When a body tissue or an organ is severely injured or largely lost, it is clinically treated with either reconstruction surgery or organ transplantation. One of the major problems for organ transplantation is the shortage of donor tissues or organs. In addition, permanent immunosuppressive medication often causes various side-effects . One promising approach to tackle these problems is to promote the self-healing potential of patients themselves for induction of body tissues and organ regeneration. The technology and methodology of biomedical sciences with the potential to induce tissue regeneration are called ‘Tissue engineering’.
Tissue engineering (TE) is a relatively new, interdisciplinary, and multidisciplinary field that has witnessed intense development in recent years. The term ‘Tissue engineering’ had been defined at a National Science Foundation workshop as ‘the application of the principles and methods of engineering and life sciences toward the fundamental understanding of structure–function relationships in normal and pathological mammalian tissues and the development of biological substitutes that restore, maintain, or improve tissue function’ .
One of the main motivations for TE research is the chronic shortage of organ donors and other limitations related to organ and tissue transplantation. The idea that tissues, and ultimately organs, can be ‘engineered’ for use in patients requiring transplantation is revolutionary and stimulating. TE and regenerative medicine offer solutions to a number of compelling clinical problems that have not been adequately addressed through the use of permanent replacement devices. The basic concept of TE was originally introduced by Langer and Vacanti . They were the first to promote the concept that cells may be seeded on artificial matrices to support the formation of functional tissue-like structures that may be used to repair diseased organs or to screen for wanted and unwanted effects of drugs. Several technologies and methodologies of TE to induce the regeneration of various tissues have been reported thus far to demonstrate their scientific and clinical feasibility [3,4].
However, TE is a discipline still at its infancy, an intricate puzzle that is far from complete .
TE is a rapidly growing new interdisciplinary field of applied research that probably represents a prototype of future developments in science. Primarily, such separate fields of science as engineering and life sciences have combined to overcome traditional barriers in medical research. TE crosses numerous medical and technical specialties. Cell biologists, molecular biologists, biomaterial engineers, microscopic imaging specialists, robotics engineers, computer-assisted designers, and developers of equipment such as bioreactors, in which tissues are grown and nurtured, and finally legal advisors and marketing research specialists (product development and medical implementation) are all involved in the practical process of TE .
General strategies to replace tissue loss
The primary objective of all approaches in TE is the functional or structural restoration of tissue through the delivery of living elements that become integrated into the patient. Most of the techniques of guided tissue restoration developed during the last two decades have mainly only been cell or matrix based. However, investigators in the rapidly emerging field of TE currently use a combination of both to promote new tissue formation .
In other words, TE combines engineering and biosciences together with clinical application, mainly in the various surgical specialties, to develop living substitutes for tissues and organs. This may allow the induction of true regeneration of tissue defects, and may well help improve the treatment of many innate and acquired chronic diseases and tissue defects by replacing or supporting the function of defective or injured body parts.
The key to its progress is an understanding between basic scientists, biochemical engineers, clinicians, and industry. To this end, the main successful approaches so far have been progresses in the understanding of cell–cell interactions, the selection of appropriate matrices (cell–matrix interaction), and chemical signaling (growth factors). Therefore, the classical approach for engineering tissue has been the seeding of more or less biodegradable scaffolds with donor cells and/or growth factors, then culturing and implanting the scaffolds to induce and direct the growth of new, healthy tissue.
In particular, the potential to use engineered tissues as organ replacements in patients has intrigued scientists and clinicians alike. Independent of the scientific and intellectual challenge, there is a clear medical necessity to develop alternatives to current organ transplantation, given the mismatch between patients waiting for organ transplants and the availability of donor organs worldwide . Thus far, clinically successful applications include skin, cartilage, and bone replacement.
Intensive experimental research for reconstructive purposes has addressed peripheral nerves, adipose tissue, muscle, urinary tubular structures, connective tissue (tendons, abdominal wall) cartilage, bone, osteocartilagenous constructs (joints), heart valves, blood vessels, breast, small intestine, esophagus, pancreas, liver, and tracheal constructs [9,10].
