Concepts of scaffold-based tissue engineering--the rationale to use solid free-form fabrication techniques

A paradigm shift is taking place in orthopaedic and reconstructive surgery from using medical devices and tissue grafts to a tissue engineering approach that uses biodegradable scaffolds combined with cells or biological molecules to repair and/or regenerate tissues. One of the potential benefits offered by solid free-form fabrication technology (SFF) is the ability to create scaffolds with highly reproducible architecture and compositional variation across the entire scaffold, due to its tightly controlled computer-driven fabrication. In this review, we define scaffold properties and attempt to provide some broad criteria and constraints for scaffold design in bone engineering.We also discuss the application-specific modifications driven by surgeon's requirements in vitro and/or in vivo. Next, we review the current use of SFF techniques in scaffold fabrication in the context of their clinical use in bone regeneration. Lastly, we comment on future developments in our groups, such as the functionalization of novel composite scaffolds with combinations of growth factors; and more specifically the promising area of heparan sulphate polysaccharide immobilization within the bone tissue engineering arena.     

Esophageal tissue engineering: an in-depth review on scaffold design

Treatment of esophageal cancer often requires surgical procedures that involve removal. The current approaches to restore esophageal continuity however, are known to have limitations which may not result in full functional recovery. In theory, using a tissue engineered esophagus developed from the patient's own cells to replace the removed esophageal segment can be the ideal method of reconstruction. One of the key elements involved in the tissue engineering process is the scaffold which acts as a template for organization of cells and tissue development. While a number of scaffolds range from traditional non-biodegradable tubing to bioactive decellularized matrix have been proposed to engineer the esophagus in the past decade, results are still not yet favorable with many challenges relating to tissue quality need to be met improvements. The success of new esophageal tissue formation will ultimately depend on the success of the scaffold being able to meet the essential requirements specific to the esophageal tissue. Here, the design of the scaffold and its fabrication approaches are reviewed. In this paper, we review the current state of development in bioengineering the esophagus with particular emphasis on scaffold design.     

Nanoparticles for bone tissue engineering

Tissue engineering (TE) envisions the creation of functional substitutes for damaged tissues through integrated solutions, where medical, biological, and engineering principles are combined. Bone regeneration is one of the areas in which designing a model that mimics all tissue properties is still a challenge. The hierarchical structure and high vascularization of bone hampers a TE approach, especially in large bone defects. Nanotechnology can open up a new era for TE, allowing the creation of nanostructures that are comparable in size to those appearing in natural bone. Therefore, nanoengineered systems are now able to more closely mimic the structures observed in naturally occurring systems, and it is also possible to combine several approaches ‐ such as drug delivery and cell labeling ‐ within a single system. This review aims to cover the most recent developments on the use of different nanoparticles for bone TE, with emphasis on their application for scaffolds improvement; drug and gene delivery carriers, and labeling techniques. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:590–611, 2017     

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     

基于组织工程研究的可降解支架材料选择策略

目前,器官或组织移植是治疗器官衰竭或大范围组织缺损唯一长期有效的方法,但存在供体短缺、免疫排斥等问题。组织工程技术作为一种潜在的替代治疗方法,支架材料的选择是其中具有决定意义的组成部分。组织工程支架材料按其来源可分为天然及其改性修饰材料、人工合成与复合支架材料3种。组织工程目的就是修复临床上的病损组织或器官,并达到较理想的结构和功能的恢复。因此组织工程支架也必须从基本性质上具有一定的仿生化结构及功能,即"活"支架,这样才能彻底代替病损组织或器官。通过多种支架材料的优化组合(即材料的复合),对材料进行表面改性、制备工艺优化及添加细胞因子缓释微球等技术,模拟病损器官组织的特性及周围环境,有望打开组织工程的新局面。理想的组织工程支架应当以临床需要为根本目的,依靠材料学、分子生物学、工程学等多学科间的交叉研究,取各家之长,优化配比组合,达到仿生的目的。本课题组前期工作已经将骨髓间充质干细胞体外诱导分化为胆管上皮样细胞,并设计出左旋聚乳酸/聚己内酯共聚物(PLCL)胆道支架,内部混有包含生长因子的纳米缓释微球,供细胞因子的远期释放,支架内表面涂有基质胶/胶原混合层,且胶内加入bFGF、EGF,提供诱导因子的早期释放。将诱导细胞与PLCL胆道支架复合,制备组织工程胆管。文中综述了现存各类支架材料的研究状况,简单介绍了制备工艺、表面修饰等影响支架性能的因素,力求探索组织工程支架材料的选择策略。     

