The term tissue engineering was introduced in 1987 during a meeting of the National Science Foundation. It is the application of principles and methods of engineering and life sciences toward the fundamental understanding of structure-function relationships in normal and pathological mammalian tissue, and the development of biological substitutes to restore, maintain, or improve tissue function.1 Tissue engineering can be considered a multidisciplinary technology used to reach a universal goal—to grow and expand tissues in vitro from donor cells (grow-your-own).
The success of tissue engineering relies on mimicking the composition and structure of the original tissue. To achieve this goal, it is necessary to understand the fundamentals of cells and cell-to-cell interactions in vivo. It is also important to understand the behavior of cell lines and cultures in vitro. Much research focuses on the use of tissue engineering in wound care and dermal tissue regeneration.
This review provides an overview of the history and techniques of tissue engineering, current wound healing-related research, available tissue-engineered wound dressings, and future challenges.

History of Tissue Engineering

Providing a history of tissue engineering it is inevitable to return to the basics, starting in 1665, when Hooke2 (1635–1703) discovered small holes in cross-sections, which he called cells and described in his book Micrographia. In 1805, Oken3 stated,“All life is based on individual cells.”
In 1838–1839, Schleiden4 and Schwann5 formulated the so-called “Cell Theory” based on their microscopic findings.This theory summarized their findings as:

• The cell is the unit of structure,physiology, and organization in living things
• The cell retains a dual existence as a distinct entity and a building block in the construction of organisms
• Cells form by free-cell formation, similar to the formation of crystals (spontaneous generation).

In 1858, Virchow6 (1821–1902) described his ideas about cell formation with the now famous words, “Omnis cellula e cellula…” saying that cells arise from pre-existing cells, confuting Schleiden4 and Schwann’s5 idea of spontaneous cell generation. Virchow6 presented his ideas about regeneration stating that tissue regeneration is dependent on cell proliferation. This led research to focus attention toward the more fundamental cellular level. Thiersch7 attempted to grow skin cells into granulating wounds. In doing this, he discovered the important influence of granulation tissue on wound healing in 1874.7
Loeb8 first reported the idea of growing cells outside the human body in 1897. From that point on many researchers experimented with growing cells in vitro. While experimenting with different media, survival was estalished, despite no growth. Harrison9 (1870–1959) was the first to grow frog ectodermal cells in vitro in 1907, thus developing the first neuronal tissue culture line. This was followed in 1912 by Carrel10 who was able to grow pieces of chick embryo in various media, which he initially maintained for 85 days, and subsequently for years. Later, interest arose in growing cells instead of complete tissues. In 1916, Rous and Jones11 discovered that trypsin is capable of degrading matrix proteins, thus separating cells. Trends changed toward expansion of cell types throughout the 1940s and soon after, much research was performed, which led to the ability to grow tissue-specific cell lines in vitro. Enders12 contributed greatly to the use of human embryonic cells in 1952. In 1998, the development of stem cell lines formed the basis of modern tissue engineering.13 Research since 1998 has focused on identifying the differential capacities of embryonic and adult stem cells and how to influence and accompany this differentiating pathway.Aside from this, research focuses on the problems associated with expansion of autologous and allogeneic differentiated cells, as a large number of cells are needed to generate a relatively small volume of tissue. Furthermore, fundamental research is continually being performed to gain insight into the cellular interactions that are of great importance in tissue engineering.