Although TE investigations have yielded promising results, it is important to point out that no ‘tissue engineering’ procedure, or any other treatment, has as yet been successful in fully regenerating a tissue that does not have the capability to regenerate spontaneously (Fig. 1) . Engineering of fully functional and transplantable organs such as the heart, liver, kidneys, or pancreas has not yet been achieved and is complicated by their structural and functional complexities. Consequently, most tissue engineers have focused on developing organ units with a defined function rather than complete organs . Ultimately, these units may be used to restore and enhance a defined organ function in vivo. Examples for this concept are the development of hepatocyte spheroids, which are metabolically active and may function as detoxifying units , or β-cell aggregates, which may eventually produce insulin in patients with diabetes .
Tissue engineering versus regenerative medicine
The term ‘tissue engineering’ was initially introduced to describe the technology for producing tissue in vitro . More recently, the term ‘regenerative medicine’ has been used to describe the development of technology and surgical procedures for the regeneration of tissue in vivo. There are advantages and disadvantages to both strategies. One advantage of the synthesis of tissue in vitro is the ability to examine the tissue as it forms and perform certain nondestructive measurements to establish its functions before implantation. However, a disadvantage is the absence of a physiological mechanical environment during tissue formation in vitro. Another disadvantage is the necessary incorporation of the tissues after implantation, which requires remodeling–degradation and new tissue formation–at the interfaces of the implant with the host tissues. However, in most cases, no distinction is made between TE and regenerative medicine, with both being referred to as TE .
Currently, TE has three cornerstones that are the three components of any tissue: a cell population capable of proliferation and differentiation into mature cells; a scaffold that can host these cells, provide a suitable environment for cellular functioning, and serve as a sustained-release delivery vehicle of growth factors; and finally, signaling molecules and growth factors that stimulate the cellular response and the production of an extracellular matrix (ECM) (Fig. 2) [11,14].
Tissue engineering components
Primary organ-specific cells have been widely used for the generation of bioartificial tissues. Despite many advances, efficient expansion of these cells remains a challenge today.
Scientists have investigated almost every tissue type in the human body in terms of TE. For clinical applications, at present, cells have been derived from the patients themselves, from family members or close relatives, or other individuals. However, although this may be the most ideal source of cells, availability and accessibility may often be quite difficult. One approach to overcome the cell-source difficulty could be isolation of human stem cells. Stem cells, by definition, are cells able to self-generate, with a single cell able to differentiate into multiple, functional cell types . Certain adult stem cell types are pluripotent, meaning that they can differentiate into cells derived from three germ layers [16–18]. This ability to differentiate into multiple cell types is called plasticity or transdifferentiation. Hematopoietic stem cells are stem cells that give rise to all the blood cells including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells) and lymphoid lineages (T-cells, B-cells, NK-cells). Hematopoietic stem cells may differentiate into another three major cell types: brain cells (neurons, oligodendrocytes, and astrocytes); skeletal and cardiac muscle cells; and liver cells. Bone marrow stromal cells (mesenchymal stem cells), in addition to differentiating into skeletal muscle cells, may also differentiate into osteoblasts, chondrocytes, adipocytes, and other myocytes such as cardiac muscle cells, etc . The plasticity provides the basic possibility for multiple-TE using a certain type of stem cells. The induction of differentiation is a highly programmed lineage-specific process at the molecular level, and several studies have provided considerable insights into the microenvironment affecting the differentiation of multipotential human mesenchymal stem cells, including hormones, cytokines, and growth factors [e.g., transforming growth factor-β, insulin, steroids, bonemorphogenetic proteins (BMPs)], as well as other factors like cell density, scaffold materials, etc .
The stem cell field is advancing rapidly, providing new options for therapy. Stem cells also fulfill a second requirement: they can functionally reconstitute a given tissue in vivo. Human stem cells can be induced to proliferate through multiple generations and made to differentiate into the appropriate cell type. These stem cells identified can be signaled to turn into different morphological cell types by altering the culturing conditions of the cells before implantation . Although there are a number of technical hurdles such as the need for pure stem-cell preparations, methods to reduce adhesion of cells during cell culture, and processes to generate large number of cells to create tissue, work on stem cells involved in the generation of muscle, bone, and cartilage has demonstrated promising results in TE applications .
