Using embryonic stem cells to form a biological pacemaker via tissue engineering technology

Biological pacemakers can be achieved by various gene‐based and cell‐based approaches. Embryonic stem cells (ESCs)‐derived pacemaker cells might be the most promising way to form biological pacemakers, but there are challenges as to how to control the differentiation of ESCs and to overcome the neoplasia, proarrhythmia, or immunogenicity resulting from the use of ESCs. As a potential approach to solve these difficult problems, tissue‐engineering techniques may provide a precise control on the different cell components of multicellular aggregates and the forming of a construct with‐defined architectures and functional properties. The combined interactions between ESC‐derived pacemaker cells, supporting cells, and matrices may completely reproduce pacemaker properties and result in a steady functional unit to induce rhythmic electrical and contractile activities. As ESCs have a high capability for self‐renewal, proliferation, and potential differentiation, we hypothesize that ESCs can be used as a source of pacemaker cells for tissue‐engineering applications and the ambitious goal of biological cardiac pacemakers may ultimately be achieved with ESCs via tissue‐engineering technology.     

工程化骨软骨生理微环境构建方法及研究进展

骨组织工程是通过在体外构建有正常组织功能或疾病生理特点的临床模型,用以药物筛选,或研究疾病发生发展过程。骨骼肌肉系统是载重系统,其功能与组织结构、细胞外基质等密切相关。在构建骨组织体外模型时,需要结合骨、软骨及其他构成成分的生理微环境,表现关节骨软骨接合处的生理特点及作用机制,进而模拟正常及病理状态下骨组织系统对刺激的反应。本综述从骨软骨组织的生理构造入手,阐述了骨软骨连接处在退行性关节病变发生发展过程中的作用,并系统的论述了体外构建三维骨软骨组织的方法及这些方法的优势和局限性,为体外构建骨软骨组织工程在临床上应用提供支持。     

Generation of tissue constructs for cardiovascular regenerative medicine: From cell procurement to scaffold design

The ability of the human body to naturally recover from coronary heart disease is limited because cardiac cells are terminally differentiated, have low proliferation rates, and low turn-over rates. Cardiovascular tissue engineering offers the potential for production of cardiac tissue ex vivo, but is currently limited by several challenges: (i) Tissue engineering constructs require pure populations of seed cells, (ii) Fabrication of 3-D geometrical structures with features of the same length scales that exist in native tissue is non-trivial, and (iii) Cells require stimulation from the appropriate biological, electrical and mechanical factors. In this review, we summarize the current state of microfluidic techniques for enrichment of subpopulations of cells required for cardiovascular tissue engineering, which offer unique advantages over traditional plating and FACS/MACS-based enrichment. We then summarize modern techniques for producing tissue engineering scaffolds that mimic native cardiac tissue.     

Directed assembly of cell-laden hydrogels for engineering functional tissues

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.     

Directed assembly of cell-laden hydrogels for engineering functional tissues

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.     

Functional tissue engineering: the role of biomechanics

"Tissue engineering" uses implanted cells, scaffolds, DNA, protein, and/or protein fragments to replace or repair injured or diseased tissues and organs. Despite its early success, tissue engineers have faced challenges in repairing or replacing tissues that serve a predominantly biomechanical function. An evolving discipline called "functional tissue engineering" (FTE) seeks to address these challenges. In this paper, the authors present principles of functional tissue engineering that should be addressed when engineering repairs and replacements for load-bearing structures. First, in vivo stress/strain histories need to be measured for a variety of activities. These in vivo data provide mechanical thresholds that tissue repairs/replacements will likely encounter after surgery. Second, the mechanical properties of the native tissues must be established for subfailure and failure conditions. These "baseline data" provide parameters within the expected thresholds for different in vivo activities and beyond these levels if safety factors are to be incorporated. Third, a subset of these mechanical properties must be selected and prioritized. This subset is important, given that the mechanical properties of the designs are not expected to completely duplicate the properties of the native tissues. Fourth, standards must be set when evaluating the repairs/replacements after surgery so as to determine, "how good is good enough?" Some aspects of the repair outcome may be inferior, but other mechanical characteristics of the repairs and replacements might be suitable. New and improved methods must also be developed for assessing the function of engineered tissues. Fifth, the effects of physical factors on cellular activity must be determined in engineered tissues. Knowing these signals may shorten the iterations required to replace a tissue successfully and direct cellular activity and phenotype toward a desired end goal. Finally, to effect a better repair outcome, cell-matrix implants may benefit from being mechanically stimulated using in vitro "bioreactors" prior to implantation. Increasing evidence suggests that mechanical stress, as well as other physical factors, may significantly increase the biosynthetic activity of cells in bioartificial matrices. Incorporating each of these principles of functional tissue engineering should result in safer and more efficacious repairs and replacements for the surgeon and patient.     

