Engineering a clinically-useful matrix for cell therapy

The design criteria for matrices for encapsulation of cells for cell therapy include chemical, biological, engineering, marketing, regulatory, and financial constraints. What is required is a biocompatible material for culture of cells in three-dimensions (3-D) that offers ease of use, experimental flexibility to alter composition and compliance, and a composition that would permit a seamless transition from in vitro to in vivo use. The challenge is to replicate the complexity of the native extracellular matrix (ECM) environment with the minimum number of components necessary to allow cells to rebuild a given tissue. Our approach is to deconstruct the ECM to a few modular components that can be reassembled into biomimetic materials that meet these criteria. These semi-synthetic ECMs (sECMs) employ thiol-modified derivatives of hyaluronic acid (HA) that can form covalently crosslinked, biodegradable hydrogels. These sECMs are “living” biopolymers, meaning that they can be crosslinked in the presence of cells or tissues to enable cell therapy and tissue engineering. Moreover, the sECMs allow inclusion of the appropriate biological cues needed to simulate the complexity of the ECM of a given tissue. Taken together, the sECM technology offers a manufacturable, highly reproducible, flexible, FDA-approvable, and affordable vehicle for cell expansion and differentiation in 3-D.Key words: hyaluronan, 3-D cell culture, tissue engineering, extracellular matrix, crosslinked hydrogel, design criteria, commercial utility, stem cells, regenerative medicine     

Modular extracellular matrices: solutions for the puzzle

The common technique of growing cells in two-dimensions (2-D) is gradually being replaced by culturing cells on matrices with more appropriate composition and stiffness, or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm has been constrained by the absence of a commercially available, biocompatible material that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. The challenge-the puzzle that needs a solution-is to replicate the complexity of the native extracellular matrix (ECM) environment with the minimum number of components necessary to allow cells to rebuild and replicate a given tissue. For use in drug discovery, toxicology, cell banking, and ultimately in reparative medicine, the ideal matrix would therefore need to be highly reproducible, manufacturable, approvable, and affordable. Herein we describe the development of a set of modular components that can be assembled into biomimetic materials that meet these requirements. These semi-synthetic ECMs, or sECMs, are based on hyaluronan derivatives that form covalently crosslinked, biodegradable hydrogels suitable for 3-D culture of primary and stem cells in vitro, and for tissue formation in vivo. The sECMs can be engineered to provide appropriate biological cues needed to recapitulate the complexity of a given ECM environment. Specific applications for different sECM compositions include stem cell expansion with control of differentiation, scar-free wound healing, growth factor delivery, cell delivery for osteochondral defect and liver repair, and development of vascularized tumor xenografts for personalized chemotherapy.     

Simplifying the extracellular matrix for 3-D cell culture and tissue engineering: a pragmatic approach

The common technique of growing cells on tissue culture plastic (TCP) is gradually being supplanted by methods for culturing cells in two-dimensions (2-D) on matrices with more appropriate physical and biological properties or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm is currently constrained by the lack of a biocompatible material in the marketplace that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. In this Prospect, I argue that the standard for 3-D cell culture should be bio-inspired, biomimetic materials that can be used "as is" in drug discovery, toxicology, cell banking, and ultimately in medicine. Such biomaterials must therefore be highly reproducible, manufacturable, approvable, and affordable. To obtain integrated, functional, multicellular systems that recapitulate tissues and organs, the needs of the true end-users-physicians and patients-must dictate the key design criteria. Herein I describe the development of one such material that meets these requirements: a covalently crosslinked, biodegradable, simplified mimic of the extracellular matrix (ECM) that permits 3-D culture of cells in vitro and enables tissue formation in vivo. In contrast to materials that were designed for in vitro cell culture and then found unsuitable for clinical use, these semi-synthetic hyaluronan-derived materials were developed for in vivo tissue repair, and are now being re-engineered for in vitro applications in research.     

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications.     

Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.     

The interplay between tissue growth and scaffold degradation in engineered tissue constructs