In-vitro culture systems
Tissue engineering using open systems of cell transplantation
The primary goal of the open system of cell transplantation is to engineer new tissues by placing the transplanted cells in direct contact with the host with the aim of providing a permanent natural solution to the replacement of lost tissue. Cells for transplantation using this technique are attached to matrices consisting of natural materials or synthetic polymers and are then implanted into the host. This cell–polymer construct then incorporates itself into the recipient's own tissue. In cell culture experiments, it has been observed that dissociated mature cells tend to reform their original structures when provided with the appropriate environmental cues. This has been demonstrated by the formation of tubular structures by capillary endothelial cells in culture, as well as the formation of milk-secreting acini by mammary epithelial cells in vitro . Isolated cells also have the capability to reform their structure, but only up to a limited degree when placed as a suspension into the host. This is mainly because of the absence of an intrinsic tissue ground substance framework. Implantation of tissue in larger volumes is also severely restricted because diffusion limitations restrict interaction with the host environment for nutrition, gas exchange, and waste elimination. On the basis of these observations, approaches to engineer tissue by attaching isolated cells to porous polymeric templates have been developed (Fig. 3).
Static cell cultures
Early work in the field of TE was based on the use of standard static cell-culture conditions for the in-vitro fabrication of tissues before implantation. However, there have been certain limitations to the exchange of nutrients and gases. These limitations are being overcome by advanced application of mechanical engineering expertise in the field of biotechnology by designing and producing dynamic cell culture systems, better known as bioreactors.
Bioreactors are dynamic cell culture systems that allow more control to generate larger volumes of cells when compared with conventional static-culture techniques (Fig. 4). The flow of tissue culture medium and their mixing within bioreactors can be controlled to enhance the mass transfer of nutrients, gases, and metabolites to regulate the size and structure of the tissue being generated.
A scaffold can play many roles in the tissue regeneration process: (a) it can serve as a framework to support cell migration into the defect from surrounding tissues, especially important when a fibrin clot is absent. (b) Before it is absorbed, a scaffold can serve as a matrix for endogenous or exogenous cell adhesion and can facilitate/regulate certain cell processes including mitosis, synthesis, and migration. This may be mediated by ligands for cell receptors (integrins), on the biomaterial and/or the biomaterial, which may selectively adsorb cell adhesion proteins. (c) The scaffold may serve as a delivery vehicle for exogenous cells, growth factors, and genes. This activity is enabled by a large surface area for attachment and the possible control of the density of the agents (i.e., agents/unit volume). (d) The scaffold may structurally reinforce the defect to maintain the shape of the defect and prevent distortion of surrounding tissue. (e) The scaffold can serve as a barrier to prevent the infiltration of surrounding tissue that may impede the process of regeneration .
In general, to promote cellular functions, scaffolds used for TE should have the following characteristics: biocompatibility, biodegradability, reproducibility, high porosity with interconnected pores, and no potential of serious immunological or foreign body reactions. In addition, it is also highly desirable that the scaffold has the ability to promote ECM secretion and to carry biomolecular signals [22,23]. Owing to their functional properties and design flexibility, polymers are the primary choice of materials for making scaffolds. Polymers used for making scaffolds are classified as either naturally derived polymers or synthetic polymers . The former includes collagen, gelatin, chitosan, chitin, cellulose, and starch. The latter includes frequently used biodegradable synthetic polymers such as poly(lactic acid), poly(glycolic acid), poly(lactic-coglycolic acid), poly(ε-caprolactone), and poly(lactic-cocaprolactone). These are all approved by the US Food and Drug Administration for certain biomedical applications.
Solid scaffolds originated from attempts by chemical engineers to create porous materials from biodegradable polymers as a temporal template-like instructive support for cell attachment and tissue neomorphogenesis [25,26]. Thus, the solid biodegradable scaffold-based approach in TE has its roots in biomaterial science and represents an adaptation of degradable polymers used in medical devices for the purpose of engineering living tissues . There are several basic assumptions behind solid biodegradable scaffold-based TE approaches: (a) cell growth is substrate attachment dependent; cells need a solid substrate for attachment and proliferation; (b) tissue constructs must have an organospecific shape; a solid scaffold is essential to maintain the desired shape; a tissue construct cannot maintain its shape without a solid rigid scaffold; (c) the scaffold serves not only as an attachment substrate but also as a source of inductive and instructive signals for cell differentiation, migration, proliferation, and orientation; (d) the porous structure of a solid scaffold will allow optimal cell seeding, tissue construct viability, and vascularization; and (e) the mechanical properties initially provided by the rigid solid scaffold after its biodegradation will be maintained by controlled neomorphogenesis of parenchymal and stromal tissue synthesized in vitro or in vivo in the tissue construct [2,25,26].
