Multiscale tissue engineering for liver reconstruction

The liver is a target of in vitro tissue engineering despite its capability to regenerate in vivo. The construction of liver tissues in vitro remains challenging. In this review, conventional 3D cultures of hepatocytes are first discussed. Recent advances in the 3D culturing of liver cells are then summarized in the context of in vitro liver tissue reconstruction at the micro- and macroscales. The application of microfluidics technology to liver tissue engineering has been introduced as a bottom-up approach performed at the microscale, whereas whole-organ bioengineering technology was introduced as a top-down approach performed at the macroscale. Mesoscale approaches are also discussed in considering the integration of micro- and macroscale approaches. Multiple parallel multiscale liver tissue engineering studies are ongoing; however, no tissue-engineered liver that is appropriate for clinical use has yet been realized. The integration of multiscale tissue engineering studies is essential for further understanding of liver reconstruction strategies.     

Stress and strain localization in stretched collagenous tissues via a multiscale modelling approach

Mechanobiology of cells in soft collagenous tissues is highly affected by both tissue response at the macroscale and stress/strain localization mechanisms due to features at lower scales. In this paper, the macroscale mechanical behaviour of soft collagenous tissues is modelled by a three-level multiscale approach, based on a multi-step homogenisation technique from nanoscale up to the macroscale. Nanoscale effects, related to both intermolecular cross-links and collagen mechanics, are accounted for, together with geometric nonlinearities at the microscale. Moreover, an effective submodelling procedure is conceived in order to evaluate the local stress and strain fields at the microscale, which is around and within cells. Numerical results, obtained by using an incremental finite element formulation and addressing stretched tendinous tissues, prove consistency and accuracy of the model at both macroscale and microscale, confirming also the effectiveness of the multiscale modelling concept for successfully analysing physiopathological processes in biological tissues.     

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。     

基于水凝胶的“自下而上”组织工程技术研究进展

随着微纳生物制造技术和新型生物材料(如新型水凝胶)的发展,基于模块化组装的"自下而上"组织工程技术引起了广泛的关注,在复杂微结构和血管化组织/器官构建方面显示了广阔的发展前景.本文介绍了"自下而上"组织工程技术的基本原理及模块单元的制备和组装方法,综述和讨论了"自下而上"组织工程技术在体外重构三维组织/器官方面取得的最新研究进展,并对其在生物医学领域的发展前景进行了展望.     

New frontiers in calvarial reconstruction: integrating computer-assisted design and tissue engineering in cranioplasty

Repair of large and complex calvarial defects remains a particular challenge for reconstruction. The paucity of techniques and materials emphasizes the need for alternative bone formation strategies. Recent integrative approaches suggest that successful reconstruction requires interdisciplinary teams, with surgeons interacting with imaging experts, materials scientists, and engineers. In this review, the authors present an overview of current materials used in calvarial reconstruction. Subsequently, progress in computer-designed prostheses, tissue engineering, and osteoinduction strategies is discussed. Finally, the authors discuss their experience with the integration of computer-aided fabrication of customized implants and tissue engineering for calvarial reconstruction.     

Multiscale mechanobiology: Coupling models of adhesion kinetics and nonlinear tissue mechanics

The mechanical behavior of tissues at the macroscale is tightly coupled to cellular activity at the microscale. Dermal wound healing is a prominent example of a complex system in which multiscale mechanics regulate restoration of tissue form and function. In cutaneous wound healing, a fibrin matrix is populated by fibroblasts migrating in from a surrounding tissue made mostly out of collagen. Fibroblasts both respond to mechanical cues, such as fiber alignment and stiffness, as well as exert active stresses needed for wound closure.Here, we develop a multiscale model with a two-way coupling between a microscale cell adhesion model and a macroscale tissue mechanics model. Starting from the well-known model of adhesion kinetics proposed by Bell, we extend the formulation to account for nonlinear mechanics of fibrin and collagen and show how this nonlinear response naturally captures stretch-driven mechanosensing. We then embed the new nonlinear adhesion model into a custom finite element implementation of tissue mechanical equilibrium. Strains and stresses at the tissue level are coupled with the solution of the microscale adhesion model at each integration point of the finite element mesh. In addition, solution of the adhesion model is coupled with the active contractile stress of the cell population. The multiscale model successfully captures the mechanical response of biopolymer fibers and gels, contractile stresses generated by fibroblasts, and stress-strain contours observed during wound healing. We anticipate that this framework will not only increase our understanding of how mechanical cues guide cellular behavior in cutaneous wound healing, but will also be helpful in the study of mechanobiology, growth, and remodeling in other tissues.     