Collagen type I and hyaluronic acid based hybrid scaffolds for heart valve tissue engineering

Tissue engineers have achieved limited success so far in designing an ideal scaffold for aortic valve; scaffolds lack in mechanical compatibility, appropriate degradation rate, and microstructural similarity. This paper, therefore, has demonstrated a carbodiimide-based sequential crosslinking technique to prepare aortic valve extracellular matrix mimicking (ECM) hybrid scaffolds from collagen type I and hyaluronic acid (HA), the building blocks of heart valve ECM, with tailorable crosslinking densities. Swelling studies revealed that crosslinking densities of parent networks increased with increasing the concentration of the crosslinking agents whereas crosslinking densities of hybrid scaffolds averaged from those of parent collagen and HA networks. Hybrid scaffolds also offered a wide range of pore size (66-126 μm) which fulfilled the criteria for valvular tissue regeneration. Scanning electron microscopy and images of Alcian blue-Periodic acid Schiff stained samples suggested that our crosslinking technique yielded an ECM mimicking microstructure with interlaced bands of collagen and HA in the hybrid scaffolds. The mutually reinforcing networks of collagen and HA also resulted in increased bending moduli up to 1660 kPa which spanned the range of natural aortic valves. Cardio sphere-derived cells (CDCs) from rat hearts showed that crosslinking density affected the available cell attachment sites on the surface of the scaffold. Increased bending moduli of CDCs seeded scaffolds up to two folds (2-6 kPa) as compared to the non-seeded scaffolds (1 kPa) suggested that an increase in crosslinking density of the scaffolds could not only increase the in vitro bending modulus but also prevented its disintegration in the cell culture medium.     

Numerical and experimental evaluation of TPMS Gyroid scaffolds for bone tissue engineering

The combination of computational methods with 3D printing allows for the control of scaffolds microstructure. Lately, triply periodic minimal surfaces (TPMS) have been used to design porosity-controlled scaffolds for bone tissue engineering (TE). The goal of this work was to assess the mechanical properties of TPMS Gyroid structures with two porosity levels (50 and 70%). The scaffold stiffness function of porosity was determined by the asymptotic homogenisation method and confirmed by mechanical testing. Additionally, microCT analysis confirmed the quality of the printed parts. Thus, the potential of both design and manufacturing processes for bone TE applications is here demonstrated.     

Visualizing feasible operating ranges within tissue engineering systems using a “windows of operation” approach: A perfusion‐scaffold bioreactor case study

Tissue engineering approaches to developing functional substitutes are often highly complex, multivariate systems where many aspects of the biomaterials, bio‐regulatory factors or cell sources may be controlled in an effort to enhance tissue formation. Furthermore, success is based on multiple performance criteria reflecting both the quantity and quality of the tissue produced. Managing the trade‐offs between different performance criteria is a challenge. A “windows of operation” tool that graphically represents feasible operating spaces to achieve user‐defined levels of performance has previously been described by researchers in the bio‐processing industry. This paper demonstrates the value of “windows of operation” to the tissue engineering field using a perfusion‐scaffold bioreactor system as a case study. In our laboratory, perfusion bioreactor systems are utilized in the context of bone tissue engineering to enhance the osteogenic differentiation of cell‐seeded scaffolds. A key challenge of such perfusion bioreactor systems is to maximize the induction of osteogenesis but minimize cell detachment from the scaffold. Two key operating variables that influence these performance criteria are the mean scaffold pore size and flow‐rate. Using cyclooxygenase‐2 and osteopontin gene expression levels as surrogate indicators of osteogenesis, we employed the “windows of operation” methodology to rapidly identify feasible operating ranges for the mean scaffold pore size and flow‐rate that achieved user‐defined levels of performance for cell detachment and differentiation. Incorporation of such tools into the tissue engineer's armory will hopefully yield a greater understanding of the highly complex systems used and help aid decision making in future translation of products from the bench top to the market place. Biotechnol. Bioeng. 2012; 109: 3161–3171. © 2012 Wiley Periodicals, Inc.     

