The role of bioreactors in tissue engineering

Ex vivo engineering of living tissues is a rapidly developing area with the potential to impact significantly on a wide-range of biomedical applications. Major obstacles to the generation of functional tissues and their widespread clinical use are related to a limited understanding of the regulatory role of specific physicochemical culture parameters on tissue development, and the high manufacturing costs of the few commercially available engineered tissue products. By enabling reproducible and controlled changes of specific environmental factors, bioreactor systems provide both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In addition, by automating and standardizing tissue manufacture in controlled closed systems, bioreactors could reduce production costs, thus facilitating a wider use of engineered tissues.     

The role of adipose-derived stromal cells and hydroxypropylmethylcellulose in engineering cartilage tissue in vivo

This study demonstrated a newly developed method using adipose tissue-derived stromal cells (ADSCs) and hydroxypropylmethylcellulose (HPMC) in building injectable tissue engineered cartilage in vivo. ADSCs from rabbit subcutaneous fatty tissue were cultured in chondrogenic differentiation medium and supplemented with transforming growth factor-β1 (TGF-β1) and basic fibroblast growth factor (bFGF). Histological, immunohistochemistry and RT-PCR analysis confirmed that the ADSCs differentiated into chondrocytes following induction. Induced ADSCs mixed with 15 % HPMC were injected into the subcutaneous tissue of nude mice and, after a period of 8 weeks, newly formed cartilage was observed at the site of injection. The ability of ADSCs cultured in the induction medium with TGF-β1 and bFGF to differentiate into chondrocytes and construct new cartilage indicates that ADSCs are suitable for use as seed cells in cartilage tissue engineering. HPMC, according to its good water solubility and being able to transform from liquid to solid at body temperature, was found to be an ideal scaffold for tissue engineering.     

Flow dynamics in bioreactors containing tissue engineering scaffolds

Bioreactors are widely used in tissue engineering as a way to distribute nutrients within porous materials and provide physical stimulus required by many tissues. However, the fluid dynamics within the large porous structure are not well understood. In this study, we explored the effect of reactor geometry by using rectangular and circular reactors with three different inlet and outlet patterns. Geometries were simulated with and without the porous structure using the computational fluid dynamics software Comsol Multiphysics 3.4 and/or ANSYS CFX 11 respectively. Residence time distribution analysis using a step change of a tracer within the reactor revealed non-ideal fluid distribution characteristics within the reactors. The Brinkman equation was used to model the permeability characteristics with in the chitosan porous structure. Pore size was varied from 10 to 200 microm and the number of pores per unit area was varied from 15 to 1,500 pores/mm(2). Effect of cellular growth and tissue remodeling on flow distribution was also assessed by changing the pore size (85-10 microm) while keeping the number of pores per unit area constant. These results showed significant increase in pressure with reduction in pore size, which could limit the fluid flow and nutrient transport. However, measured pressure drop was marginally higher than the simulation results. Maximum shear stress was similar in both reactors and ranged approximately 0.2-0.3 dynes/cm(2). The simulations were validated experimentally using both a rectangular and circular bioreactor, constructed in-house. Porous structures for the experiments were formed using 0.5% chitosan solution freeze-dried at -80 degrees C, and the pressure drop across the reactor was monitored.     

Growth factors in cartilage tissue engineering

Tissue engineering of cartilage consists of two steps. Firstly, the cells from a small biopsy of patient's own tissue have to be multiplied. During this multiplication process they lose their cartilage phenotype. In the second step, these cells have to be stimulated to re-express their cartilage phenotype and produce cartilage matrix. Growth factors can be used to improve cell multiplication, redifferentiation and production of matrix. The choice of growth factors should be made for each phase of the tissue engineering process separately, taking into account cell phenotype and the presence of extracellular matrix. This paper demonstrates some examples of the use of growth factors to increase the amount, the quality and the assembly of the matrix components produced for cartilage tissue engineering. In addition it shows that the "culture history" (e.g., addition of growth factors during cell multiplication or preculture period in a 3-dimensional environment) of the cells influences the effect of growth factor addition. The data demonstrate the potency as well as the limitations of the use of growth factors in cartilage tissue engineering.     

Recent progress in cartilage tissue engineering

Despite over two decades of research on cartilage tissue engineering, very few products have moved from bench to bedside and effective therapy remains lacking. This review discusses recent progress in developing novel strategies for engineering cartilage tissues with long-term functionality. Specifically we focus on the following aspects including identifying promising cell sources, designing 3D scaffolds with dynamic and spatially patterned cues to guide desired cellular processes, mimicking zonal organization, integrating with host tissue, and monitoring cell fate and tissue regeneration in situ.     