Indeed, it has been proven that cells can attach to the scaffold; the solid scaffold can maintain a tissue construct shape; functionalized scaffolds can serve as a tool for instructive morphogenetic signaling and directed cell differentiation; and finally, cells inside porous scaffolds can maintain viability and eventually neoformed tissue can replace the biodegradable scaffold [5,26,28]. However, despite the proven feasibility of solid scaffolds, this classic approach still has some limitations and challenges: (a) vascularization of thick tissue constructs is an unsolved issue, (b) precise placing of different multiple cell types inside three-dimensional porous scaffolds is technologically challenging, (c) achieving organospecific level of cell density in tissue constructs remains a huge challenge, and (d) recent reports on the effect of matrix rigidity on stem cell differentiation can undermine the value of solid rigid biodegradable scaffolds at least for certain tissue applications [29–31].
Another issue is biodegradability, in which the scaffold is ultimately destroyed by the influx of inflammatory cells associated with the degradation process or if the scaffold material stimulates the immune system as it degrades and releases antigenic material . Moreover, in-vitro cell seeding on a solid biodegradable scaffold with sequential, relatively slow, complete scaffold biodegradation, and tissue neomorphogenesis leads to laborious, expensive, time-consuming, and commercially unsuccessful TE technology. It is also not immediately obvious how a chemically modified and functionalized two-dimensional surface of a solid scaffold designed to biodegrade can maintain its functional activity in a three-dimensional tissue construct in vivo . Recent reviews on the problem of vascularization in tissue-engineered constructs based on the use of biodegradable solid scaffolds also indicate that after two decades of intensive efforts, an effective solution to this problem has not yet been found [32–34].
From previous studies, it is known that application of growth factors directly on wounds accelerates wound healing in vivo. Recombinant human BMPs (rhBMPs), BMP-2, and BMP-7 are among the most promising growth factors to improve bone healing and spinal fusion; platelet-derived growth factor and basic fibroblast growth factor are essential for wound healing and tissue generation, including angiogenesis . There are two ways to incorporate growth factors in TE: direct blending or coating on the scaffold or addition to the cell culture medium during the development of the cell/scaffold construct. The first approach could be considered as a drug delivery system, with the scaffold as a carrier for drugs. The second approach is extremely important for stem cell TE, because several growth factors are indispensable for stem cell proliferation and differentiation .
Despite the promising results of in-vitro application of growth factors, in-vivo clinical application has not been as successful. This is because of the short half-lives of these growth factors, which necessitates a sustained delivery system that ensures the supply of therapeutic doses of these growth factors over prolonged periods to negate the need for their repeated local administration for maintenance of effective levels .
Present status of tissue engineering
Seeding of cells on three-dimensional biodegradable polymers in a cell culture or a flow bioreactor provided the opportunity to study tissue development, tissue regeneration, and tissue repair in vitro. Investigators have attempted to study the properties of almost all mammalian cell types under in-vitro conditions. Implantation of the cell polymer constructs allows further study in vivo. Knowledge gained from the different disciplines in active TE research has promoted the evolution of this new field from primary animal research to clinical trials. The present status of different structural and functional tissue has been described below:
Liver and intestinal bioengineering
Liver transplantation is currently the only cure for patients with end-stage liver disease. A variety of strategies in liver engineering have used three-dimensional structures for culturing adult primary or fetal hepatocytes. A three-dimensional environment upregulates the expression of leukotriene metabolism, cholesterol metabolism, and glutathione synthesis genes . Furthermore, it enhances hepatocytic P450 and albumin synthesis . The microenvironment in vitro enables tissue restructuring capable of yielding functional cells desirable for therapeutic applications and at the same time preserves a pool of stem cells as a source for self-renewal. Patients with acute and chronic liver failure not eligible or waiting for organ donations have been treated with hybrid devices using hepatocytes and artificial constructs in clinical trials . These bioartificial liver devices have been reported to lower the risk for encephalopathy, reduce blood ammonium levels, improve hemodynamic status, and reduce mortality [39,40]. The cells engineered for the multiple trials were primary adult hepatocytes, porcine hepatocytes, or the HC3A cell line, all cultured in three-dimensional bioreactors. Stephenne et al.  reported hepatocyte transplantation in a child with urea cycle disorder poorly equilibrated by conventional therapy. After the first seven hepatocyte infusions, the mean blood ammonia level decreased significantly.