In vitro evaluation of natural marine sponge collagen as a scaffold for bone tissue engineering

The selection of a suitable scaffold matrix is critical for cell-based bone tissue engineering. This study aimed to identify and characterize natural marine sponges as potential bioscaffolds for osteogenesis. Callyspongiidae marine sponge samples were collected from the Fremantle coast of Western Australia. The sponge structure was assessed using scanning electron microscopy (SEM) and Hematoxylin and eosin. Mouse primary osteoblasts were seeded onto the sponge scaffold and immunostained with F-actin to assess cell attachment and aggregation. Alkaline phosphatase expression, von Kossa staining and real-time PCR were performed to examine the osteogenic potential of sponge samples. SEM revealed that the sponge skeleton possessed a collagenous fibrous network consisting of interconnecting channels and a porous structure that support cellular adhesion, aggregation and growth. The average pore size of the sponge skeleton was measured 100 to 300 μm in diameter. F-actin staining demonstrated that osteoblasts were able to anchor onto the surface of collagen fibres. Alkaline phosphatase expression, a marker of early osteoblast differentiation, was evident at 7 days although expression decreased steadily with long term culture. Using von Kossa staining, mineralisation nodules were evident after 21 days. Gene expression of osteoblast markers, osteocalcin and osteopontin, was also observed at 7, 14 and 21 days of culture. Together, these results suggest that the natural marine sponge is promising as a new scaffold for use in bone tissue engineering.     

Fabrication of agar-gelatin hybrid scaffolds using a novel entrapment method for in vitro tissue engineering applications

Scaffolds of agar and gelatin were developed using a novel entrapment method where agar and gelatin molecules mutually entrapped one another forming stable cell adhesive matrices. Glutaraldehyde was used as a crosslinking agent for gelatin. Three types of hybrid matrices were prepared using agar and gelatin in different proportions in the weight ratio of 1:1, 2:1, and 3:1. Surface characterization of dry scaffolds was carried out by scanning electron microscope. Swelling studies were carried out in phosphate buffer saline (PBS) at physiological pH 7.4. The integral stability of the scaffolds was evaluated by estimating the released disintegrated gelatin from them in PBS at pH 7.4. The attachment kinetics of the cells was evaluated by culturing mouse fibroblast cell line NIH 3T3 on films. The cytocompatibility of these matrices was determined by studying growth kinetics of NIH 3T3 cells on them and morphology of cells was observed through optical photographs taken at various days of culture. It was found that the matrices containing agar and gelatin in 2:1 weight ratio exhibited best growth kinetics. The results obtained from these studies have suggested that the above-described method is a cheap and easy way to fabricate agar-gelatin hybrid scaffolds to grow cells which can be used in various in vitro tissue engineering applications like screening of drugs.     

CAD/CAM bilateral ear prostheses construction for Treacher Collins syndrome patients using laser scanning and rapid prototyping

Ear defects in patients affected by Treacher Collins syndrome necessitate the replacement of the existing anatomic residuals of the ears with custom-made prostheses. This paper describes a multidisciplinary protocol involving both medicine and computer-aided design/computer-aided manufacturing for manufacturing ear prostheses. Using innovative prototyping technologies together with conventional silicone processing procedures, a step-by-step procedure is presented. The complete workflow includes laser scanning of the defective regions of a patient's face, the use of 3D anatomic models from an ear digital library and rapid prototyping of both substructures for bar anchoring and moulds for silicone processing.     

Osteochondral tissue engineering: scaffolds, stem cells and applications

Osteochondral tissue engineering has shown an increasing development to provide suitable strategies for the regeneration of damaged cartilage and underlying subchondral bone tissue. For reasons of the limitation in the capacity of articular cartilage to self-repair, it is essential to develop approaches based on suitable scaffolds made of appropriate engineered biomaterials. The combination of biodegradable polymers and bioactive ceramics in a variety of composite structures is promising in this area, whereby the fabrication methods, associated cells and signalling factors determine the success of the strategies. The objective of this review is to present and discuss approaches being proposed in osteochondral tissue engineering, which are focused on the application of various materials forming bilayered composite scaffolds, including polymers and ceramics, discussing the variety of scaffold designs and fabrication methods being developed. Additionally, cell sources and biological protein incorporation methods are discussed, addressing their interaction with scaffolds and highlighting the potential for creating a new generation of bilayered composite scaffolds that can mimic the native interfacial tissue properties, and are able to adapt to the biological environment.     

Helical spring template fabrication of cell‐laden microfluidic hydrogels for tissue engineering

Cell‐laden microfluidic hydrogels find great potential applications in microfluidics, tissue engineering, and drug delivery, due to their ability to control mass transport and cell microenvironment. A variety of methods have been developed to fabricate hydrogels with microfluidic channels, such as molding, bioprinting, and photopatterning. However, the relatively simple structure available and the specific equipment required limit their broad applications in tissue engineering. Here, we developed a simple method to fabricate microfluidic hydrogels with helical microchannels based on a helical spring template. Results from both experimental investigation and numerical modeling revealed a significant enhancement on the perfusion ability and cell viability of helical microfluidic hydrogels compared to those with straight microchannels. The feasibility of such a helical spring template method was also demonstrated for microfluidic hydrogels with complex three‐dimensional channel networks such as branched helical microchannels. The method presented here could potentially facilitate the development of vascular tissue engineering and cell microenvironment engineering. Biotechnol. Bioeng. 2013; 110: 980–989. © 2012 Wiley Periodicals, Inc.     

