92e: Tissue Engineering

Tissue engineering is a field that applies principles of regenerative medicine to restore the function of various organs by combining cells with biomaterials. It is multidisciplinary, often combining the skills of physicians, cell biologists, bioengineers, and material scientists, to recapitulate the native three-dimensional architecture of an organ, the appropriate cell types, and the supportive nutrients and growth factors that allow normal cell growth, differentiation, and function. Tissue engineering is a relatively new field, originating in the late 1970s. Early studies focused on efforts to create skin substitutes using biomaterials and epithelial skin cells with a goal of providing barrier protection for patients with burns. The early strategies employed a tissue biopsy, followed by ex vivo expansion of cells seeded on scaffolds. The cell–scaffold composite was later implanted back into the same patient, where the new tissue would mature. However, there were many hurdles to overcome. The three major challenges in the field of tissue engineering involved: (1) the ability to grow and expand normal primary human cells in large quantities; (2) the identification of appropriate biomaterials; and (3) the requirement for adequate vascularization and innervation of the engineered constructs.

The original model for tissue engineering focused largely on the isolation of tissue from the organ of interest, the growth and expansion of the tissue-specific cells, and the seeding of these cells onto three-dimensional scaffolds. Just a few decades ago, most primary cultures of human cells could not be grown and expanded in large quantities, representing a major impediment to the engineering of human tissues. However, the identification of specific tissue progenitor cells in the 1990s allowed expansion of multiple cell types, and progress has occurred steadily since then. Some cell types are more amenable to expansion than others, reflecting in part their native regenerative capacity but also varying requirements for nutrients, growth factors, and cell–cell contacts. As an example of progress, after years of effort, protocols for the growth and expansion of human cardiomyocytes are now available. However, there are still many tissue-specific cell types that cannot be expanded from tissue sources, including the pancreas, liver, and nerves. The discovery of pluripotent or highly multipotent stem cells (Chap. 88) may ultimately allow most human cell types to be used for tissue engineering. The stem cell characteristics depend on their origin and their degree of plasticity, with cells from the earliest developmental stages, such as embryonic stem cells, having the greatest plasticity. Induced pluripotent stem cells have the advantage that they can be derived from individual patients, allowing autologous transplants. They can also be differentiated, in vitro, along cell-specific lineages, although these protocols are still at an early stage of development. Human embryonic and induced pluripotent stem cells have a very high replicative potential, but they also have the potential for rejection and tumor formation (e.g., teratomas). The more recently described amniotic fluid and placental stem cells have a high replicative potential but without an apparent propensity for tumor formation. Moreover, they have the potential to be used in an autologous manner without rejection. Adult stem cells, such as those derived from bone marrow, also have less propensity for tumor formation and, if used in an autologous manner, will not be rejected, but their replicative potential is limited, especially for endoderm and ectoderm cells.

Stem cells can be derived from autologous or heterologous sources. Heterologous cells can be used when only temporary coverage is needed, such as replacing skin after a burn or wound. However, if a more permanent construct is required, autologous cells are preferred to avoid rejection. There are also practical issues related to tissue sources. For example, if a patient presents with end-stage heart disease, obtaining a cardiac tissue biopsy for cell expansion is unlikely to be feasible, and bone marrow–derived mesenchymal cells may provide an alternative.

The biomaterials used to create the scaffolds for tissue engineering require specific properties to enhance the long-term success of the implanted constructs. Ideally, the biomaterials should be biocompatible; elicit minimal inflammatory responses; have appropriate biomechanical properties; and promote cell attachment, viability, proliferation, and differentiated function. Ideally, the scaffolds should replicate the biomechanical and structural properties of the tissue being replaced. In addition, biodegradation should be controlled such that the scaffold retains its structural integrity until the cells deposit their own matrix. If the scaffolds degrade too quickly, the constructs may collapse. If the scaffolds degrade too slowly, fibrotic tissue may form. Also, the degradation of the scaffolds should not alter the local environment unfavorably, because this can impair the function of cells or newly formed tissue.