Technique

Ingredients. Four components are essential for tissue engineering: (stem) cells, a matrix or scaffold, a bioreactor, and cytokines. In tissue engineering, a scaffold becomes embedded with living cells and specific regulatory cytokines, and is placed into a bioreactor. Ideally, a suitable biochemical and biomechanical microenvironment is created and cell multiplication fills the scaffold with tissue and allows the cells to grow into the correct shape. When implanted into the body, the seeded scaffold becomes integrated concomitantly supporting and directing cell proliferation. As the cells proliferate the scaffold slowly biodegrades, gradually allowing blood vessels and host cytokines to make contact with the cells. Through this process, the scaffold further biodegrades while the cells proliferate and differentiate into the desired tissue. Finally, the scaffold completely dissolves and the formed tissue starts functioning in its new surrounding.
Stem cells. Because every tissue consists of a variety of cell types, it is desirable to use common progenitor cells that have the embryological ability to differentiate into multiple cell types. Pluripotent cells are more often referred to as stem cells. Stem cells are also able to recreate themselves indefinitely and maintain their own population. Stem cells can be isolated from an embryo, fetus, or an adult.
The use of embryonic stem cells in medicine is controversial. Many religious and ethical groups express concerns with their use.
A benefit of adult stem cells (ASC) use is that it does not require destruction of embryos.
Moreover, in regards to potential skin substitutes, recent research14 has shown that epidermal adult stem cells have a greater plasticity than was initially expected. In an animal model, adult epidermal stem cells were able to differentiate into different functional cell types.14 One additional advantage of ASC (and especially epithelial) use is the ease of collection from organisms.

Extracellular Matrix and Scaffold

A matrix or culture is needed to facilitate cell growth. In-vivo cells are arranged in the extracellular matrix (ECM) compromising a complex structural entity surrounding and supporting cells.The ECM is necessary in order for certain cells to carry out their functions. The ECM has 3 major components: structural proteins (mainly collagen); proteoglycans and hyaluronan; and specialized multiadhesive proteins.15 Every tissue contains itsown type of ECM specialized for its particular function. The amount of specific components varies according to the function of the tissue.As a result of the minimal availability of extracellular space in the epidermis, cells are tightly bound to the basal lamina, which is a thin matrix overlying the loose connective tissue in the dermis.
Proteoglycans are macromolecules consisting of a core protein attached to several polysaccharides (glycosaminoglycans). Proteoglycans are able to bind cells to the matrix and bind growth factors (eg, fibroblast growth factor [FGF], transforming growth factor beta [TGF-β] to glycosaminoglycans, thus preventing extracellular protease degradation of growth factors.
Organization of all ECM components depends on the presence of binding proteins in the ECM. Fibronectins are another class of matrix proteins that can bind to integrins and play an important role in attaching cells to the ECM.
In tissue engineering, much research focuses on developing artificial matrices or scaffolds. Scaffolds can be permanent or temporary depending on the application. Permanent scaffolds are not biodegradable and remain incorporated in the body. These scaffolds are necessary in situations where continuous strength is needed to allow tissue to retain its shape. Today, mostly temporary and biodegradable scaffolds are used ultimately leaving only the engineered tissue in situ.
Research has shown that combining collagen and hyaluronic acids in scaffolds, in addition to using hyaluronic acid matrices, has been successful in tissueengineering of cartilage and dermal tissue regeneration.16,17

Bioreactor

A bioreactor can be defined as any apparatus that attempts to mimic and reproduce physiological conditions in order to maintain and encourage cell culture for tissue regeneration.18 Cell-culture parameters such as temperature, pH, biochemical gradients, and mechanical stresses should be continuously controlled during the maturation period.18
Bioreactors have already improved the processing and the final results of skin regeneration. It is essential that bioreactors are designed and fabricated following specifications that differ from tissue to tissue.19