Analysis of cell growth and diffusion in a scaffold for cartilage tissue engineering

Developments in tissue engineering over the past decade have offered promising future for the repair and reconstruction of damaged tissues. To regenerate three dimensional and weight-bearing implants, advances in biomaterials and manufacturing technologies prompted cell cultivations with natural or artificial scaffolds, in which cells are allowed to proliferate, migrate, and differentiate in vitro. In this article, we develop a mathematical model for cell growth in a porous scaffold. By treating the cell-scaffold construct as a porous medium, a continuum model is set up based on basic principles of mass conservation. In addition to cell growth kinetics, we incorporate cell diffusion in the model to describe the effects of cell random walks. Computational results are compared to experimental data found in the literature. With this model, we are able to investigate cell motility, heterogeneous cell distributions, and non-uniform seeding for tissue engineering applications. Results show that random walks tend to enhance uniform cell spreads in space, which in turn increases the probabilities for cells to acquire nutrients; therefore random walks are likely to be a positive contribution to the overall cell growth on scaffolds.     

A cell leakproof PLGA‐collagen hybrid scaffold for cartilage tissue engineering

A cell leakproof porous poly(DL ‐lactic‐co‐glycolic acid) (PLGA)‐collagen hybrid scaffold was prepared by wrapping the surfaces of a collagen sponge except the top surface for cell seeding with a bi‐layered PLGA mesh. The PLGA‐collagen hybrid scaffold had a structure consisting of a central collagen sponge formed inside a bi‐layered PLGA mesh cup. The hybrid scaffold showed high mechanical strength. The cell seeding efficiency was 90.0% when human mesenchymal stem cells (MSCs) were seeded in the hybrid scaffold. The central collagen sponge provided enough space for cell loading and supported cell adhesion, while the bi‐layered PLGA mesh cup protected against cell leakage and provided high mechanical strength for the collagen sponge to maintain its shape during cell culture. The MSCs in the hybrid scaffolds showed round cell morphology after 4 weeks culture in chondrogenic induction medium. Immunostaining demonstrated that type II collagen and cartilaginous proteoglycan were detected in the extracellular matrices. Gene expression analyses by real‐time PCR showed that the genes encoding type II collagen, aggrecan, and SOX9 were upregulated. These results indicated that the MSCs differentiated and formed cartilage‐like tissue when being cultured in the cell leakproof PLGA‐collagen hybrid scaffold. The cell leakproof PLGA‐collagen hybrid scaffolds should be useful for applications in cartilage tissue engineering. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Oxygen mapping: Probing a novel seeding strategy for bone tissue engineering

Bone tissue engineering (BTE) utilizing biomaterial scaffolds and human mesenchymal stem cells (hMSCs) is a promising approach for the treatment of bone defects. The quality of engineered tissue is crucially affected by numerous parameters including cell density and the oxygen supply. In this study, a novel oxygen‐imaging sensor was introduced to monitor the oxygen distribution in three dimensional (3D) scaffolds in order to analyze a new cell‐seeding strategy. Immortalized hMSCs, pre‐cultured in a monolayer for 30–40% or 70–80% confluence, were used to seed demineralized bone matrix (DBM) scaffolds. Real‐time measurements of oxygen consumption in vitro were simultaneously performed by the novel planar sensor and a conventional needle‐type sensor over 24 h. Recorded oxygen maps of the novel planar sensor revealed that scaffolds, seeded with hMSCs harvested at lower densities (30–40% confluence), exhibited rapid exponential oxygen consumption profile. In contrast, harvesting cells at higher densities (70–80% confluence) resulted in a very slow, almost linear, oxygen decrease due to gradual achieving the stationary growth phase. In conclusion, it could be shown that not only the seeding density on a scaffold, but also the cell density at the time point of harvest is of major importance for BTE. The new cell seeding strategy of harvested MSCs at low density during its log phase could be a useful strategy for an early in vivo implantation of cell‐seeded scaffolds after a shorter in vitro culture period. Furthermore, the novel oxygen imaging sensor enables a continuous, two‐dimensional, quick and convenient to handle oxygen mapping for the development and optimization of tissue engineered scaffolds. Biotechnol. Bioeng. 2017;114: 894–902. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.