Protein engineering approaches to biomaterials design

Biomaterials play crucial roles in reconstructive surgery, tissue engineering and regenerative medicine. Protein engineering offers powerful solutions to the challenges posed by the creation of well-defined, multifunctional materials that guide cell and tissue behavior. Especially challenging is the complex interplay between mechanical and biological properties in determining the success or failure of biomaterials designed for clinical use.     

Parts plus pipes: synthetic biology approaches to metabolic engineering

Synthetic biologists combine modular biological "parts" to create higher-order devices. Metabolic engineers construct biological "pipes" by optimizing the microbial conversion of basic substrates to desired compounds. Many scientists work at the intersection of these two philosophies, employing synthetic devices to enhance metabolic engineering efforts. These integrated approaches promise to do more than simply improve product yields; they can expand the array of products that are tractable to produce biologically. In this review, we explore the application of synthetic biology techniques to next-generation metabolic engineering challenges, as well as the emerging engineering principles for biological design.     

Biological matrices and bionanotechnology

A crucial step towards the goal of tissue engineering a heart valve will be the choice of scaffold onto which an appropriate cell phenotype can be seeded. Successful scaffold materials should be amenable to modification, have a controlled degradation, be compatible with the cells, lack cytotoxicity and not elicit an immune or inflammatory response. In addition, the scaffold should induce appropriate responses from the cells seeded onto it, such as cell attachment, proliferation and remodelling capacity, all of which should promote the formation of a tissue construct that can mimic the structure and function of the native valve. This paper discusses the various biological scaffolds that have been considered and are being studied for use in tissue engineering a heart valve. Also, strategies to enhance the biological communication between the scaffold and the cells seeded onto it as well as the use of bionanotechnology in the manufacture of scaffolds possessing the desired properties will be discussed.     

A modular approach to creating large engineered cartilage surfaces

Native articular cartilage has limited capacity to repair itself from focal defects or osteoarthritis. Tissue engineering has provided a promising biological treatment strategy that is currently being evaluated in clinical trials. However, current approaches in translating these techniques to developing large engineered tissues remains a significant challenge. In this study, we present a method for developing large-scale engineered cartilage surfaces through modular fabrication. Modular Engineered Tissue Surfaces (METS) uses the well-known, but largely under-utilized self-adhesion properties of de novo tissue to create large scaffolds with nutrient channels. Compressive mechanical properties were evaluated throughout METS specimens, and the tensile mechanical strength of the bonds between attached constructs was evaluated over time. Raman spectroscopy, biochemical assays, and histology were performed to investigate matrix distribution. Results showed that by Day 14, stable connections had formed between the constructs in the METS samples. By Day 21, bonds were robust enough to form a rigid sheet and continued to increase in size and strength over time. Compressive mechanical properties and glycosaminoglycan (GAG) content of METS and individual constructs increased significantly over time. The METS technique builds on established tissue engineering accomplishments of developing constructs with GAG composition and compressive properties approaching native cartilage. This study demonstrated that modular fabrication is a viable technique for creating large-scale engineered cartilage, which can be broadly applied to many tissue engineering applications and construct geometries.     

Cell–scaffold mechanical interplay within engineered tissue

Effective tissue engineering requires appropriate selection of cells and scaffold, where the latter serves as a mechanical and biological support for cell growth and functionality. The optimal combination of cell source and scaffold properties can vary for each desired application. Such preconditions necessitate enhanced understanding of the interactions between cells and scaffold within engineered tissue. Several studies have examined the deforming effects cells induce in scaffolds via exertion of contractile forces. In contrast, other studies focus on the scaffold's biochemical and mechanical properties and their effects on cell behavior.This review summarizes the mechanical interplay between cells and scaffold within engineered tissue. We present evidence for contractile forces exerted by cells on three-dimensional (3D) scaffolds and discuss existing methods for their quantification. In addition, we address some theories related to the effects of scaffold stiffness and mechanical stimulation on cell behavior. Further understanding of the reciprocal effects between cells and scaffold will provide both enhanced knowledge regarding the expected properties of engineered tissue and more competent tissue regeneration techniques.     

