Preparation of 3D fibrin scaffolds for stem cell culture applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue. A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach.     

Musculoskeletal tissue engineering by endogenous stem/progenitor cells

From its inception, tissue engineering has had three tenets: cells, biomaterial scaffolds and signaling molecules. Among the triad, cells are the center piece, because cells are the building blocks of tissues. For decades, cell therapies have focused on the procurement, manipulation and delivery of healthy cells for the treatment of diseases or trauma. Given the complexity and potential high cost of cell delivery, there is recent and surging interest to orchestrate endogenous cells for tissue regeneration. Biomaterial scaffolds are vital for many but not all, tissue-engineering applications and serve to accommodate or promote multiple cellular functions. Signaling molecules can be produced by transplanted cells or endogenous cells, or delivered specifically to regulate cell functions. This review highlights recent work in tissue engineering and cell therapies, with a focus on harnessing the capacity of endogenous cells as an alternative or adjunctive approach for tissue regeneration.     

Cell culture in autologous fibrin scaffolds for applications in tissue engineering

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth.     

Fibrin as a scaffold for cardiac tissue engineering

Fibrin is a natural biopolymer with many interesting properties, such as biocompatibility, bioresorbability, ease of processing, ability to be tailored to modify the conditions of polymerization, and potential for incorporation of both cells and cell mediators. Moreover, the fibrin network has a nanometric fibrous structure, mimicking extracellular matrix, and it can also be used in autologous applications. Therefore, fibrin has found many applications in tissue engineering, combined with cells, growth factors, or drugs. Because a major limitation of cardiac cell therapy is low cell engraftment, the use of biodegradable scaffolds for specific homing and in situ cell retention is desirable. Thus, fibrin-based injectable cardiac tissue engineering may enhance cell therapy efficacy. Fibrin-based biomaterials can also be used for engineering heart valves or cardiac patches. The aim of this review is to show cardiac bioengineering uses of fibrin, both as a cell delivery vehicle and as an implantable biomaterial.     

Purpose-driven biomaterials research in liver-tissue engineering

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.     

Adipose-derived adult stem cells for cartilage tissue engineering

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

Microscale methods to assemble mammalian cells into tissue-like structures

Different cell types make up tissues and organs hierarchically and communicate within a complex, three-dimensional (3D) environment. The in vitro recapitulation of tissue-like structures is meaningful, not only for fundamental cell biology research, but also for tissue engineering (TE). Currently, TE research adopts either the top-down or bottom-up approach. The top-down approach involves defining the macroscopic tissue features using biomaterial scaffolds and seeding cells into these scaffolds. Conversely, the bottom-up approach aims at crafting small tissue building blocks with precision-engineered structural and functional microscale features, using physical and/or chemical approaches. The bottom-up strategy takes advantage of the repeating structural and functional units that facilitate cell-cell interactions and cultures multiple cells together as a functional unit of tissue. In this review, we focus on currently available microscale methods that can control mammalian cells to assemble into 3D tissue-like structures.     

Composite scaffolds for cartilage tissue engineering

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

Adipose-derived stem cells: isolation, expansion and differentiation

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in adipose-derived stem cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.     

A multiscale computational fluid dynamics approach to simulate the micro-fluidic environment within a tissue engineering scaffold with highly irregular pore geometry

Mechanical stimulation can regulate cellular behavior, e.g., differentiation, proliferation, matrix production and mineralization. To apply fluid-induced wall shear stress (WSS) on cells, perfusion bioreactors have been commonly used in tissue engineering experiments. The WSS on cells depends on the nature of the micro-fluidic environment within scaffolds under medium perfusion. Simulating the fluidic environment within scaffolds will be important for gaining a better insight into the actual mechanical stimulation on cells in a tissue engineering experiment. However, biomaterial scaffolds used in tissue engineering experiments typically have highly irregular pore geometries. This complexity in scaffold geometry implies high computational costs for simulating the precise fluidic environment within the scaffolds. In this study, we propose a low-computational cost and feasible technique for quantifying the micro-fluidic environment within the scaffolds, which have highly irregular pore geometries. This technique is based on a multiscale computational fluid dynamics approach. It is demonstrated that this approach can capture the WSS distribution in most regions within the scaffold. Importantly, the central process unit time needed to run the model is considerably low.

     

Self-assembly of elastin-based peptides into the ECM: the importance of integrins and the elastin binding protein in elastic fiber assembly

The formation of a suitable extracellular matrix (ECM) that promotes cell adhesion, organization, and proliferation is essential within biomaterial scaffolds for tissue engineering applications. In this work, short elastin mimetic peptide sequences, EM-19 and EM-23, were engineered to mimic the active motifs of human elastin in hopes that they can stimulate ECM development in synthetic polymer scaffolds. Each peptide was incubated with human aortic smooth muscle cells (SMCs) and elastin and desmosine production were quantified after 48 h. EM-19 inhibited elastin production through competitive binding phenomena with the elastin binding protein (EBP), whereas EM-23, which contains an RGDS domain, induces recovery of elastin production at higher concentrations, alluding to a higher binding affinity for the integrins than for the EBP and the involvement of integrins in elastin production. Colocalization of each peptide with the elastin matrix was confirmed using immunofluorescent techniques. Our data suggest that with appropriate cell-binding motifs, we can simulate the cross-linking of tropoelastin into the developing elastin matrix using short peptide sequences. The potential for increased cell adhesion and the incorporation of elastin chains into tissue engineering scaffolds make these peptides attractive bioactive moieties that can easily be incorporated into synthetic biomaterials to induce ECM formation.     