Cytokines

Cytokines are polypeptides that can bind to cell-specific receptors present in cell membranes of target cells triggering cell-specific responses. These polypeptides usually exist as inactive precursors that need to be cleaved and bound to the ECM to become active. By binding receptors cytokines can exert a signal transduction cascade via second messengers leading to changes in gene expression mediating specific cellular responses.15 Responses of binding a cytokine can include altering the expression of membrane proteins, secretion of effector molecules, and proliferation. Cytokines are not cell type specific—a single cytokine can act on multiple cell types and different cell types can secrete the same cytokine, despite differing responses between different cell types.
Epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and TGF-β are the 4 most important growth factors in wound healing. EGF stimulates growth and migration of keratinocytes in vitro and epidermal regeneration in vivo.20 Epidermal growth factor has already been applied to delivery systems leading to the enhancement of epidermal regeneration in partial-thickness wounds and second- degree burns. Once applied to a hydrogel, significantly faster wound healing was observed.21
Fibroblast growth factor (FGF) acts on fibroblasts. Of the 20 FGF family members,FGF-β is especially known as a potent stimulator of endothelial cells that induces angiogenesis. Platelet-derived growth factor (PDGF) is also important in wound healing as it attracts various cells to the lesion, including fibroblasts, smooth muscle cells, neutrophils, and macrophages.22 It also stimulates secretion of growth factors by macrophages and matrix products, such as fibronectin, hyaluronan, collagens, and proteoglycans.23 PDGF plays an important role during the remodeling phase by stimulating the fibroblast production of collagenases. Recombinant PDGF is therapeutically applied to chronic diabetic ulcers enhancing wound healing.24
The transforming growth factor-β (TGF-β) super family consists of more than 40 members with a similar structure. Almost all cells in the human body secrete inactive TGF-β. In order to bind its receptor,TGF-β must be activated. Binding to its receptor initiates a cascade mediated by activation of a protein kinase.15 Research has shown that incorporation of TGF-β in a collagen scaffold resulted in a faster epithelialization and contraction rates in full-thickness skin defects in rabbits.20 Research focuses on the development of delivery systems for TGF-β that can gradually release TGF-β.20
TGF-β1 is a pleiotropic growth factor, which has reguvanlatory effects on many different cell types. For instance, it plays an important role in cell proliferation and differentiation,25 bone formation,26,27 angiogenesis,28,29 neuroprotection,30 and wound repair.31,32 However, long exposure to high doses of TGF-β1 results in fibroses and hypertrophic scars.22
Literature regarding cytokine manipulation of proliferative scars has shown that TGF-β2 may be involved in the development of tissue fibrosis.33
In wound healing, TGF-β3 has been demonstrated to attenuate type 1 collagen synthesis and reduce scar tissue formation.34–36 Despite previous meritorious efforts to investigate the therapeutic potential of TGF-β3, its effective use is limited by a number of common shortcomings, such as a short half-life, in-vivo instability, and relatively inaccuracy of the delivery system.37

Wound Healing and Tissue Engineering

History. When considering wound healing historically, a trend can be seen throughout the centuries representing a shift from dry wound healing to wet wound healing.38 The oldest medical texts describing ancient medicine are written on papyrus that date from 1500 to 1000 BC and are based on much older initial documents dating from approximately 3000 BC.39 These documents indicate that in 3000 BC hemorrhages were cauterized to stop the bleeding.40 Around 2000 BC bandages began being used for approximating the wound edges. Later, around 1000 BC, Homer described 147 wounds in the Iliad.41
Throughout history different materials have been used for bandaging wounds. The indication of bandages shifted as Celsus in 25 AD described signs and symptoms of inflammation. Since then, bandages were used for prevention of the described symptoms and signs. In the Middle Ages, Paracelsus wrote of dressing wounds made with cotton and gauze.41 In the 19th century, Pasteur favored the use of dry dressings to help prevent infection.42
In 1962,Winter43 demonstrated that partial-thickness wounds reepithelialized more rapidly under occlusive dressings because occlusive dressings maintained a moist wound surface.This landmark study showed that a moist environment accelerated the re-epithelialization process.43 Numerous studies followed that demonstrated wound occlusion and moisture increased all phases of healing. Occlusive dressings are designed to keep the wound bed moist with exudate. The basic concept behind moist wound healing is that the presence of exudate in a wound will provide an environment that stimulates healing by delivering a range of cells and cytokines necessary for wound repair. With these findings, use of dry dressings became less popular, and lead to the development of occlusive dressings such as, hydrocolloids, hydrogels, semipermeable films, alginates, and foams.
Since 1992, research indicates that wet wound healing might also accelerate the healing process when compared to dry wound healing.44 Later, in 1995 it was shown that wet wound healing showed less subepidermal inflammatory cells when compared to moist wound healing represented by a hydrocolloid dressing.45 Wet wound healing uses a plastic transparent chamber filled with a medium that is attached to the wound.The chamber makes sure that the wound is constantly in contact with the medium and allows visible sight of the wound bed.38 Fluid chambers function as a quasi mini-incubator that make it possible to inject antibiotics and analgesics to the medium. With developments in tissue engineering emphasizing the importance of adding growth factors, wet wound healing is suitable for allowing cytokines and cells to be added to the medium.