Functional tissue engineering of chondral and osteochondral constructs

Due to the prevalence of osteoarthritis (OA) and damage to articular cartilage, coupled with the poor intrinsic healing capacity of this avascular connective tissue, there is a great demand for an articular cartilage substitute. As the bearing material of diarthrodial joints, articular cartilage has remarkable functional properties that have been difficult to reproduce in tissue-engineered constructs. We have previously demonstrated that by using a functional tissue engineering approach that incorporates mechanical loading into the long-term culture environment, one can enhance the development of mechanical properties in chondrocyte-seeded agarose constructs. As these gel constructs begin to achieve material properties similar to that of the native tissue, however, new challenges arise, including integration of the construct with the underlying native bone. To address this issue, we have developed a technique for producing gel constructs integrated into an underlying bony substrate. These osteochondral constructs develop cartilage-like extracellular matrix and material properties over time in free swelling culture. In this study, as a preliminary to loading such osteochondral constructs, finite element modeling (FEM) was used to predict the spatial and temporal stress, strain, and fluid flow fields within constructs subjected to dynamic deformational loading. The results of these models suggest that while chondral ("gel alone") constructs see a largely homogenous field of mechanical signals, osteochondral ("gel bone") constructs see a largely inhomogeneous distribution of mechanical signals. Such inhomogeneity in the mechanical environment may aid in the development of inhomogeneity in the engineered osteochondral constructs. Together with experimental observations, we anticipate that such modeling efforts will provide direction for our efforts aimed at the optimization of applied physical forces for the functional tissue engineering of an osteochondral articular cartilage substitute.     

Rapid prototyping in tissue engineering: challenges and potential

Tissue engineering aims to produce patient-specific biological substitutes in an attempt to circumvent the limitations of existing clinical treatments for damaged tissue or organs. The main regenerative tissue engineering approach involves transplantation of cells onto scaffolds. The scaffold attempts to mimic the function of the natural extracellular matrix, providing a temporary template for the growth of target tissues. Scaffolds should have suitable architecture and strength to serve their intended function. This paper presents a comprehensive review of the fabrication methods, including conventional, mainly manual, techniques and advanced processing methods such as rapid prototyping (RP) techniques. The potential and challenges of scaffold-based technology are discussed from the perspective of RP technology.     

Design and engineering of allosteric communications in proteins

Allostery in proteins plays an important role in regulating protein activities and influencing many biological processes such as gene expression, enzyme catalysis, and cell signaling. The process of allostery takes place when a signal detected at a site on a protein is transmitted via a mechanical pathway to a functional site and, thus, influences its activity. The pathway of allosteric communication consists of amino acids that form a network with covalent and non-covalent bonds. By mutating residues in this allosteric network, protein engineers have successfully established novel allosteric pathways to achieve desired properties in the target protein. In this review, we highlight the most recent and state-of-the-art techniques for allosteric communication engineering. We also discuss the challenges that need to be overcome and future directions for engineering protein allostery.     

Hyaluronic acid and dextran-based semi-IPN hydrogels as biomaterials for bioprinting

Bioprinting is a recent technology in tissue engineering used for the design of porous constructs through layer-by-layer deposition of cell-laden material. This technology would benefit from new biomaterials that can fulfill specific requirements for the fabrication of well-defined 3D constructs, such as the preservation of cell viability and adequate mechanical properties. We evaluated the suitability of a novel semi-interpenetrating network (semi-IPN), based on hyaluronic acid and hydroxyethyl-methacrylate-derivatized dextran (dex-HEMA), to form 3D hydrogel bioprinted constructs. The rheological properties of the solutions allowed proper handling during bioprinting, whereas photopolymerization led to stable constructs of which their mechanical properties matched the wide range of mechanical strengths of natural tissues. Importantly, excellent viability was observed for encapsulated chondrocytes. The results demonstrate the suitability of hyaluronic acid/dex-HEMA semi-IPNs to manufacture bioprinted constructs for tissue engineering.     

Mechanical characterization of anisotropic planar biological soft tissues using finite indentation: experimental feasibility

Heart valve tissue engineering offers a promising alternative for current treatment and replacement strategies, e.g., synthetic or bioprosthetic heart valves. In vitro mechanical conditioning is an important tool for engineering strong, implantable heart valves. Detailed knowledge of the mechanical properties of the native tissue as well as the developing tissue construct is vital for a better understanding and control of the remodeling processes induced by mechanical conditioning. The nonlinear, anisotropic and inhomogeneous mechanical behavior of heart valve tissue necessitates a mechanical characterization method that is capable of dealing with these complexities. In a recent computational study we showed that one single indentation test, combining force and deformation gradient data, provides sufficient information for local characterization of nonlinear soft anisotropic tissue properties. In the current study this approach is validated in two steps. First, indentation tests with varying indenter sizes are performed on linear elastic PDMS rubbers and compared to tensile tests on the same specimen. For the second step, tissue constructs are engineered using uniaxial or equibiaxial static constrained culture conditions. Digital image correlation (DIC) is used to quantify the anisotropy in the tissue constructs. For both validation steps, material parameters are estimated by inverse fitting of a computational model to the experimental results.     