Integrating Cellular Metabolism into a Multiscale Whole-Body Model

Cellular metabolism continuously processes an enormous range of external compounds into endogenous metabolites and is as such a key element in human physiology. The multifaceted physiological role of the metabolic network fulfilling the catalytic conversions can only be fully understood from a whole-body perspective where the causal interplay of the metabolic states of individual cells, the surrounding tissue and the whole organism are simultaneously considered. We here present an approach relying on dynamic flux balance analysis that allows the integration of metabolic networks at the cellular scale into standardized physiologically-based pharmacokinetic models at the whole-body level. To evaluate our approach we integrated a genome-scale network reconstruction of a human hepatocyte into the liver tissue of a physiologically-based pharmacokinetic model of a human adult. The resulting multiscale model was used to investigate hyperuricemia therapy, ammonia detoxification and paracetamol-induced toxication at a systems level. The specific models simultaneously integrate multiple layers of biological organization and offer mechanistic insights into pathology and medication. The approach presented may in future support a mechanistic understanding in diagnostics and drug development.     

Multiscale model predicts tissue-level failure from collagen fiber-level damage

Excessive tissue-level forces communicated to the microstructure and extracellular matrix of soft tissues can lead to damage and failure through poorly understood physical processes that are multiscale in nature. In this work, we propose a multiscale mechanical model for the failure of collagenous soft tissues that incorporates spatial heterogeneity in the microstructure and links the failure of discrete collagen fibers to the material response of the tissue. The model, which is based on experimental failure data derived from different collagen gel geometries, was able to predict the mechanical response and failure of type I collagen gels, and it demonstrated that a fiber-based rule (at the micrometer scale) for discrete failure can strongly shape the macroscale failure response of the gel (at the millimeter scale). The model may be a useful tool in predicting the macroscale failure conditions for soft tissues and engineered tissue analogs. In addition, the multiscale model provides a framework for the study of failure in complex fiber-based mechanical systems in general.     

Empirical Study of an Adaptive Multiscale Model for Simulating Cardiac Conduction

We modify and empirically study an adaptive multiscale model for simulating cardiac action potential propagation along a strand of cardiomyocytes. The model involves microscale partial differential equations posed over cells near the action potential upstroke and macroscale partial differential equations posed over the remainder of the tissue. An important advantage of the modified model of this paper is that, unlike our original model, it does not require perfect alignment between myocytes and the macroscale computational grid. We study the effects of gap-junctional coupling, ephaptic coupling, and macroscale grid spacing on the accuracy of the multiscale model. Our simulations reveal that the multiscale method accurately reproduces both the wavespeed and the waveform, including both upstroke and recovery, of fully microscale models. They also reveal that perfect alignment between myocytes and the macroscale grid is not necessary to reproduce the dynamics of a traveling action potential. Further, our simulations suggest that the macroscale grid spacing used in an adaptive multiscale model need not be much finer than the spatial width of an action potential. These results are demonstrated to hold under high, low, and zero gap-junctional coupling regimes.     

Integrating intracellular dynamics using CompuCell3D and Bionetsolver: applications to multiscale modelling of cancer cell growth and invasion

In this paper we present a multiscale, individual-based simulation environment that integrates CompuCell3D for lattice-based modelling on the cellular level and Bionetsolver for intracellular modelling. CompuCell3D or CC3D provides an implementation of the lattice-based Cellular Potts Model or CPM (also known as the Glazier-Graner-Hogeweg or GGH model) and a Monte Carlo method based on the metropolis algorithm for system evolution. The integration of CC3D for cellular systems with Bionetsolver for subcellular systems enables us to develop a multiscale mathematical model and to study the evolution of cell behaviour due to the dynamics inside of the cells, capturing aspects of cell behaviour and interaction that is not possible using continuum approaches. We then apply this multiscale modelling technique to a model of cancer growth and invasion, based on a previously published model of Ramis-Conde et al. (2008) where individual cell behaviour is driven by a molecular network describing the dynamics of E-cadherin and β-catenin. In this model, which we refer to as the centre-based model, an alternative individual-based modelling technique was used, namely, a lattice-free approach. In many respects, the GGH or CPM methodology and the approach of the centre-based model have the same overall goal, that is to mimic behaviours and interactions of biological cells. Although the mathematical foundations and computational implementations of the two approaches are very different, the results of the presented simulations are compatible with each other, suggesting that by using individual-based approaches we can formulate a natural way of describing complex multi-cell, multiscale models. The ability to easily reproduce results of one modelling approach using an alternative approach is also essential from a model cross-validation standpoint and also helps to identify any modelling artefacts specific to a given computational approach.     