The first scaffolds designed for tissue regeneration were naturally derived materials, such as collagen. The first artificially derived material for tissue engineering used a biodegradable scaffold made of polyglycolic acid. Naturally derived scaffolds have properties very similar to the native matrix, but there is an inherent batch-to-batch variability, whereas the production of artificially derived biomaterials can be better controlled, allowing for more uniform results. More recently, combination scaffolds, made of both naturally and artificially derived biomaterials, have been used for tissue engineering.

An emerging area is the use of peptide nanostructures to facilitate tissue engineering. Some of these are self-assembling peptide amphiphiles that allow scaffolds to form in vivo, for example at sites of spinal cord injury where they have been used experimentally to prevent scar formation and facilitate nerve and blood vessel regeneration. Peptide nanostructures can be combined with other biomaterials, and they can be linked to growth factors, antibodies, and various signaling molecules that can modulate cell behavior during organ regeneration.

Implanted tissue-engineered constructs require adequate vascularity and innervation. Judah Folkman, a pioneer in the field of angiogenesis, made the observation that cells could survive in volumes up to 3 mm³ via nutrient diffusion alone, but larger cell volumes required vascularization for survival. Adequate vascularity was also essential for normal innervation to occur. This was a major challenge in the field of tissue engineering, which largely depended on the patient’s native angiogenesis and innervation. Even if sufficient cell quantities are available, there is a theoretical limit on the types of tissue constructs that could be created. In response to this challenge, material scientists designed scaffolds with much greater porosity and architecture. Scaffold designs included the creation of thin, porous sponges comprised of 95% air, markedly increasing the surface area for the resident cells. These properties promoted increased vascularity and innervation. The addition of growth factors, such as vascular endothelial growth factor and nerve growth factor, has been used to enhance angiogenesis and innervation.

All human tissues are complex. However, from an architectural aspect, tissues can be categorized under four levels. Flat tissue structures, such as skin, are the least complex (level 1), comprised predominantly of a single epithelial cell type. Tubular structures, such as blood vessels and the trachea, are more complex architecturally (level 2) and must be constructed to ensure that the structure does not collapse over time. These tissues typically have two major cell types. They are designed to act as a conduit for air or fluid at a steady state within a defined physiologic range. Hollow nontubular organs, such as the stomach, bladder, or uterus, are more complex architecturally (level 3). The cells are functionally more complex, and these cell types often have a functional interdependence. By far, the most complex are the solid organs (level 4), because the amount of cells per cm² are exponentially greater than any of the other tissue types.

For the first three tissue levels (1–3), when the constructs are initially implanted, the cell layering on the scaffolds is thin, not unlike that seen in tissue culture matrices. The cell layering continues to mature, in concert with the recipient’s native angiogenesis and neo-innervation. For level 4 (solid) organs, the vascularity requirements are substantial, and native tissue angiogenesis is not sufficient. The engineering strategies for tissues vary according to their complexity level.

The basic principles of tissue engineering involve the use of the relevant cell populations, where the cell biology is well understood and the cells can be reproducibly retrieved and expanded, and the use of optimized biomaterials and scaffold designs. Cell seeding can be performed using various techniques, including static or flow-based systems that use bioreactors.

Most techniques for the engineering of tissues fall under one of five strategies (Fig. 92e-1):

1. Scaffolds can be used alone, without cells, and implanted, where they depend on native cell migration onto the scaffold from the adjacent tissue for regeneration. The first use of decellularized scaffolds for tissue regeneration was for urethral reconstruction. These techniques are most optimal when the size of the defect is relatively small, usually <0.5 cm from each tissue edge. Larger defects tend to heal by scarring, due to the deposition of fibroblasts, and eventual fibrosis. Scaffolds alone have also been used for other applications, including for wound coverage, soft tissue coverage after joint surgery, urogynecologic applications for sling surgery, and as materials for hernia repair.