In vitro tissue engineering is emerging as a potential tool to meet the high demand for replacement tissue, caused by the increased incidence of tissue degeneration and damage. A key challenge in this field is ensuring that the mechanical properties of the engineered tissue are appropriate for the in vivo environment. Achieving this goal will require detailed understanding of the interplay between cell proliferation, extracellular matrix (ECM) deposition and scaffold degradation. In this paper, we use a mathematical model (based upon a multiphase continuum framework) to investigate the interplay between tissue growth and scaffold degradation during tissue construct evolution in vitro. Our model accommodates a cell population and culture medium, modelled as viscous fluids, together with a porous scaffold and ECM deposited by the cells, represented as rigid porous materials. We focus on tissue growth within a perfusion bioreactor system, and investigate how the predicted tissue composition is altered under the influence of (1) differential interactions between cells and the supporting scaffold and their associated ECM, (2) scaffold degradation, and (3) mechanotransduction-regulated cell proliferation and ECM deposition. Numerical simulation of the model equations reveals that scaffold heterogeneity typical of that obtained from $\mu $ CT scans of tissue engineering scaffolds can lead to significant variation in the flow-induced mechanical stimuli experienced by cells seeded in the scaffold. This leads to strong heterogeneity in the deposition of ECM. Furthermore, preferential adherence of cells to the ECM in favour of the artificial scaffold appears to have no significant influence on the eventual construct composition; adherence of cells to these supporting structures does, however, lead to cell and ECM distributions which mimic and exaggerate the heterogeneity of the underlying scaffold. Such phenomena have important ramifications for the mechanical integrity of engineered tissue constructs and their suitability for implantation in vivo.     

Decellularized adipose matrix provides an inductive microenvironment for stem cells in tissue regeneration

Stem cells play a key role in tissue regeneration due to their self-renewal and multidirectional differentiation, which are continuously regulated by signals from the extracellular matrix (ECM) microenvironment. Therefore, the unique biological and physical characteristics of the ECM are important determinants of stem cell behavior. Although the acellular ECM of specific tissues and organs (such as the skin, heart, cartilage, and lung) can mimic the natural microenvironment required for stem cell differentiation, the lack of donor sources restricts their development. With the rapid development of adipose tissue engineering, decellularized adipose matrix (DAM) has attracted much attention due to its wide range of sources and good regeneration capacity. Protocols for DAM preparation involve various physical, chemical, and biological methods. Different combinations of these methods may have different impacts on the structure and composition of DAM, which in turn interfere with the growth and differentiation of stem cells. This is a narrative review about DAM. We summarize the methods for decellularizing and sterilizing adipose tissue, and the impact of these methods on the biological and physical properties of DAM. In addition, we also analyze the application of different forms of DAM with or without stem cells in tissue regeneration (such as adipose tissue), repair (such as wounds, cartilage, bone, and nerves), in vitro bionic systems, clinical trials, and other disease research.     

Regulation of valvular interstitial cell phenotype and function by hyaluronic acid in 2-D and 3-D culture environments

Disruption of the extracellular matrix (ECM) is frequently found in calcific aortic valve disease (CAVD), yet the role of ECM components in valvular interstitial cell (VIC) function and dysfunction remains poorly understood. This study examines the contributions of exogenous and endogenous hyaluronic acid (HA), in both two-dimensional (2-D) and 3-D environments, in regulating the phenotype and calcification of VICs. VIC calcification was first assessed in a 2-D setting in which the cells were exposed to different molecular weights of exogenous HA presented in either an immobilized or soluble form. Delivery of HA suppressed nodule formation in a molecular weight-dependent manner, while blocking VIC recognition of HA via an antibody to CD44 abolished these nodule-suppressive effects and stimulated other hallmarks of valvular dysfunction. These 2-D results were then validated in a more physiologically-relevant setting, using an approach that allowed the characterization of VIC phenotype in response to HA alterations in the native 3-D environment. In this approach, leaflet organ cultures were analyzed following treatment with anti-CD44 or with hyaluronidase to specifically remove HA. Disruption of VIC-HA interactions upregulated markers of VIC disease and induced leaflet mineralization. Similarly, HA-deficient leaflets exhibited numerous hallmarks of CAVD, including increased VIC proliferation, apoptosis, increased expression of disease-related markers, and mineralization. These findings suggest that VIC-HA interactions are crucial in maintaining a healthy VIC phenotype. Identification ECM components that can regulate VIC phenotype and function has significant implications for understanding native valve disease, investigating possible treatments, and designing new biomaterials for valve tissue engineering.     

Synthesis of hyaluronan haloacetates and biology of novel cross-linker-free synthetic extracellular matrix hydrogels

Hyaluronan (HA) derivatives containing thiol-reactive electrophilic esters were prepared to react with thiol-modified macromolecules to give cross-linker-free hydrogels. Specifically, HA was converted to two haloacetate derivatives, HA bromoacetate (HABA) and HA iodoacetate (HAIA). In cytotoxicity assays, these reactive macromolecules predictably induced cell death in a dose-dependent manner. Cross-linker-free synthetic extracellular matrix (sECM) hydrogels were prepared by thiol alkylation using HAIA and HABA as polyvalent electrophiles and thiol-modified HA (CMHA-S) with or without thiol-modified gelatin (Gtn-DTPH) as polyvalent nucleophiles. When primary human fibroblasts were seeded on the surface of the sECMs containing only the electrophilic HA haloacetate and nucleophilic CMHA-S components, no significant cytoadherence was observed. Cell attachment and viability was 17% (HABA) to 30% (HAIA) lower on HA haloacetate cross-linked hydrogels than on CMHA-S that had been oxidatively cross-linked via disulfide-bonds. In contrast, sECMs that included Gtn-DTPH allowed fibroblasts to attach, spread, and proliferate. Taken together, the HA haloacetates are attractive candidates for producing cross-linker-free sECM biomaterials that can function either as anti-adhesive barriers or as cytoadhesive sECMs for cell culture in pseudo-3-D.     

