Design and development of tissue engineered lung

Before we can realize our long term goal of engineering lung tissue worthy of clinical applications, advances in the identification and utilization of cell sources, development of standardized procedures for differentiation of cells, production of matrix tailored to meet the needs of the lung and design of methods or techniques of applying the engineered tissues into the injured lung environment will need to occur. Design of better biomaterials with the capacity to guide stem cell behavior and facilitate lung lineage choice as well as seamlessly integrate with living lung tissue will be achieved through advances in the development of decellularized matrices and new understandings related to the influence of extracellular matrix on cell behavior and function. We have strong hopes that recent developments in the engineering of conducting airway from decellularized trachea will lead to similar breakthroughs in the engineering of distal lung components in the future.     

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.     

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.     

Decellularized tissue and cell-derived extracellular matrices as scaffolds for orthopaedic tissue engineering

The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic.     

Tissue engineering: current state and perspectives

Tissue engineering is an interdisciplinary field that involves cell biology, materials science, reactor engineering, and clinical research with the goal of creating new tissues and organs. Significant advances in tissue engineering have been made through improving singular aspects within the overall approach, e.g., materials design, reactor design, or cell source. Increasingly, however, advances are being made by combining several areas to create environments which promote the development of new tissues whose properties more closely match their native counterparts. This approach does not seek to reproduce all the complexities involved in development, but rather seeks to promote an environment which permits the native capacity of cells to integrate, differentiate, and develop new tissues. Progenitors and stem cells will play a critical role in understanding and developing new engineered tissues as part of this approach.     

Engineering stem cell niches in bioreactors

Stem cells, including embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells and amniotic fluid stem cells have the potential to be expanded and differentiated into various cell types in the body. Efficient differentiation of stem cells with the desired tissue-specific function is critical for stem cell-based cell therapy, tissue engineering, drug discovery and disease modeling. Bioreactors provide a great platform to regulate the stem cell microenvironment, known as “niches”, to impact stem cell fate decision. The niche factors include the regulatory factors such as oxygen, extracellular matrix (synthetic and decellularized), paracrine/autocrine signaling and physical forces (i.e., mechanical force, electrical force and flow shear). The use of novel bioreactors with precise control and recapitulation of niche factors through modulating reactor operation parameters can enable efficient stem cell expansion and differentiation. Recently, the development of microfluidic devices and microbioreactors also provides powerful tools to manipulate the stem cell microenvironment by adjusting flow rate and cytokine gradients. In general, bioreactor engineering can be used to better modulate stem cell niches critical for stem cell expansion, differentiation and applications as novel cell-based biomedicines. This paper reviews important factors that can be more precisely controlled in bioreactors and their effects on stem cell engineering.     

Lung tissue engineering: An update

Pulmonary disease is a worldwide public health problem that reduces the life quality and increases the need for hospital admissions as well as the risk of premature death. A common problem is the significant shortage of lungs for transplantation as well as patients must also take immunosuppressive drugs for the rest of their lives to keep the immune system from attacking transplanted organs. Recently, a new strategy has been proposed in the cellular engineering of lung tissue as decellularization approaches. The main components for the lung tissue engineering are: (1) A suitable biological or synthetic three-dimensional (3D) scaffold, (2) source of stem cells or cells, (3) growth factors required to drive cell differentiation and proliferation, and (4) bioreactor, a system that supports a 3D composite biologically active. Although a number of synthetic as well biological 3D scaffold suggested for lung tissue engineering, the current favorite scaffold is decellularized extracellular matrix scaffold. There are a large number of commercial and academic made bioreactors, the favor has been, the one easy to sterilize, physiologically stimuli and support active cell growth as well as clinically translational. The challenges would be to develop a functional lung will depend on the endothelialized microvascular network and alveolar–capillary surface area to exchange gas. A critical review of the each components of lung tissue engineering is presented, following an appraisal of the literature in the last 5 years. This is a multibillion dollar industry and consider unmet clinical need.     

Immobilization of heparin on decellularized kidney scaffold to construct microenvironment for antithrombosis and inducing reendothelialization

In recent years, rapid development of tissue engineering technology provides possibilities for the construction of artificial tissues or organs. In construction of engineered kidneys, researchers used native decellularized extracellular matrix (ECM) as the scaffolds to recellularization. However, thrombosis has been a great issue that hinders the progress of transplantation in vivo. In this study, heparin was immobilized to the collagen part of decellularized scaffold with collagen-binding peptide (CBP). Through the anticoagulant and endothelial cell reperfusion experiments, it can be demonstrated that the heparinized scaffolds absorbed less platelets and red blood cells which can effectively reduce the formation of thrombosis. Moreover, it is conducive to long-term adhesion of endothelial cells which is important for the formation of subsequent vascularization. Taken together, our results reveal that the whole kidney can be modified by CBP-heparin composite to reduce the thrombosis and provide the better conditions for neovascularization.     

Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development

Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.     

Human breast cancer decellularized scaffolds promote epithelial-to-mesenchymal transitions and stemness of breast cancer cells in vitro

Breast cancer, with unsatisfactory survival rates, is the leading cause of cancer-related death in women worldwide. Recent advances in the genetic basis of breast cancer have benefitted the development of gene-based medicines and therapies. Tissue engineering technologies, including tissue decellularizations and reconstructions, are potential therapeutic alternatives for cancer research and tissue regeneration. In our study, human breast cancer biopsies were decellularized by a detergent technique, with sodium lauryl ether sulfate (SLES) solution, for the first time. And the decellularization process was optimized to maximally maintain tissue microarchitectures and extracellular matrix (ECM) components with minimal DNA compounds preserved. Histology analysis and DNA quantification results confirmed the decellularization effect with maximal genetic compounds removal. Quantification, immunofluorescence, and histology analyses demonstrated better preservation of ECM components in 0.5% SLES-treated scaffolds. Scaffolds seeded with MCF-7 cells demonstrated the process of cell recellularization in vitro, with increased cell migration, proliferation, and epithelial-to-mesenchymal transition (EMT) process. When treated with 5-fluorouracil, the expressions of stem cell markers, including Oct4, Sox2, and CD49F, were maximally maintained in the recellularized scaffold with decreased apoptosis rates compared with monolayer cells. These results showed that the decellularized breast scaffold model with SLES treatments would help to simulate the pathogenesis of breast cancer in vitro. And we hope that this model could further accelerate the development of effective therapies for breast cancer and benefit drug screenings.     

Bioconjugation of hydrogels for tissue engineering

Success of tissue engineered constructs in regenerative medicine is limited by the lack of cellmatrix interactions to guide devleopment of the seeded cells into the desired tissue. This review highlights the most exciting developments in bioconjugation of synthetic hydrogels targeted to tissue engineering. Application of conjugation techniques has resulted in the synthesis of novel biomimetic cell-responsive hydrogels to control the cascade of cell migration, adhesion, survival, differentiation, and maturation to the desired lineage concurrent with matrix remodeling. The future outlook includes developing conjugated patterned hydrogel matrices, developing novel hydrogel matrices to support self-renewal and pluripotency of embryonic and adult stem cells, and merging 3D printing with bioconjugation to fabricate hydrogels with anatomical arrangement of cells and biomolecules.     

Scale-dependent mechanical properties of native and decellularized liver tissue

Decellularization, a technique used in liver regenerative medicine, is the removal of all the cellular components from a tissue or organ, leaving behind an intact structure of extracellular matrix. The biomechanical properties of this novel scaffold material are currently unknown and are important due to the mechanosensitivity of liver cells. Characterizing this material is important for bioengineering liver tissue from this decellularized scaffold as well as creating new 3-dimensional mimetic structures of liver extracellular matrix. This study set out to characterize the biomechanical properties of perfused liver tissue in its native and decellularized states on both a macro- and nano-scale. Poroviscoelastic finite element models were then used to extract the fluid and solid mechanical properties from the experimental data. Tissue-level spherical indentation-relaxation tests were performed on 5 native livers and 8 decellularized livers at two indentation rates and at multiple perfusion rates. Cellular-level spherical nanoindentation was performed on 2 native livers and 1 decellularized liver. Tissue-level results found native liver tissue to possess a long-term Young’s modulus of 10.5 kPa and decellularized tissue a modulus of 1.18 kPa. Cellular-level testing found native tissue to have a long-term Young’s modulus of 4.40 kPa and decellularized tissue to have a modulus of 0.91 kPa. These results are important for regenerative medicine and tissue engineering where cellular response is dependent on the mechanical properties of the engineered scaffold.     

Engineering a bilayered hydrogel to control ASC differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM). Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.     

Immune response to stem cells and strategies to induce tolerance

Although recent progress in cardiovascular tissue engineering has generated great expectations for the exploitation of stem cells to restore cardiac form and function, the prospects of a common mass-produced cell resource for clinically viable engineered tissues and organs remain problematic. The refinement of stem cell culture protocols to increase induction of the cardiomyocyte phenotype and the assembly of transplantable vascularized tissue are areas of intense current research, but the problem of immune rejection of heterologous cell type poses perhaps the most significant hurdle to overcome. This article focuses on the potential advantages and problems encountered with various stem cell sources for reconstruction of the damaged or failing myocardium or heart valves and also discusses the need for integrating advances in developmental and stem cell biology, immunology and tissue engineering to achieve the full potential of cardiac tissue engineering. The ultimate goal is to produce 'off-the-shelf' cells and tissues capable of inducing specific immune tolerance.     

