Design concepts and strategies for tissue engineering scaffolds

In the emerging field of tissue engineering and regenerative medicine, new viable and functional tissue is fabricated from living cells cultured on an artificial matrix in a simulated biological environment. It is evident that the specific requirements for the three main components, cells, scaffold materials, and the culture environment, are very different, depending on the type of cells and the organ-specific application. Identifying the variables within each of these components is a complex and challenging assignment, but there do exist general requirements for designing and fabricating tissue engineering scaffolds. Therefore, this review explores one of the three main components, namely, the key concepts, important parameters, and required characteristics related to the development and evaluation of tissue engineering scaffolds. An array of different design strategies will be discussed, which include mimicking the extra cellular matrix, responding to the need for mass transport, predicting the structural architecture, ensuring adequate initial mechanical integrity, modifying the surface chemistry and topography to provide cell signaling, and anticipating the material selection so as to predict the required rate of bioresorption. In addition, this review considers the major challenge of achieving adequate vascularization in tissue engineering constructs, without which no three-dimensional thick tissue such as the heart, liver, and kidney can remain viable.     

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。     

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.     

Cells for tissue engineering

Recent advances in stem-cell technology have improved the prognosis for tissue engineering. The use of cultured stem and/or progenitor cells has the potential to improve the extent of regeneration, and also increases the likelihood that the transplanted tissue will integrate with the surrounding tissue. It could eventually even reduce or eliminate the need for immunosuppressive drugs.     

组织工程产品的种类及应用——组织工程连载之六

组织工程产品包括人造皮肤、血管、软骨、骨、角膜、心脏瓣膜、气管、肌腱、韧带、神经、肌肉、骨髓、生殖道、尿道、肠、乳房、肝脏、肾脏、胰脏、心脏、膀胱、手等,但绝大部分处于实验室研究探索阶段,正在进行临床实验或批准应用还不多。已经获得批准的主要是皮肤产品、软骨及骨产品、心血管产品、神经系统产品、人工器官等,其临床应用较多。今后将会有越来越多的组织工程产品面世,其临床应用也会越来越广泛。     

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.     

Microscale culture of human liver cells for drug development

Tissue function depends on hierarchical structures extending from single cells ( approximately 10 microm) to functional subunits (100 microm-1 mm) that coordinate organ functions. Conventional cell culture disperses tissues into single cells while neglecting higher-order processes. The application of semiconductor-driven microtechnology in the biomedical arena now allows fabrication of microscale tissue subunits that may be functionally improved and have the advantages of miniaturization. Here we present a miniaturized, multiwell culture system for human liver cells with optimized microscale architecture that maintains phenotypic functions for several weeks. The need for such models is underscored by the high rate of pre-launch and post-market attrition of pharmaceuticals due to liver toxicity. We demonstrate utility through assessment of gene expression profiles, phase I/II metabolism, canalicular transport, secretion of liver-specific products and susceptibility to hepatotoxins. The combination of microtechnology and tissue engineering may enable development of integrated tissue models in the so-called 'human on a chip'.     

Tendon tissue engineering: progress, challenges, and translation to the clinic

The tissue engineering field has made great strides in understanding how different aspects of tissue engineered constructs (TECs) and the culture process affect final tendon repair. However, there remain significant challenges in developing strategies that will lead to a clinically effective and commercially successful product. In an effort to increase repair quality, a better understanding of normal development, and how it differs from adult tendon healing, may provide strategies to improve tissue engineering. As tendon tissue engineering continues to improve, the field needs to employ more clinically relevant models of tendon injury such as degenerative tendons. We need to translate successes to larger animal models to begin exploring the clinical implications of our treatments. By advancing the models used to validate our TECs, we can help convince our toughest customer, the surgeon, that our products will be clinically efficacious. As we address these challenges in musculoskeletal tissue engineering, the field still needs to address the commercialization of products developed in the laboratory. TEC commercialization faces numerous challenges because each injury and patient is unique. This review aims to provide tissue engineers with a summary of important issues related to engineering tendon repairs and potential strategies for producing clinically successful products.     

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.     

Design and development of tissue engineered lung: Progress and challenges

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.Key words: adult and embryonic stem cells, tissue engineered lung, lung stem cells, lung matrices, lung development     

Hepatic tissue engineering: from transplantation to customized cell-based liver directed therapies from the laboratory

Today, liver transplantation is still the only curative treatment for liver failure due to end-stages liver diseases. Donor organ shortage, high cost and the need of immunosuppressive medications are still the major limitations in the field of liver transplantation. Thus, alternative innovative cell-based liver directed therapies, e.g. liver tissue engineering, are under investigation with the aim, that in future an artificial liver tissue could be created and be used for the replacement of the liver function in patients. Using cells instead of organs in this setting should permit (i) expansion of cells in an in vitro phase, (ii) genetic or immunological manipulation of cells for transplantation, (iii) tissue typing and cryopreservation in a cell bank, and (iv) the ex vivo genetic modification of patient's own cells prior re-implantation. Function and differentiation of liver cells are influenced by the three-dimensional organ architecture. The use of polymeric matrices permits the three dimensional formation of a neo-tissue and specific stimulation by adequate modification of the matrix-surface which might be essential for appropriate differentiation of transplanted cells. Additionally, culturing hepatocytes on three dimensional matrices permits culture in a flow bioreactor system with increased function and survival of the cultured cells. Based on bioreactor technology, bioartificial liver devices (BAL) are developed for extracorporeal liver support. Although BALs improved clinical and metabolic conditions, increased patient survival rates have not been proven yet. For intra-corporeal liver replacement, a concept which combines Tissue Engineering using three-dimensional, highly porous matrices with cell transplantation could be useful. In such a concept, whole liver mass transplantation, long term engraftment and function as well as correction of a metabolic defect in animal models could be achieved with a principally reversible procedure. Future studies have to investigate, which environmental conditions and transplantation system would be most suitable for the development of artificial functional liver tissue including blood supply for a potential use in a clinical setting.     