Biocompatibility of hydrogel-based scaffolds for tissue engineering applications

Recently, understanding of the extracellular matrix (ECM) has expanded rapidly due to the accessibility of cellular and molecular techniques and the growing potential and value for hydrogels in tissue engineering. The fabrication of hydrogel-based cellular scaffolds for the generation of bioengineered tissues has been based on knowledge of the composition and structure of ECM. Attempts at recreating ECM have used either naturally-derived ECM components or synthetic polymers with structural integrity derived from hydrogels. Due to their increasing use, their biocompatibility has been questioned since the use of these biomaterials needs to be effective and safe. It is not surprising then that the evaluation of biocompatibility of these types of biomaterials for regenerative and tissue engineering applications has been expanded from being primarily investigated in a laboratory setting to being applied in the multi-billion dollar medicinal industry. This review will aid in the improvement of design of non-invasive, smart hydrogels that can be utilized for tissue engineering and other biomedical applications. In this review, the biocompatibility of hydrogels and design criteria for fabricating effective scaffolds are examined. Examples of natural and synthetic hydrogels, their biocompatibility and use in tissue engineering are discussed. The merits and clinical complications of hydrogel scaffold use are also reviewed. The article concludes with a future outlook of the field of biocompatibility within the context of hydrogel-based scaffolds.     

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.     

The evolution of extracellular matrix

We present a perspective on the molecular evolution of the extracellular matrix (ECM) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries. ECM components do not function in isolation, and the biological ECM system or "adhesome" also depends on posttranslational processing enzymes, cell surface receptors, and extracellular proteases. We focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage. The analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa. Integrin, CD36, and certain domains predate the metazoa, and some ECM-related proteins are identified in choanoflagellates as predicted sequences. Modern deuterostomes and vertebrates have many novelties and elaborations of ECM as a result of domain shuffling, domain innovations and gene family expansions. Knowledge of the evolution of metazoan ECM is important for understanding how it is built as a system, its roles in normal tissues and disease processes, and has relevance for tissue engineering, the development of artificial organs, and the goals of synthetic biology.     

Decellularized extracellular matrices derived from cultured cells at stepwise myogenic stages for the regulation of myotube formation

The regulation of stem cell differentiation is key for muscle tissue engineering and regenerative medicine. To this end, various substrates mimicking the native extracellular matrix (ECM) have been developed with consideration of the mechanical, topological, and biochemical properties. However, mimicking the biochemical properties of the native ECM is difficult due to its compositional complexity. To develop substrates that mimic the native ECM and its biochemical properties, decellularization is typically used. Here, substrates mimicking the native ECM at each myogenic stage are prepared as stepwise myogenesis-mimicking matrices via the in vitro myogenic culture of C2C12 myoblasts and decellularization. Cells adhered to the stepwise myogenesis-mimicking matrices at similar levels. However, the matrices derived from cells at the myogenic early stage suppressed cell growth and promoted myogenesis. This promotion of myogenesis was potentially due to the suppression of the activation of endogenous BMP signaling following the suppression of the expression of the myogenic-inhibitory factors, Id2 and Id3. Our stepwise myogenesis-mimicking matrices will be suitable ECM models for basic biological research and myogenesis of stem cells. Further, these matrices will provide insights that improve the efficacy of decellularized ECM for muscle repair.     

Extracellular matrix and matrix receptors in blood-brain barrier formation and stroke

The blood-brain barrier (BBB) is formed primarily to protect the brain microenvironment from the influx of plasma components, which may disturb neuronal functions. The BBB is a functional unit that consists mainly of specialized endothelial cells (ECs) lining the cerebral blood vessels, astrocytes, and pericytes. The BBB is a dynamic structure that is altered in neurologic diseases, such as stroke. ECs and astrocytes secrete extracellular matrix (ECM) proteins to generate and maintain the basement membranes (BMs). ECM receptors, such as integrins and dystroglycan, are also expressed at the brain microvasculature and mediate the connections between cellular and matrix components in physiology and disease. ECM proteins and receptors elicit diverse molecular signals that allow cell adaptation to environmental changes and regulate growth and cell motility. The composition of the ECM is altered upon BBB disruption and directly affects the progression of neurologic disease. The purpose of this review is to discuss the dynamic changes of ECM composition and integrin receptor expression that control BBB functions in physiology and pathology.     