Massive resection of the intestine leads to compensatory hyperplasia. Some patients do not fully recover absorptive functions and must be given parenteral nutrition . The feasibility of the intestinal engineering begins with the remarkable regenerative ability of the intestinal epithelium. Experiments with intestinal TE to this date have focused primarily on rodents, with successful results. Neonatal rat intestinal organoid units (partially digested pieces of the intestine) were harvested by enzymatic digestion and density gradient cell separation . Organoid units were seeded in a tubular biocompatible scaffold and implanted into the omentum of adult rats that had 85% intestinal resection, mimicking short-bowel syndrome. The organoid units survived, and became vascularized and anastomosed with the host's intestine . The implants formed rudimentary cyst-like structures and villi. The rodents experienced less weight loss and improved surgical recovery compared with nonimplanted groups. The current approach in rodents requires intestinal organoids procured from neonatal animals, as those derived from adult have not yielded encouraging results. In the clinical setting, it would be very difficult to obtain human neonatal samples for intestinal organoids. Furthermore, the multicellular complexity of the intestinal unit, from clinical-scale dimensions to enteric pacemaker regeneration, complicates the human situation. The intestine exhibits autonomous peristaltic movements, controlled by the enteric nervous system. The intestinal grafts used in rodents lack the proper intestinal motility to prevent obstruction. Continuous progress to overcome these obstacles may make this treatment a clinically viable alternative in the future.
Bone and cartilage confer shape, mechanical support, and protection to the body. In addition, bones contribute to the mineral homeostasis of the body and participate in endocrine regulation of energy metabolism. Bone structures form by two processes: intramembraneous and endochondral ossification . Intramembraneous ossification involves condensed mesenchymal tissue differentiating into osteoblasts, whereas endochondral ossification requires the formation of cartilaginous anlage to then undergo calcification and invasion by blood vessels, thereby resulting in new bone . Throughout an organism's lifespan, bones undergo constant remodeling for stress adaptation. In clinical situations in which congenital malformations or extensive injury occur, tissue grafting is necessary. The first human trial using cell transplantation technologies to repair bone defects was published in 2001 in the New England Journal of Medicine by Quarto et al. . The researchers transplanted osteoprogenitor cells isolated from the bone marrow and grown in hydroxyapatite scaffolds into three patients, resulting in recovered limb function. Since then, several more studies have focused on osteogenic engineering, reconstruction and bone augmentation. However, the long-term benefits are not yet clear and future research must focus on mechanical and stability characterization for clinical implementation . Chondrocytes cannot migrate to the site of injury because of the dense ECM surrounding them and the absence of vasculature . Therefore, repair of articular defects caused by degenerative joint diseases or traumatic injuries represents an open clinical challenge. Several nutrients have been used to induce chondrogenesis in vitro: hyaluronan , agarose , polylactic acid , and collagen type I-III membranes [52–55]. However, no approach has led to the generation of long-term cartilage replacement tissue in vivo. Preclinical and clinical results have been mixed. It has been reported that cells can be lost from the site of injury by leakage, insufficient integration, or induced dedifferentiation of chondrogenic cells by inflammatory stress [56–58]. A prospective, randomized study identified fibrin glue as superior to abrasion measurements when compared with collagen or hyaluronan . This effort has not been followed with sufficient clinical studies. Ongoing research to address these hurdles is needed to provide valid engineering alternatives to the current clinical management of cartilage injuries or defects.