Advances in polymeric systems for tissue engineering and biomedical applications

The characteristics of tissue engineered scaffolds are major concerns in the quest to fabricate ideal scaffolds for tissue engineering applications. The polymer scaffolds employed for tissue engineering applications should possess multifunctional properties such as biocompatibility, biodegradability and favorable mechanical properties as it comes in direct contact with the body fluids in vivo. Additionally, the polymer system should also possess biomimetic architecture and should support stem cell adhesion, proliferation and differentiation. As the progress in polymer technology continues, polymeric biomaterials have taken characteristics more closely related to that desired for tissue engineering and clinical needs. Stimuli responsive polymers also termed as smart biomaterials respond to stimuli such as pH, temperature, enzyme, antigen, glucose and electrical stimuli that are inherently present in living systems. This review highlights the exciting advancements in these polymeric systems that relate to biological and tissue engineering applications. Additionally, several aspects of technology namely scaffold fabrication methods and surface modifications to confer biological functionality to the polymers have also been discussed. The ultimate objective is to emphasize on these underutilized adaptive behaviors of the polymers so that novel applications and new generations of smart polymeric materials can be realized for biomedical and tissue engineering applications.     

Contribution of biomechanics to management of ligament and tendon injuries

The contribution of biomechanics to the advancement of management of ligament and tendon injuries has been significant. Thanks to Professor Y.C. Fung's writing and guidance, our field of research has done fundamental work on anatomy and biology of ligaments and tendons, developed methods to accurately determine mechanical properties, identified various experimental factors which could change the outcome measurements as well as examined biological factors that change tissue properties in-vivo. Professor Fung also gave us his quasi-linear viscoelastic theory for soft tissues so that the time and history dependent properties of ligaments and tendons could be properly described. We have further adopted Professor Fung's eight steps on methods of approach for biomechanical investigation to understand as well as enhance the treatment of ligament and tendon injuries during work or sports related activities. Examples on how to better treat the tears of the medial collateral ligament of the knee, as well as how to improve reconstruction procedures for the anterior cruciate ligament are presented in detail. Currently the use of functional tissue engineering for ligament and tendon healing is a topic of great interest. Here the use of biological scaffolds, such as porcine small intestinal submucosa, has shown promise. For the last 35 to 40 years, the field of biomechanics has made great strides in the treatment of ligament and tendon injuries, and many patients have benefited. The future is even brighter because of what has been done properly in the past. Exciting advances can be made in the field of tissue engineering through novel in-vitro culture and bioscaffold fabrication techniques. Recent technology can also allow the collection of in-vivo data so that ligament and tendon injuries can be better understood. Yet, solving new and more complex problems must still follow the stepwise methods of approach as taught by Professor Fung.     

Tailoring of the rheological properties of bioinks to improve bioprinting and bioassembly for tissue replacement

BackgroundTissue replacement is among the most important challenges in biotechnology worldwide.Scope of reviewWe aim to highlight the importance of the intricate feedback between rheological properties and materials science and cell biological parameters in order to obtain an efficient bioink design, supported by various practical examples.Major conclusionsViscoelastic properties of bioink formulas, rheological properties, injection speed and printing nozzle diameter must be considered in bioink design. These properties are related to cell behavior and the survival rate during and after printing. Mechanosensing can strongly influence epigenetics to modify the final cell phenotype, which can affect the replacement tissue.General significanceIn tissue engineering, biotechnologists must consider the biophysical properties and biological conditions of the materials used, as well as the material delivery mode (in a case or tissue) and maturation mode (curing or biomass), to ensure the development off appropriate materials mimicking the native tissue.     

细胞打印技术及应用

细胞打印技术是一种在体外构造具有生物活性的三维多细胞体系的先进技术。近年来,有关细胞打印技术的研究引起广泛的关注,其原因在于该领域具有明显的学科交叉与渗透融合的特点,它处于生命科学与快速成型技术、生物制造技术、生物科学和材料科学的交汇点。更加值得关注的是它为组织工程学突破二维研究的局限性,在三维尺度上精确控制与人体组织或器官相似的三维构造体方面的研究提供了一种新的思路。基于这一技术不仅在三维组织工程,还对细胞生物学、高通量药物筛选及细胞传感器等方面的前沿问题均有广阔的研究应用前景,介绍了近年来开发用于细胞打印的技术及其潜在的应用。