Hollow fibre membrane bioreactors for tissue engineering applications

Hollow fibre membrane bioreactors (HFB) provide a novel approach towards tissue engineering applications in the field of regenerative medicine. For adherent cell types, HFBs offer an in vivo-like microenvironment as each fibre replicates a blood capillary and the mass transfer rate across the wall is independent from the shear stresses experienced by the cell. HFB also possesses the highest surface area to volume ratio of all bioreactor configurations. In theory, these factors enable a high quantity of the desired cellular product with less population variation, and favourable operating costs. Experimental analyses of different cell types and bioreactor designs show encouraging steps towards producing a clinically relevant device. This review discusses the basic HFB design for cell expansion and in vitro models; compares data produced on commercially available systems and addresses the operational differences between theory and practice. HFBs are showing some potential for mammalian cell culture but further work is needed to fully understand the complexities of cell culture in HFBs and how best to achieve the high theoretical cell yields.     

Current state of cartilage tissue engineering

Damage to cartilage is of great clinical consequence given the tissue's limited intrinsic potential for healing. Current treatments for cartilage repair are less than satisfactory, and rarely restore full function or return the tissue to its native normal state. The rapidly emerging field of tissue engineering holds great promise for the generation of functional cartilage tissue substitutes. The general approach involves a biocompatible, structurally and mechanically sound scaffold, with an appropriate cell source, which is loaded with bioactive molecules that promote cellular differentiation and/or maturation. This review highlights aspects of current progress in cartilage tissue engineering.     

Composite scaffolds for cartilage tissue engineering

Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.     

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.     

Optimizing the medium perfusion rate in bone tissue engineering bioreactors

There is a critical need to increase the size of bone grafts that can be cultured in vitro for use in regenerative medicine. Perfusion bioreactors have been used to improve the nutrient and gas transfer capabilities and reduce the size limitations inherent to static culture, as well as to modulate cellular responses by hydrodynamic shear. Our aim was to understand the effects of medium flow velocity on cellular phenotype and the formation of bone‐like tissues in three‐dimensional engineered constructs. We utilized custom‐designed perfusion bioreactors to culture bone constructs for 5 weeks using a wide range of superficial flow velocities (80, 400, 800, 1,200, and 1,800 µm/s), corresponding to estimated initial shear stresses ranging from 0.6 to 20 mPa. Increasing the flow velocity significantly affected cell morphology, cell–cell interactions, matrix production and composition, and the expression of osteogenic genes. Within the range studied, the flow velocities ranging from 400 to 800 µm/s yielded the best overall osteogenic responses. Using mathematical models, we determined that even at the lowest flow velocity (80 µm/s) the oxygen provided was sufficient to maintain viability of the cells within the construct. Yet it was clear that this flow velocity did not adequately support the development of bone‐like tissue. The complexity of the cellular responses found at different flow velocities underscores the need to use a range of evaluation parameters to determine the quality of engineered bone. Bioeng. 2011; 108:1159–1170. © 2010 Wiley Periodicals, Inc.     

Protein-based tissue engineering in bone and cartilage repair

Bioactive proteins signal host or transplanted cells to form the desired tissue type. Matrix systems are utilized to locally deliver the proteins and to maintain effective protein concentrations. For some indications, a matrix is required to define the physical form of the regenerated tissue. Substantial progress has been made in bone tissue engineering in recent years, based on the results of controlled clinical studies using bone morphogenetic proteins. Ongoing research in this area centers on the design of additional delivery matrices to expand the clinical indications, using synthetic delivery systems that mimic biological qualities of the natural materials currently in use. Although a similar rationale exists for the regeneration of articular cartilage with bioactive factors, advancement in this area has not been as substantial.     

Design considerations and challenges for mechanical stretch bioreactors in tissue engineering

With the increase in average life expectancy and growing aging population, lack of functional grafts for replacement surgeries has become a severe problem. Engineered tissues are a promising alternative to this problem because they can mimic the physiological function of the native tissues and be cultured on demand. Cyclic stretch is important for developing many engineered tissues such as hearts, heart valves, muscles, and bones. Thus a variety of stretch bioreactors and corresponding scaffolds have been designed and tested to study the underlying mechanism of tissue formation and to optimize the mechanical conditions applied to the engineered tissues. In this review, we look at various designs of stretch bioreactors and common scaffolds and offer insights for future improvements in tissue engineering applications. First, we summarize the requirements and common configuration of stretch bioreactors. Next, we present the features of different actuating and motion transforming systems and their applications. Since most bioreactors must measure detailed distributions of loads and deformations on engineered tissues, techniques with high accuracy, precision, and frequency have been developed. We also cover the key points in designing culture chambers, nutrition exchanging systems, and regimens used for specific tissues. Since scaffolds are essential for providing biophysical microenvironments for residing cells, we discuss materials and technologies used in fabricating scaffolds to mimic anisotropic native tissues, including decellularized tissues, hydrogels, biocompatible polymers, electrospinning, and 3D bioprinting techniques. Finally, we present the potential future directions for improving stretch bioreactors and scaffolds. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:543–553, 2016     

深低温冻存组织工程化软骨在关节软骨缺损修复的应用

目的：探讨经深低温冻存组织工程化软骨修复关节软骨缺损的可行性。方法：分离收集3周龄新西兰大白兔关节软骨细胞进行体外培养,接种于PGA三维支架材料上,复合物体外培养1周后冻存,冻存1个月后解冻、复苏及体外培养,1周后接种于已建立的双侧兔膝关节软骨缺损模型的膝关节软骨缺损处,并设对照组。分别于手术后4周、8周、12周行大体标本及组织观察。结果：大体观察结果表明,实验组与对照组缺损处均由软骨组织修复;组织学观察可以见到实验组和对照组关节软骨缺损处有密集的软骨细胞,均有软骨生成及基质分泌,两组差异无统计学意义。结论：应用深低温冻存组织工程化软骨修复关节软骨缺损的方法是有效可行的,为其进一步临床应用提供了实验依据。     