Toward engineering of vascularized three-dimensional liver tissue equivalents possessing a clinically significant mass

In this review, we focus on how to develop and rear liver tissue equivalents that can be finally used as liver tissues as a substitute for the original liver. The size should be over 500 cm³ and its per-volume-based functionalities should be those comparable to the in vivo liver. As can easily be imagined, it will necessitate continuous efforts and we cannot predict when it becomes feasible at present. However, we need to set up an appropriate road map based on the latest knowledge concerning various related areas and to make efficient and integrative efforts to address the issues. The efforts that are currently required include design and fabrication of scaffolds, procurement of large mass of mature hepatocytes, rearing of the liver tissue equivalents in vitro and proof-of-concept studies in large animals such as pigs. Through the establishment of fundamental methodologies in such preclinical studies, we will know whether we can proceed to human clinical trials of such tissue equivalents. According to the possible road map, we summarized latest related approaches, with consistently stressing the two important but sometimes conflicting standpoints, that is, optimization of oxygenation supply to the cells in both micro- and macro-scale and three-dimensional (3D) culture of hepatocyte progenitors or stem cells toward hepatic lineages. In addition, we tried to clear up the remaining issues and the clues to overcome them.     

Tools for studying and modulating (cardiac muscle) cell mechanics and mechanosensing across the scales

Cardiomyocytes generate force for the contraction of the heart to pump blood into the lungs and body. At the same time, they are exquisitely tuned to the mechanical environment and react to e.g. changes in cell and extracellular matrix stiffness or altered stretching due to reduced ejection fraction in heart disease, by adapting their cytoskeleton, force generation and cell mechanics. Both mechanical sensing and cell mechanical adaptations are multiscale processes. Receptor interactions with the extracellular matrix at the nanoscale will lead to clustering of receptors and modification of the cytoskeleton. This in turn alters mechanosensing, force generation, cell and nuclear stiffness and viscoelasticity at the microscale. Further, this affects cell shape, orientation, maturation and tissue integration at the microscale to macroscale. A variety of tools have been developed and adapted to measure cardiomyocyte receptor-ligand interactions and forces or mechanics at the different ranges, resulting in a wealth of new information about cardiomyocyte mechanobiology. Here, we take stock at the different tools for exploring cardiomyocyte mechanosensing and cell mechanics at the different scales from the nanoscale to microscale and macroscale.     

Fetal and adult liver stem cells for liver regeneration and tissue engineering

For the development of innovative cell-based liver directed therapies, e.g. liver tissue engineering, the use of stem cells might be very attractive to overcome the limitation of donor liver tissue. Liver specific differentiation of embryonic, fetal or adult stem cells is currently under investigation. Different types of fetal liver (stem) cells during development were identified, and their advantageous growth potential and bipotential differentiation capacity were shown. However, ethical and legal issues have to be addressed before using fetal cells. Use of adult stem cells is clinically established, e.g. transplantation of hematopoietic stem cells. Other bone marrow derived liver stem cells might be mesenchymal stem cells (MSC). However, the transdifferentiation potential is still in question due to the observation of cellular fusion in several in vivo experiments. In vitro experiments revealed a crucial role of the environment (e.g. growth factors and extracellular matrix) for specific differentiation of stem cells. Co-cultured liver cells also seemed to be important for hepatic gene expression of MSC. For successful liver cell transplantation, a novel approach of tissue engineering by orthotopic transplantation of gel-immobilized cells could be promising, providing optimal environment for the injected cells. Moreover, an orthotopic tissue engineering approach using bipotential stem cells could lead to a repopulation of the recipients liver with healthy liver and biliary cells, thus providing both hepatic functions and biliary excretion. Future studies have to investigate, which stem cell and environmental conditions would be most suitable for the use of stem cells for liver regeneration or tissue engineering approaches.     

Engineering of human hepatic tissue with functional vascular networks

Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.     

Engineering of human hepatic tissue with functional vascular networks

Although absolute organ shortage highlights the needs of alternative organ sources for regenerative medicine, the generation of a three-dimensional (3D) and complex vital organ, such as well-vascularized liver, remains a challenge. To this end, tissue engineering holds great promise; however, this approach is significantly limited by the failure of early vascularization in vivo after implantation. Here, we established a stable 3D in vitro pre-vascularization platform to generate human hepatic tissue after implantation in vivo. Human fetal liver cells (hFLCs) were mixed with human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (hMSCs) and were implanted into a collagen/fibronectin matrix composite that was used as a 3-D carrier. After a couple of days, the fluorescent HUVECs developed premature vascular networks in vitro, which were stabilized by hMSCs. The establishment of functional vessels inside the pre-vascularized constructs was proven using dextran infusion studies after implantation under a transparency cranial window. Furthermore, dynamic morphological changes during embryonic liver cell maturation were intravitaly quantified with high-resolution confocal microscope analysis. The engineered human hepatic tissue demonstrated multiple liver-specific features, both structural and functional. Our new techniques discussed here can be implemented in future clinical uses and industrial uses, such as drug testing.     