Renal and cardiac grafting
The transplantation of precursor kidney structures (metanephros), isolated from human embryonic tissue, into immunodeficient mice has resulted in the partial restoration of kidney structure and function (Fig. 5) .
As the transplants cannot use the host's excretory system, fluid was collected through microcatheterization. Urea nitrogen and creatinine levels were detected, thereby demonstrating a functional excretory unit. It has been reported that fetal kidney cells grown in a three-dimensional fibrin gel scaffold were able to reconstitute glomeruli and tubular excretory structures . However, the kidneys excrete other substances such as glucose, amino acids, and xenobiotics. They also regulate electrolyte concentration, acid–base balance, blood volume, and vascular pressure. In addition, the kidneys exhibit endocrine functions by producing erythropoietin, renin, prostaglandins, and vitamin D3. Therefore, recapitulation of these complex functions must be achieved in engineered renal grafts before clinical implementation can be considered. In addition, the kidneys have endocrine functions. Bioartificial kidneys are currently in phase I/II clinical trials for patients with end-stage renal failure. Bioartificial kidneys use a hemofilter combined with renal tubular cells to serve reabsorptive and secretory as well as metabolic and endocrinologic functions . In 2004, a phase I/II clinical trial concluded that the device was well tolerated. . No significant change was observed in the pH of the ultrafiltrate or in vitamin D3 levels, but there were reductions in the levels of granulocyte colony-stimulating factor, interleukin-6, and interleukin-10. In 2006, the trial was discontinued because of the unproven efficacy of the device . Thereafter, modifications to the composition of bioartificial devices with renal epithelial cells and immunomodulatory membranes yielded encouraging results in phase I trials . Altogether, these studies suggest that there are significant challenges with cell-containing devices. Maintenance of the derived cells in a viable and functional state is yet to be achieved.
The first bioartificial heart was reported by the group of Doris Taylor in 2008 at the University of Minnesota . A perfused heart was enzymatically digested to decellularize cadaveric hearts. A construct composed of collagens type I-III and fibronectin remained within the thinned, decellularized heart matrix, preserving the orientation and cardiac chamberization. Rat neonatal cardiomyocytes were cultured in a bioreactor to recellularize the heart. After 8 days, the new organ exhibited preload and afterload pump properties, electrocardiographic recordings, and physiologic pressure volume loops. Tissue patches with murine embryonic stem cells have been developed and used in mouse studies of ischemic cardiomyopathy . Engraftment of the patches at the site of fibrosis resulted in an improved ejection fraction and reduction of scarred tissue. However, embryonic stem cell-derived cardiomyocytes exhibited immature sarcoplasmic reticulum function and calcium handling. Other studies have used different cell types to improve the mechanics of cardioengineering or deliver stem cells embedded in stents with positive results, but current cardiac tissue grafts have significant limitations: incompatible biomechanical properties, atypical anisotropic and fibrous structural properties, and poor cell engrafting within the constructs . To date, cardiac TE is still in the preclinical stage for human studies.
Future of tissue engineering and regenerative medicine
Just as several technologies facilitated the development of TE as a viable discipline, new technologies will aid its progress and development. One of these emerging technologies is the isolation and expansion of stem cells and the identification of the signals required for their differentiation into specific cell types. Another related technology is the genetic modification of cells in vitro or in vivo. These technologies will address the difficulties that are often encountered in obtaining a sufficient amount of tissue for the isolation of autologous cells.
New matrix materials will likely be developed with selected chemical compositions that allow them to better serve as insoluble regulators of cell function. Finally, methods will likely be introduced to control the mechanical environment of the cells in vitro to better regulate their biosynthetic behavior. Collectively, these approaches will enable the synthesis of tissue in vitro that better replicates the native material and will be valuable in preparing implants to facilitate tissue regeneration in vivo.
The proliferation of cells in monolayer culture and their subsequent growth in three-dimensional scaffolds for TE continue to provide unique opportunities to observe selected cell behavior. TE science will thus provide critical new knowledge that will deepen our understanding of the phenotype of many cell types and this will likely facilitate meaningful advances in TE and regenerative medicine.
The vision to create whole organs in the laboratory is intriguing. If successful, it may provide hope to patients with so far incurable disabling diseases.
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