Tissue-engineered Wound Healing Products

 

Tissue-engineered wound healing products can be classified as acellular or cellular (Table 1).46 Acellular products contain, as the name implies, no cells and consist of a matrix that functions by binding to the host, allowing matrix-cell interactions. Due to its porous nature, the matrix allows host cells to infiltrate.Today, a matrix can contain virus vectors or plasmids, which can transcribe and translate the in-built DNA leading to secretion of specific growth factors.47–50 These growth factors carry out their specific functions by stimulating host cells to enhance wound healing. In specific situations it is even possible to manufacture matrices containing plasmids that can release hormones. The matrix contains ECM-proteins such as collagen, hyaluronic acid, and fibronectin, thus ensuring biocompatibility. Examples of these products are: E-Matrix (Encelle Inc, Greenville, NC), OASIS® (Healthpoint Ltd, Fort Worth,Tex), Integra® (Integra LifeSciences, Plainsboro, NJ), Permacol® (Tissue Science Laboratories Inc,Andover, Md), Matriderm® (Dr. Suwelack Skin & Health Care AG, Germany), and EZDerm® (Brennen Medical, St. Paul, Minn).
Restoring function after hand burns plays a major role in the restitution of quality of life.The grafted areas are of utmost importance for good hand function.51 Haslik et al51 evaluated the collagen-elastin matrix Matriderm as a possible alternative for 2-stage substitutes after hand burns.This matrix was evaluated in several trials by van Zuijlen et al52 and showed the sufficient survival of autografts on top of the dermal substitutes in a 1-step procedure. Haslik et al51 concluded that Matriderm is a promising dermal substitute for the treatment of severe hand burns, with an overall take rate of 97%.
Cellular products on the other hand do contain living cells, often fibroblasts and keratinocytes embedded in a collagen or polyglactin scaffold forming an epidermal layer skin substitute.46 Autologous cells are used in these products to minimize the risk of rejection. Autologous keratinocytes are derived from progenitor cells from dermal sheets in the outer root surrounding hair follicles53 or from epithelial cells obtained via a biopsy of the recipient’s skin. A permanent autologous epidermal skin graft is applied, which functions as a reliable barrier and promotes the formation of granulation tissue. Examples of tissue-engineered epidermal substitutes are: Epicel® (Genzyme Biosurgery, Cambridge, Mass), Laserskin® (Fidia Advanced Biopolymers, Abano Terme, Italy), Myskin (Celltran Ltd, Sheffield, UK), and EpiDex (Modex Therapeutics, Switzerland).
Laserskin autograft is an epidermal substitute. There are orderly arrays of laser-perforated microholes for the ingrowth and proliferation of keratinocyte.54 As keratinocytes are directly cultivated on Laserskin, the graft can easily be peeled off from the skin. Lam et al55 showed in an animal experiment that composite Laserskin grafts are good human skin substitutes in terms of durability, biocompatibility, high seeding efficacy for keratinocytes, high graft take rate, and low infection rate.
Aside from epidermal substitutes, tissue-engineered dermal substitutes have also been developed. The dermis is composed of loose connective tissue containing collagen and fibrils, anchoring the dermis to the epidermis.56 The upper layer of the dermis, the papillary layer, is a cell-rich layer containing fibroblasts and macrophages allowing dermis-epidermis interactions. These interactions trigger synthesis of ECM components and stimulation of differentiation and growth of keratinocytes. Inclusion of living fibroblasts leads to active release of cytokines. However, until now, only allogeneic tissue-engineered dermal wound products have been developed, and unfortunately always carry the risk of (chronic) graft rejection. Examples of these products include: Dermagraft® (Advanced BioHealing, La Jolla, Calif), Alloderm® (LifeCell Inc, Branchburg, NJ), TransCyte® (Advanced BioHealing, La Jolla, Calif), and ICX-SKN® (Intercytex, Cambridge, UK).
Dermagraft is a cryopreserved human fibroblastderived dermal substitute. Fibroblasts incorporated with Dermagraft secreted VEGF, PDGF, insulin-like growth factor I (IGF-I),colony stimulating factors (CSF), interleukins (IL), tumor necrosis factor (TNF), and TGF-β.46
Alloderm is a dermal collagen matrix derived from banked human skin that is specially treated to remove most cellular components. Alloderm has been successvanfully used in the resurfacing of full-thickness burn wounds in combination with an ultra-thin autograft that replaces the epidermis.57
TransCyte is a laboratory-grown extracellular matrix of allogeneic human dermal fibroblasts. Noordenbos et al58 reported on the safety and efficacy of TransCyte for treatment of partial-thickness burns. Wounds treated with TransCyte healed faster, with no infection and less hypertrophic scarring,compared to wounds treated with silver sulfadiazine.
More recently, ICX-SKN, an autosynthesized human collagen-based extracellular matrix with human dermal fibroblasts vascularized during healing of acute surgical wounds and showed integration and persistence during the healing process.59
Currently, (allogeneic) bilayered products are available— eg, Apligraf® (Organogenesis, Canton, Mass) and OrCel® (OrCel International, New York, NY). Apligraf, a living bilayered skin substitute, is capable of regenerating tissue in response to an injury.This is apparent because of the good interaction between Apligraf and the wound, thus making it suitable for wide application.46
OrCel is a bilayered cellular matrix in which normal human allogeneic skin cells (epidermal keratinocytes and dermal fibroblasts) are cultured in 2 separate layers into a type I bovine collagen sponge. Like Apligraf, OrCel is a bilayer dressing resembling normal skin. OrCel was developed as a tissue-engineered biological dressing. OrCel delivers ECM components and growth factors and creates an environment conducive to wound healing—it was never intended as an artificial skin for grafting.