To engineer is to create: the link between engineering and regeneration

Tissue engineering is a radically different approach to reconstruction of the body following degenerative diseases, trauma or chronic debilitating conditions. Although there have been some successes, tissue engineering is not yet delivering significant progress in terms of clinical outcomes and commercialization. Part of the problem is that we have failed to understand what tissue engineering really means and to appreciate that engineering is synonymous with creation. These processes involve many different phases and there has been minimal integration of these phases within tissue-engineering paradigms. The conventional concept, based upon a temporal sequence from sourcing cells through to the incorporation of generated tissue into a host, has to be transformed by a systems engineering approach in which all biological and technological phases, and the inter-relationships between them, are fully integrated. It might be that real success will not be achieved until systems biology is superimposed onto this systems engineering paradigm.     

Top down and bottom up engineering of bone

The goal of this retrospective article is to place the body of my lab's multiscale mechanobiology work in context of top-down and bottom-up engineering of bone. We have used biosystems engineering, computational modeling and novel experimental approaches to understand bone physiology, in health and disease, and across time (in utero, postnatal growth, maturity, aging and death, as well as evolution) and length scales (a single bone like a femur, m; a sample of bone tissue, mm-cm; a cell and its local environment, μm; down to the length scale of the cell's own skeleton, the cytoskeleton, nm). First we introduce the concept of flow in bone and the three calibers of porosity through which fluid flows. Then we describe, in the context of organ-tissue, tissue-cell and cell-molecule length scales, both multiscale computational models and experimental methods to predict flow in bone and to understand the flow of fluid as a means to deliver chemical and mechanical cues in bone. Addressing a number of studies in the context of multiple length and time scales, the importance of appropriate boundary conditions, site specific material parameters, permeability measures and even micro-nanoanatomically correct geometries are discussed in context of model predictions and their value for understanding multiscale mechanobiology of bone. Insights from these multiscale computational modeling and experimental methods are providing us with a means to predict, engineer and manufacture bone tissue in the laboratory and in the human body.     

Freestanding stacked mesh‐like hydrogel sheets enable the creation of complex macroscale cellular scaffolds

Hydrogel‐based bottom‐up tissue engineering depends on assembly of cell‐laden modules for complex three‐dimensional tissue reconstruction. Though sheet‐like hydrogel modules enable rapid and controllable assembly, they have limitations in generating spatial microenvironments and mass transport. Here, we describe a simple method for forming large‐scale cell‐hydrogel assemblies via stacking cell‐embedded mesh‐like hydrogel sheets to create complex macroscale cellular scaffolds. Freestanding stacked hydrogel sheets were fabricated for long‐term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets (8.0 mm × 8.7 mm) were aligned using a polydimethylsiloxane drainage well. The stacked hydrogel sheets were precisely aligned so that the openings could facilitate mass transport through the stacked sheets. Despite the relatively large height of the stacked structure (400–700 μm), which is larger than the diffusion limit thickness of 150–200 μm, the freestanding cell‐ydrogel assemblies maintained cell viability and exhibited enhanced cellular function compared with single hydrogel sheets. Furthermore, a three‐dimensional co‐culture system was constructed simply by stacking different cell‐containing hydrogel sheets. These results show that stacked hydrogel sheets have significant potential as a macroscale cell‐culture and assay platform with complex microenvironments for biologically relevant in vitro tissue‐level drug assays and physiological studies.     

Vital roles of stem cells and biomaterials in skin tissue engineering

Tissue engineering essentially refers to technology for growing new human tissue and is distinct from regenerative medicine. Currently, pieces of skin are already being fabricated for clinical use and many other tissue types may be fabricated in the future.Tissue engineering was first defined in 1987 by the United States National Science Foundation which critically discussed the future targets of bioengineering research and its consequences. The principles of tissue engineering are to initiate cell cultures in vitro, grow them on scaffolds in situ and transplant the composite into a recipient in vivo. From the beginning, scaffolds have been necessary in tissue engineering applications. Regardless, the latest technology has redirected established approaches by omitting scaffolds. Currently, scientists from diverse research institutes are engineering skin without scaffolds. Due to their advantageous properties, stem cells have robustly transformed the tissue engineering field as part of an engineered bilayered skin substitute that will later be discussed in detail. Additionally, utilizing biomaterials or skin replacement products in skin tissue engineering as strategy to successfully direct cell proliferation and differentiation as well as to optimize the safety of handling during grafting is beneficial. This approach has also led to the cells’ application in developing the novel skin substitute that will be briefly explained in this review.