces in relative motion. Both the models and in-situ pressure measurements in the bioreactor demonstrate that controlled cyclic stresses of up to 10?kPa can be applied to tissue constructs. Initial tests on three-dimensional scaffolds seeded with chondrocytes show that glycosaminoglycan production is increased with regard to controls after 24 and 48?h of cyclic noncontact stimulation in the bioreactor.     

Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells

We aimed to determine whether three-dimensional (3D) cartilage could be engineered from umbilical cord blood (CB) cells and compare it with both engineered fetal cartilage and native tissue. Ovine mesenchymal progenitor cells were isolated from CB samples (n=4) harvested at 80-120 days of gestation by low-density fractionation, expanded, and seeded onto polyglycolic acid scaffolds. Constructs (n=28) were maintained in a rotating bioreactor with serum-free medium supplemented with transforming growth factor-beta1 for 4-12 weeks. Similar constructs seeded with fetal chondrocytes (n=13) were cultured in parallel for 8 weeks. All specimens were analyzed and compared with native fetal cartilage samples (n=10). Statistical analysis was by analysis of variance and Student's t-test (p<.01). At 12 weeks, CB constructs exhibited chondrogenic differentiation by both standard and matrix-specific staining. In the CB constructs, there was a significant time-dependent increase in extracellular matrix levels of glycosaminoglycans (GAGs) and type-II collagen (C-II) but not of elastin (EL). Fetal chondrocyte and CB constructs had similar GAG and C-II contents, but CB constructs had less EL. Compared with both hyaline and elastic native fetal cartilage, C-II and EL levels were, respectively, similar and lower in the CB constructs, which had correspondingly lower and similar GAG levels than native hyaline and elastic fetal cartilage. We conclude that CB mesenchymal progenitor cells can be successfully used for the engineering of 3D cartilaginous tissue in vitro, displaying select histological and functional properties of both native and engineered fetal cartilage. Cartilage engineered from CB may prove useful for the treatment of select congenital anomalies.     

Cell-nanofiber-based cartilage tissue engineering using improved cell seeding, growth factor, and bioreactor technologies

Biodegradable nanofibrous scaffolds serving as an extracellular matrix substitute have been shown to be applicable for cartilage tissue engineering. However, a key challenge in using nanofibrous scaffolds for tissue engineering is that the small pore size limits the infiltration of cells, which may result in uneven cell distribution throughout the scaffold. This study describes an effective method of chondrocyte loading into nanofibrous scaffolds, which combines cell seeding, mixing, and centrifugation to form homogeneous, packed cell-nanofiber composites (CNCs). When the effects of different growth factors are compared, CNCs cultured in medium containing a combination of insulin-like growth factor-1 and transforming growth factor-beta1 express the highest mRNA levels of collagen type II and aggrecan. Radiolabeling analyses confirm the effect on collagen and sulfated-glycosaminoglycans (sGAG) production. Histology reveals chondrocytes with typical morphology embedded in lacuna-like space throughout the entire structure of the CNC. Upon culturing using a rotary wall vessel bioreactor, CNCs develop into a smooth, glossy cartilage-like tissue, compared to a rough-surface tissue when maintained in a static environment. Bioreactor-grown cartilage constructs produce more total collagen and sGAG, resulting in greater gain in net tissue weight, as well as express cartilage-associated genes, including collagen types II and IX, cartilage oligomeric matrix protein, and aggrecan. In addition, dynamic culture enhances the mechanical property of the engineered cartilage. Taken together, these results indicate the applicability of nanofibrous scaffolds, combined with efficient cell loading and bioreactor technology, for cell-based cartilage tissue engineering.     

Effects of co-culturing BMSCs and auricular chondrocytes on the elastic modulus and hypertrophy of tissue engineered cartilage

Co-culture of BMSCs and chondrocytes is considered as a promising strategy to generate tissue engineered cartilage as chondrocytes induce the chondrogenesis of BMSCs and inhibit the hypertrophy of engineered cartilage. Because the tissue specific stem/progenitor cells have been isolated from mature tissues including auricular cartilage, we hypothesized that adding stem cells to auricular chondrocytes in co-culture would also enhance the quality of engineered cartilage. In the present study, using the histological assay, biomechanical evaluation, and quantitative analysis of gene expression, we compared different strategies of auricular chondrocytes, BMSCs induction, and co-culture at different ratios on PGA/PLA scaffolds to construct tissue engineered elastic cartilage in vitro and in vivo. The up-regulation of RUNX2 and down-regulation of SOX9 were found in BMSCs chondrogenic induction group, which might imply a regulatory mechanism for the hypertrophy and potential osteogenic differentiation. Engineered cartilage in co-culture 5:5 group showed the densest elastic fibers and the highest Young's modulus, which were consistent with the expression profile of cartilage matrix-related genes including DCN and LOXL2 genes. Moreover, the better proliferative and chondrogenic potentials of engineered cartilage in co-culture 5:5 group were demonstrated by the stronger expression of Ki67 and Dlk1.