2. A more recent strategy in tissue engineering involves the use of proteins, cytokines, genes, or small molecules that induce in situ tissue regeneration, either alone or with the use of scaffolds. For example, gene transcription factors used in the mouse pancreas led to tissue regeneration. Surgically implanted decellularized heart valve scaffolds, coated with proteins that attract vascular stem cells, led to the creation of in situ cell-seeded functional heart valves in sheep. Drugs that induce muscle regeneration are being tested clinically. Small molecules that induce tissue regeneration are currently under investigation for multiple applications, including growth of skin and hair and for musculoskeletal applications.

3. The most common strategy for the engineering of tissues uses scaffolds seeded with cells. The most direct and established type of tissue engineering uses flat scaffolds, either artificial or naturally derived, that are seeded with cells and used for the replacement or repair of flat tissue structures. The flat scaffolds can also be sized and molded at the time of surgical implantation, or they can be shaped prior to cell seeding, for example, for tubular organs such as blood vessels or nontubular hollow tissues such as bladders. Bioreactors are often used to expose the cell–scaffold construct to mechanical forces, such as, stress, strain, and pulsatile flow that aid in the normal development of the cells into tissues (Video 92e-1, engineered heart valve in a pulsatile bioreactor showing the valves opening and closing). This strategy is the most common method used for tissue regeneration to date, and tissues and organs, such as skin, blood vessels, urethras, tracheas, vaginas, and bladders, have been engineered and implanted in patients using these techniques.

4. The fourth strategy in tissue engineering is applicable for solid organs, where discarded organs are exposed to mild detergents and are decellularized, leaving behind a three-dimensional scaffold that preserves its vascular tree. The scaffold can then be reseeded with the patient’s own expanded vascular and tissue-specific cells. This strategy was used initially to create solid phallic structures in rabbits that were functional and able to produce offspring. Similar strategies were also used to recellularize miniature heart, liver, and kidney structures, with limited functionality to date, but with an established proof of concept (Video 92e-2, a dye is injected through the portal artery of a decellularized liver showing an intact vascular tree). These techniques are currently under investigation and have not been used clinically to date.

5. The fifth strategy for tissue engineering involves the use of bioprinting. These technologies arose through the use of modified desktop inkjet printers over a decade ago. The inkjet cartridges were filled with a cell–hydrogel combination instead of ink. A rudimentary three-dimensional elevator was lowered each time the cartridge deposited the cells and hydrogel, thus building miniature solid structures, such as two-chambered heart organoids, one layer at a time. More sophisticated bioprinters have now been built that have additional computer-aided design (CAD) and three-dimensional printing technologies. The information to print the organ can be personalized using the patient’s own imaging studies that help to define the size and shape of the particular tissue (Video 92e-3, a modified inkjet printer shows the three-dimensional construction of a two-chambered heart and how the structure beats with the cardiomyocytes in synchrony). Bioprinting is a tool that allows a scale-up option for the production of engineered tissues. Its use is still experimental and has not been applied clinically to date.

A number of engineered tissues, including architecturally flat, tubular, and hollow nontubular organs, have been implanted in patients dating back to the 1990s. These include bladders, blood vessels, urethras, vaginal organs, tracheas, and skin for permanent replacement (Table 92e-1). Various types of skin substitutes, which were used as temporary “living wound dressings” to cover burn areas until skin grafts could be obtained from the same patient, were implanted starting in the 1990s. However, the use of engineered skin as a permanent replacement occurred only recently. Many engineered tissues are still being used in patients under regulatory guidelines for clinical trials. To date, solid organs have not yet been engineered for clinical use.

Tissue engineering is a rapidly evolving field where new technologies are continuously being applied to achieve success. The field still has many challenges ahead, including the long regulatory timelines required for the approval of widespread use, the need for improved scale-up production technologies, and the cost of the technologies, which include multiple processes involving biologics. Nonetheless, the list of tissues and organs being implanted in patients keeps growing, and the ability of these technologies to improve health has been demonstrated. More patients should be able to benefit from these technologies in the coming years.