Conclusion

When considering the number of available wound healing products, achievements made within the tissueengineering field become clear. A wide range of products are commercially available today.60 Progress has been made in the tissue engineering field resulting in a variety of skin substitute products, yet no autologous bilayered skin substitutes are currently available—ideally to further minimize the risk of host versus graft reactions.
Research still focuses on stem cells.Although much is known about these unique cells, much remains unclear. In this respect, research is needed to reveal more sources of harvestable ASC in the human body, as the differentiation potential of ASC appears highly underestimated.61 In the future, ASC will be the main cell source in tissue engineering. Further research is also needed to elucidate the mechanisms of differentiation and how to manipulate and control these cells.Through learning more about the fundamentals of stem cells, they might some day be used as cellular therapy for local tissue repair.
Additional research on scaffolds is needed because, as engineering of different tissues continues to grow, so will the need for tissue-specific scaffolds. In the future, scaffolds will become more sophisticated, supplying cells in all their needs.
Another field of research deals with the development of biomaterials for scaffolds incorporating intrinsic activity mediated by cytokines or immobilized peptides. In the future, more fundamental research must be carried out to learn more about the interactions of cytokines on a cellular and molecular level. More research is required to prevent adverse host reactions on the immunological level. Many of the identified growth factors have not even been characterized, thus restraining the development of defined culture media.
Future developments will gain more insight into the complexity of interactions of cytokines and cells on the cellular level.Within the next decade,the complex mechanism underlying the differentiation of stem cells will be clearly understood and eventually this process will be able to be controlled in vitro, as well as in vivo. This, together with the ongoing developments of tissue-engineering, tissue-specific scaffolds, and bioreactors, will lead to a thorough fundamental basis from which further developments on a macroscopic level can be achieved. Eventually, skin, cartilage, and bone will be substituted to every imaginable defect. Cellular therapies might become available by substituting genetically engineered cells that are bound to specific carriers to carry out their function at a specific target organ. Far into the future it might become possible to substitute whole organs that can carry out the same complex functions as natural organs.