The effect of amino density on the attachment, migration, and differentiation of rat neural stem cells In Vitro

Artificial extracellular matrices play important roles in the regulation of stem cell behavior. To generate materials for tissue engineering, active functional groups, such as amino, carboxyl, and hydroxyl, are often introduced to change the properties of the biomaterial surface. In this study, we chemically modified coverslips to create surfaces with different amino densities and investigated the adhesion, migration, and differentiation of neural stem cells (NSCs) under serum-free culture conditions. We observed that a higher amino density significantly promoted NSCs attachment, enhanced neuronal differentiation and promoted excitatory synapse formation in vitro. These results indicate that the amino density significantly affected the biological behavior of NSCs. Thus, the density and impact of functional groups in extracellular matrices should be considered in the research and development of materials for tissue engineering.     

Engineering tissue from human embryonic stem cells

Recent advances in human embryonic stem cell (hESC) biology now offer an alternative cell source for tissue engineers, as these cells are capable of proliferating indefinitely and differentiating to many clinically relevant cell types. Novel culture methods capable of exerting spatial and temporal control over the stem cell microenvironment allow for more efficient expansion of hESCs, and significant advances have been made toward improving our understanding of the biophysical and biochemical cues that direct stem cell fate choices. Effective production of lineage specific progenitors or terminally differentiated cells enables researchers to incorporate hESC derivatives into engineered tissue constructs. Here, we describe current efforts using hESCs as a cell source for tissue engineering applications, highlighting potential advantages of hESCs over current practices as well as challenges which must be overcome.     

Adipose tissue engineering with cells in engineered matrices

Tissue engineering has shown promise for the development of constructs to facilitate large volume soft tissue augmentation in reconstructive and cosmetic plastic surgery. This article reviews the key progress to date in the field of adipose tissue engineering. In order to effectively design a soft tissue substitute, it is critical to understand the native tissue environment and function. As such, the basic physiology of adipose tissue is described and the process of adipogenesis is discussed. In this article, we have focused on tissue engineering using a cell-seeded scaffold approach, where engineered extracellular matrix substitutes are seeded with exogenous cells that may contribute to the regenerative response. The strengths and limitations of each of the possible cell sources for adipose tissue engineering, including adipose-derived stem cells, are detailed. We briefly highlight some of the results from the major studies to date, involving a range of synthetic and naturally derived scaffolds. While these studies have shown that adipose tissue regeneration is possible, more research is required to develop optimized constructs that will facilitate safe, predictable and long-term augmentation in clinical applications.Key words: tissue engineering, regenerative medicine, adipose tissue, adipose-derived stem cells, adipogenesis, cell culture, scaffolds, cell-biomaterial interactions     

Patterned vascularization in a directional ice‐templated scaffold of decellularized matrix

Vascularization is fundamental for large‐scale tissue engineering. Most of the current vascularization strategies including microfluidics and three‐dimensional (3D) printing aim to precisely fabricate microchannels for individual microvessels. However, few studies have examined the remodeling capacity of the microvessels in the engineered constructs, which is important for transplantation in vivo. Here we present a method for patterning microvessels in a directional ice‐templated scaffold of decellularized porcine kidney extracellular matrix. The aligned microchannels made by directional ice templating allowed for fast and efficient cell seeding. The pure decellularized matrix without any fixatives or cross‐linkers maximized the potential of tissue remodeling. Dramatical microvascular remodeling happened in the scaffold in 2 weeks, from small primary microvessel segments to long patterned microvessels. The majority of the microvessels were aligned in parallel and interconnected with each other to form a network. This method is compatible with other engineering techniques, such as microfluidics and 3D printing, and multiple cell types can be co‐cultured to make complex vascularized tissue and organ models.     

Decellularized Wharton's jelly extracellular matrix as a promising scaffold for promoting hepatic differentiation of human induced pluripotent stem cells

Liver tissue engineering as a therapeutic option for restoring of damaged liver function has a special focus on using native decellularized liver matrix, but there are limitations such as the shortage of liver donor. Therefore, an appropriate alternative scaffold is needed to circumvent the donor shortage. This study was designed to evaluate hepatic differentiation of human induced pluripotent stem cells (hiPSCs) in decellularized Wharton's jelly (WJ) matrix as an alternative for native liver matrix. WJ matrices were treated with a series of detergents for decellularization. Then hiPSCs were seeded into decellularized WJ scaffold (DWJS) for hepatic differentiation by a defined induction protocol. The DNA quantitative assay and histological evaluation showed that cellular and nuclear materials were efficiently removed and the composition of extracellular matrix was maintained. In DWJS, hiPSCs-derived hepatocyte-like cells (hiPSCs-Heps) efficiently entered into the differentiation phase (G1) and gradually took a polygonal shape, a typical shape of hepatocytes. The expression of hepatic-associated genes (albumin, TAT, Cytokeratin19, and Cyp7A1), albumin and urea secretion in hiPSCs-Heps cultured into DWJS was significantly higher than those cultured in the culture plates (2D). Altogether, our results suggest that DWJS could provide a proper microenvironment that efficiently promotes hepatic differentiation of hiPSCs.