An inverse method for predicting tissue-level mechanics from cellular mechanical input

Extracellular matrix (ECM) provides a dynamic three-dimensional structure which translates mechanical stimuli to cells. This local mechanical stimulation may direct biological function including tissue development. Theories describing the role of mechanical regulators hypothesize the cellular response to variations in the external mechanical forces on the ECM. The exact ECM mechanical stimulation required to generate a specific pattern of localized cellular displacement is still unknown. The cell to tissue inverse problem offers an alternative approach to clarify this relationship. Developed for structural dynamics, the inverse dynamics problem translates measurements of local state variables (at the cell level) into an unknown or desired forcing function (at the tissue or ECM level). This paper describes the use of eigenvalues (resonant frequencies), eigenvectors (mode shapes), and dynamic programming to reduce the mathematical order of a simplified cell–tissue system and estimate the ECM mechanical stimulation required for a specified cellular mechanical environment. Finite element and inverse numerical analyses were performed on a simple two-dimensional model to ascertain the effects of weighting parameters and a reduction of analytical modes leading toward a solution. Simulation results indicate that the reduced number of mechanical modes (from 30 to 14 to 7) can adequately reproduce an unknown force time history on an ECM boundary. A representative comparison between cell to tissue (inverse) and tissue to cell (boundary value) modeling illustrates the multiscale applicability of the inverse model.     

Niche interactions in epidermal stem cells

Within the epidermis and dermis of the skin, cells secrete and are surrounded by the extracellular matrix(ECM), which provides structural and biochemical support. The ECM of the epidermis is the basement membrane, and collagen and other dermal components constitute the ECM of the dermis. There is significant variation in the composition of the ECM of the epidermis and dermis, which can affect "cell to cell" and "cell to ECM" interactions. These interactions, in turn, can influence biological responses, aging, and wound healing; abnormal ECM signaling likely contributes toskin diseases. Thus, strategies for manipulating cellECM interactions are critical for treating wounds and a variety of skin diseases. Many of these strategies focus on epidermal stem cells, which reside in a unique niche in which the ECM is the most important component; interactions between the ECM and epidermal stem cells play a major role in regulating stem cell fate. As they constitute a major portion of the ECM, it is likely that integrins and type Ⅳ collagens are important in stem cell regulation and maintenance. In this review, we highlight recent research-including our previous work-exploring the role that the ECM and its associated components play in shaping the epidermal stem cell niche.     

Microstructured extracellular matrices in tissue engineering and development

Microscale heterogeneity in the extracellular matrix (ECM) provides spatial information that allows tissues to develop and function properly in vivo. This heterogeneity in composition (chemistry) and structure (geometry) creates distinct microenvironments for the cells that comprise a tissue. In response, populations of cells can coordinate their behaviors across micrometer-to-millimeter length scales to function as a unified whole. We believe techniques to mimic the microscale heterogeneity of the ECM in vitro will revolutionize studies that examine how large groups of cells interact. Micropatterned ECMs used for engineering perfused microvascular networks and functional epidermis and for understanding symmetry-breaking events in epithelial morphogenesis illustrate potential applications in tissue engineering and development.     

Gene therapy for tissue regeneration

Tissue repair and regeneration are the normal biological responses of many different tissues in the body to injury. During the healing process, profound changes occur in cell composition and extracellular matrix (ECM) formation. Fibroblasts and equivalent reparative cells migrate to the wounded area and subsequently proliferate. These cells and reparative cells from the surrounding tissue are responsible for the rapid repair which results in tissue regeneration. Growth factors, one of which is transforming growth factor-beta (TGF-beta), stimulate fibroblasts and smooth muscle cells to proliferate and synthesize ECM proteins. This process of early repair provides a rapid way to restore new tissue and mechanical integrity. This early tissue repair process is normally followed by involution, which requires the production and activation of proteases, tissue maturation and remodeling, reorganization and finally regeneration. Alternately, failure to replace the critical components of the ECM, including elastin and basement membrane, results in abnormal regeneration of the epithelial cell layer. Although remodeling should occur during healing, provisional repair may be followed by excessive synthesis and deposition of collagen, which results in irreversible fibrosis and scarring. This excessive fibrosis which occurs in aberrant healing is at least in part mediated by persistent TGF-beta. Because of the central role of collagen in the wound healing process, the pharmacological control of collagen synthesis has been of paramount importance as a possible way to abrogate aberrant healing and prevent irreversible fibrosis. Fibrosis is an abnormal response to tissue injury.