A strategy to determine operating parameters in tissue engineering hollow fiber bioreactors

The development of tissue engineering hollow fiber bioreactors (HFB) requires the optimal design of the geometry and operation parameters of the system. This article provides a strategy for specifying operating conditions for the system based on mathematical models of oxygen delivery to the cell population. Analytical and numerical solutions of these models are developed based on Michaelis–Menten kinetics. Depending on the minimum oxygen concentration required to culture a functional cell population, together with the oxygen uptake kinetics, the strategy dictates the model needed to describe mass transport so that the operating conditions can be defined. If c_min ≫ K_m we capture oxygen uptake using zero‐order kinetics and proceed analytically. This enables operating equations to be developed that allow the user to choose the medium flow rate, lumen length, and ECS depth to provide a prescribed value of c_min. When , we use numerical techniques to solve full Michaelis–Menten kinetics and present operating data for the bioreactor. The strategy presented utilizes both analytical and numerical approaches and can be applied to any cell type with known oxygen transport properties and uptake kinetics. Biotechnol. Bioeng. 2011; 108:1450–1461. © 2011 Wiley Periodicals, Inc.     

Scaffold-free cartilage by rotational culture for tissue engineering

Our objective was to investigate the hypothesis that tissue-engineered cartilage with promising biochemical, mechanical properties can be formed by loading mechanical stress under existing cell-cell interactions analogous to those that occur in condensation during embryonic development. By loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions, we could first form a scaffold-free cartilage tissue with arbitrary shapes and a large size with promising biological, mechanical properties. The cartilage tissue which constituted of chondrocytes and ECM produced by inoculated dedifferentiated chondrocytes to a high porous simple mold has arbitrary shapes, and did not need any biodegradable scaffold to control the shape. In contrast, scaffold-free cartilage tissue cultured under static conditions could not keep their shapes; it was fragile tissue. The possibility of scaffold-free organ design was suggested because the cartilage tissue increases steadily in size with culture time; indeed, the growth of cartilage tissue starting from an arbitrary shape might be predictable by mathematical expression. For tissue-engineered cartilage formation with arbitrary shapes, biochemical and mechanical properties, loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions has prominent effects. Therefore, our scaffold-free cartilage model loaded mechanical stress based on a simple mold system may be applicable for tissue-engineered cartilage.     

Adipose-derived adult stem cells for cartilage tissue engineering

Tissue engineering is a promising therapeutic approach that uses combinations of implanted cells, biomaterial scaffolds, and biologically active molecules to repair or regenerate damaged or diseased tissues. Many diverse and increasingly complex approaches are being developed to repair articular cartilage, with the underlying premise that cells introduced exogenously play a necessary role in the success of engineered tissue replacements. A major consideration that remains in this field is the identification and characterization of appropriate sources of cells for tissue-engineered repair of cartilage. In particular, there has been significant emphasis on the use of undifferentiated progenitor cells, or "stem" cells that can be expanded in culture and differentiated into a variety of different cell types. Recent studies have identified the presence of an abundant source of stem cells in subcutaneous adipose tissue. These cells, termed adipose-derived adult stem (ADAS) cells, show characteristics of multipotent adult stem cells, similar to those of bone marrow derived mesenchymal stem cells (MSCs), and under appropriate culture conditions, synthesize cartilage-specific matrix proteins that are assembled in a cartilaginous extracellular matrix. The growth and chondrogenic differentiation of ADAS cells is strongly influenced by factors in the biochemical as well as biophysical environment of the cells. Furthermore, there is strong evidence that the interaction between the cells, the extracellular biomaterial substrate, and growth factors regulate ADAS cell differentiation and tissue growth. Overall, ADAS cells show significant promise for the development of functional tissue replacements for various tissues of the musculoskeletal system.     

生物可降解材料构建组织工程软骨的研究进展

关节软骨修复困难,目前临床上治疗关节软骨损伤难以达到满意的效果。组织工程学的兴起为其提供了新的选择。本文介绍了组织工程软骨的发展历史,重点叙述了各种天然支架材料、人工合成材料、复合材料及纳米材料在软骨组织工程中的应用及其优势。目前应用的天然材料存在力学强度差及免疫源性的不足;人工合成材料降解速率快,降解产物具有细胞毒性,有待进一步完善。表面修饰等技术的应用在一定程度上克服了某些材料的不足;复合材料综合了数种材料的优点,是今后材料技术发展的方向;纳米技术的出现使新合成的材料成为纳米量级,具有了普通材料无可比拟的优势,这为组织工程材料的发展提供了新的思路。本文还对组织工程支架材料存在的问题、下一步的发展方向和前瞻性研究做了介绍。