Aging: A cell source limiting factor in tissue engineering

Tissue engineering has yet to reach its ideal goal, i.e. creating profitable off-the-shelf tissues and organs, designing scaffolds and three-dimensional tissue architectures that can maintain the blood supply, proper biomaterial selection, and identifying the most efficient cell source for use in cell therapy and tissue engineering. These are still the major challenges in this field. Regarding the identification of the most appropriate cell source, aging as a factor that affects both somatic and stem cells and limits their function and applications is a preventable and, at least to some extents, a reversible phenomenon. Here, we reviewed different stem cell types, namely embryonic stem cells, adult stem cells, induced pluripotent stem cells, and genetically modified stem cells, as well as their sources, i.e. autologous, allogeneic, and xenogeneic sources. Afterward, we approached aging by discussing the functional decline of aged stem cells and different intrinsic and extrinsic factors that are involved in stem cell aging including replicative senescence and Hayflick limit, autophagy, epigenetic changes, miRNAs, mTOR and AMPK pathways, and the role of mitochondria in stem cell senescence. Finally, various interventions for rejuvenation and geroprotection of stem cells are discussed. These interventions can be applied in cell therapy and tissue engineering methods to conquer aging as a limiting factor, both in original cell source and in the in vitro proliferated cells.     

A novel method for biomaterial scaffold internal architecture design to match bone elastic properties with desired porosity

An often-proposed tissue engineering design hypothesis is that the scaffold should provide a biomimetic mechanical environment for initial function and appropriate remodeling of regenerating tissue while concurrently providing sufficient porosity for cell migration and cell/gene delivery. To provide a systematic study of this hypothesis, the ability to precisely design and manufacture biomaterial scaffolds is needed. Traditional methods for scaffold design and fabrication cannot provide the control over scaffold architecture design to achieve specified properties within fixed limits on porosity. The purpose of this paper was to develop a general design optimization scheme for 3D internal scaffold architecture to match desired elastic properties and porosity simultaneously, by introducing the homogenization-based topology optimization algorithm (also known as general layout optimization). With an initial target for bone tissue engineering, we demonstrate that the method can produce highly porous structures that match human trabecular bone anisotropic stiffness using accepted biomaterials. In addition, we show that anisotropic bone stiffness may be matched with scaffolds of widely different porosity. Finally, we also demonstrate that prototypes of the designed structures can be fabricated using solid free-form fabrication (SFF) techniques.     

Liver tissue engineering: Recent advances in the development of a bio-artificial liver

Orthotopic liver transplantation is the most common treatment for patients with end-stage liver failure. However, liver transplantation is greatly limited by a donor shortage. Liver tissue engineering may offer a promising strategy to solve this problem by providing transplantable, bioartificial livers. Diverse types of cells, biomaterials, and growth factor delivery systems have been tested for efficient regeneration of liver tissues that possess hepatic functions comparable to native livers. This article reviews recent advances in liver tissue engineering and describes cell sources, biomaterial scaffolds, and growth factor delivery systems that are currently being used to improve the regenerative potential of tissue-engineered livers.     

In vivo tissue engineering of musculoskeletal tissues

Tissue engineering of musculoskeletal tissues often involves the in vitro manipulation and culture of progenitor cells, growth factors and biomaterial scaffolds. Though in vitro tissue engineering has greatly increased our understanding of cellular behavior and cell-material interactions, this methodology is often unable to recreate tissue with the hierarchical organization and vascularization found within native tissues. Accordingly, investigators have focused on alternative in vivo tissue engineering strategies, whereby the traditional triad (cells, growth factors, scaffolds) or a combination thereof are directly implanted at the damaged tissue site or within ectopic sites capable of supporting neo-tissue formation. In vivo tissue engineering may offer a preferential route for regeneration of musculoskeletal and other tissues with distinct advantages over in vitro methods based on the specific location of endogenous cultivation, recruitment of autologous cells, and patient-specific regenerated tissues.     

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.     

生物支架材料——组织工程连载之二

具有三维结构的支架材料是组织工程的核心内容之一。现有组织工程支架可分为天然生物材料、合成有机材料和无机材料三类。支架材料近年来研究十分活跃,不仅在组织工程的最早产品人工皮肤领域进行了更为完善的研究和开发,同时在诸如人工骨、软骨、神经、血管、皮肤、肝、脾、肾、膀胱等方面进行了大量研究和探索。与普通组织工程支架需要预先制备并在体外成型不同,近年来在骨和软骨组织工程实践中兴起的可注射支架具有许多优势,是未来组织工程支架发展的重要方向之一。     

CHITIN — A promising biomaterial for tissue engineering and stem cell technologies

Chitin, after cellulose, is the second most abundant natural polymer. With a 200-year history of scientific research, chitin is beginning to see fruitful application in the fields of stem cell and tissue engineering. To date, however, research in chitin as a biomaterial appears to lag far behind that of its close relative, chitosan, due to the perceived difficulty in processing chitin. This review presents methods to improve the processability of chitin, and goes on further to discuss the unique physicochemical and biological characteristics of chitin that favor it as a biomaterial for regenerative medicine applications. Examples of the latter are presented, with special attention on the qualities of chitin that make it inherently suitable as scaffolds and matrices for tissue engineering, stem cell propagation and differentiation.