胚胎肝干细胞的研究进展

近年来的研究表明在胚胎肝脏中存在大量的肝干细胞,它们在肝脏的发育中起着重要作用,并且受到各种时序性表达基因的调控。几个研究组采用不同的方法,分别从小鼠、大鼠和灵长类动物的胚胎肝脏分离并鉴定了具有双潜能的肝干细胞。就胚胎肝脏的发育、调控机制以及胚胎肝干细胞的分离、鉴定等方面的研究进展作一综述,并对胚胎肝干细胞的应用前景和今后的研究方向作了展望。     

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.     

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.     

Chitosan and its derivatives for tissue engineering applications

Tissue engineering is an important therapeutic strategy for present and future medicine. Recently, functional biomaterial researches have been directed towards the development of improved scaffolds for regenerative medicine. Chitosan is a natural polymer from renewable resources, obtained from shell of shellfish, and the wastes of the seafood industry. It has novel properties such as biocompatibility, biodegradability, antibacterial, and wound-healing activity. Furthermore, recent studies suggested that chitosan and its derivatives are promising candidates as a supporting material for tissue engineering applications owing to their porous structure, gel forming properties, ease of chemical modification, high affinity to in vivo macromolecules, and so on. In this review, we focus on the various types of chitosan derivatives and their use in various tissue engineering applications namely, skin, bone, cartilage, liver, nerve and blood vessel.     

Rapid genetic screening for hemochromatosis using automated SSCP-based capillary electrophoresis (SSCP-CE).

Hereditary hemochromatosis (HHC) represents an autosomal recessive disease in which increased iron absorption causes iron overload and irreversible tissue damage. The recently detected association between two point mutations in the HFE gene on chromosome 6p and HHC has made it possible to screen for the disease before the onset of irreversible tissue damage. Conventional genetic testing is based on restriction fragment-length polymorphisms (RFLP) using two endonuclease recognition sites in codon 63 or 282, respectively. In this study, we have adapted single-strand conformation polymorphism analysis for capillary electrophoresis (SSCP-CE) to detect homozygote or heterozygote point mutations. Two HFE gene fragments spanning codons 63 and 282 were amplified by a duplex PCR using genomic DNA from peripheral blood or from tissue sections of paraffin-embedded liver biopsies as template. Thereby, rapid genotyping of both HFE mutations was achieved with a single PCR, omitting the need of further analysis by restriction digest. Eighty-five patients with liver disease and/or suspected iron overload were genotyped using SSCP-CE, and all results were verified by conventional RFLP analysis. In summary, SSCP-CE proved to be a reliable, cost-effective, sensitive and rapid method for genotyping HFE mutations. This method will further facilitate high-throughput genetic screening using capillary array electrophoretic devices.     

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.     

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.     

组织工程的临床研究——组织工程连载之五

组织工程研究涉及的临床科室包括骨科、普外科、五官科、康复医学科、泌尿科、口腔颌面外科、神经外科、整形外科、胸外科、眼科、肝胆外科、血管外科;涉及的组织器官有:神经组织、肝脏组织、角膜组织、膀胱组织、血液、韧带、耳朵、生殖道、手、脂肪、乳房、心脏、肾脏、胰腺、管状组织(用于建造肠管、食管、气管、血管、肾和尿道等)等.其中皮肤组织、软骨组织、骨组织等的研究应用较为成熟.从最初工程化组织或器官的立项研究到最终批准临床应用,这是一个漫长的过程,需要众多不同学科的科研人员共同努力.随着基础研究和临床应用的深入发展,现代组织工程正在成为治疗组织、器官衰竭的有效疗法和辅助手段.     

Gene transfection of multicellular spheroid of hepatocytes on an artificial substrate

The handling of hepatocytes, a major cell population in the liver, is an important technique in both liver tissue engineering and hepatology. However, these cells are so fragile that it has been impossible to harvest hepatocytes with high viability from tissue culture dishes after a period of culture in vitro. In this study, we employed an artificial substrate for transfection of multilayer hepatocytes and harvested these cells with high viability after transfection. Hepatocytes cultured on an amphiphilic artificial substrate form multilayer aggregates (spheroids) in the presence of growth factors during gene transfection with cation liposomes. Compared to cells cultured on a collagen-coated plate, these spheroids are easily harvested with high viability by pipetting in EDTA solution. In addition, these spheroids rapidly spread on collagen after transfer from the artificial substrate, demonstrating that hepatocytes in the center of the spheroids were viable. Epidermal growth factor (EGF) increased the transfection efficiency into hepatocytes while hepatocyte growth factor (HGF) alone did not increase the efficiency. However, HGF synergestically increased the effect of EGF on transfection. Interestingly, this transfection required the process of spheroid formation because the gene was not transfected once the spheroid formation completed or under conditions where hepatocytes did not form spheroids. This method using spheroidal hepatocytes for in vitro transfection is promising for the development of ex vivo gene therapy.