Review paper: Bioengineering Skin Using Mechanisms of Regeneration and Repair

By Anthony D. Metcalfe and Mark W. J. Ferguson
on Biomaterials doi:10.1016/j.biomaterials.2007.07.031

The paper talks about the development of bioengineered skin up to the present technology, which emphasizes on the potential skin substitutes using the mechanism of regeneration and repair. The major development of bioengineered skin started about three decades ago when scientists found out about culturing keratinocytes from skin in vitro. This finding later developed into autologous skin transplantation, such as skin grafts containing all of the epidermis and part of the dermis from undamaged area of the body to a large injured area. This method, nonetheless, is not practical when the patient has a severe large damaged skin (e.g. a burn injury). Then cadaver skin is sometimes used to cover the wound temporarily although it is limited, expensive, and questionable in safety (e.g. spreading virus and other diseases). Further, both methods are not commercial off-the-shelf products, which then lead to the researches of engineered skin substitutes.

Many companies have developed and commercialized skin replacement therapy. Their products have been improving significantly, which include collagen matrix seeded with dermal fibroblasts and keratinocytes, acellular dermal substitute from bovine collagen and shark chondroitin sulfate, and cell suspension that can be sprayed onto wound. Despite the progressive technology, many companies went bankrupt and currently have financial difficulties. One of the main reasons is the high process automation costs since the companies also need to change the current medical practice to accept the usage of tissue-engineered products. This lack of usage is thought to be related to the drawbacks of skin substitutes, which include the lack of cellular mechanisms in wound healing, the lack of normal skin composition, and scarring at the margins.

The cellular mechanisms in wound healing involve inflammation, cell proliferation and migration, cell recruitment, angiogenesis, and ECM deposition. The process involves several essential cellular components such as: (1) cytokines and growth factors which determines the cell function within the wound e.g. interleukin-1 (IL-1) that transitions inflammation to remodeling phase; (2) fibroblasts that produce ECM for tissue remodeling, scar formation and repair; (3) bone marrow-derived stem cells that produce new skin cells; and (4) fibrocytes (fibroblast-like cells) that induce angiogenesis, recruit lymphocytes, and promote cell migration into the wound. A control over these cellular components is important in skin replacement therapy. An example is the promising cell resource in recreating normal skin composition: hair follicle bulge. Researchers found that the hair follicle bulge contains multipotent skin stem cells that allow a differentiation into sebaceous glands, epidermis, and hair follicles in skin transplantation. Nonetheless, inhibition of the Wnt protein is found to inhibit this folliculogenesis, whereas the overexpression shows the opposite result. Therefore, the Wnt protein may be a key factor to manipulate the wound-induced folliculogenesis.

To resolve scarring issue, fetal wound healing is an ideal model since it engages in regenerative process that does not end in scarring. Two of major differences of fetal wound healing from adult wound healing are: (1) reduced inflammatory response and (2) more molecules involved in repair. Inflammatory response of accumulating fibrocytes uncontrollably is hypothesized to cause scarring. As to engineer skin substitutes for a scar-free healing, the manipulation can be done in depositing ECM molecules as seen in embryonic wound healing.

In addition to resolving the three issues in skin substitutes, the medical interest in skin repair is mostly focusing on repair and regeneration mechanism. The regeneration process can be derived from either undifferentiated stem cells or existing tissues. Researchers have modeled mammalian wound repair and regeneration by punching the ears of three strains of mice (a crude thumb punch and a clinical biopsy punch). The MRL/MpJ mice healed faster than the others with more blastema (undifferentiated cells) formation and thickened tip epithelium. However, the regeneration only occurs when the punch is on the ear because its small dimensions with epithelium covering on both sides allow the diffusion of growth factors in a gradient similar to fetal wound healing. Transforming growth factor-b (TGF-b) is the major growth factor in wound healing. The three isoforms in mammals are expressed differently in embryo and adult wound healing. Embryonic wounds express high TGF-b3 and low TGF-b1 and TGF-b2; where adult wounds express high TGF-b1 and TGF-b2. Further studies found that neutralization of TGF-b1 and TGF-b2 or addition of TGF-b3 results in improved scarring repair or scar-free healing. Other growth factors that could be incorporated into a skin substitute are vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1). VEGF promotes angiogenesis of short, narrow, and highly branched (sprouting) vessels with normal pericyte coverage, where Ang-1 induces broader, longer vessels without increase in branching or sprouting, but with higher pericyte coverage. Combination of VEGF and Ang-1 generates larger, less branched vessels and more developed microvessels, which have a better functionality.

Another big emphasis in skin engineering is placed on the biomatrices design. The artificial ECM has been varied from naturally occurring materials including glucosaminoglycans, fibronectin, collagen, hyaluronan, and alginates, and synthetic materials including PGA, PLA, PLG, PCL, and PET. Ideal biomatrices have the characteristics of (1) non-toxic and non-immunogenic upon implantation, (2) biodegradable by proteases, (3) adherence of cells, and (4) competent in mechanical properties with the ECM in normal skin. The design of biomatrices has developed up to the design of scaffolding that has defined shapes with microporous for directing tissue growth up to when the host cells can reoccupy and resynthesize ECM. The matrix structure can be modified by varying solid content or gelation conditions, or by adding cross-linkers. Specific pore size can also be controlled using the new techniques of 3D printing and electrospinning. Synthetic materials have been shown to have a major disadvantage: the lack of cell-recognition signals. An important signal that can be lost is mechanotransduction signal (controlling cell phenotype) which is transmitted via cell adhesion to the matrix. Adding RGD sequences (Arg-Gly-Asp) is found to promote cell adhesion and migration. Latest development of biomatrices has reached to the synthetic biopolymer technology: hydrogels.

Hydrogels has a homogeneous, nanoscale microstructure that restricts cell migration to proteolytic remodeling. When it is based on polyethylene glycol (PEG) under a specific reaction, the hydrogels can be formed from aqueous precursors in the presence of cells. Therefore, important ligands can be added, for example the RGD-containing peptides for cell adhesion and protease-sensitive oligopeptides for MMP degradation. Moreover, PEG acts as an inert due to its hydrophilicity and resistance to protein adsorption so that it presents biological signals only from the attached peptides. In an experiment using the hydrogel in cell mixing and implantation to regenerate hair follicles, the researchers found that PEG hydrogels are fluid enough for cell reorganization and regeneration. Hydrogels have also been developed on fibronectin functional domain (FNfd’s) coupled to a Hyaluronan (HA) backbone. It is shown to be compatible for growing human dermal fibroblasts. Overall, hydrogels with combinations of proteins such as TGF-b, MMP, and VEGF lead to a more regenerative and anti-scarring wound healing.

Lastly, the area in skin substitutes that needs to be more focused on is biomechanics. A skin substitute needs to have appropriate mechanical properties (strength, elasticity, etc.) and handling properties (can be manipulated). Techniques to analyze the mechanical properties include torsional analysis, cutometry (elasticity), tensiometry (tension), gas-bearing electrodynamomtery, and indentometry (biological agents). Past study has shown that excessive tension by cells in biomaterials inhibits angiogenesis in fibronectin fibrils, which may explain the failure of skin substitutes to vascularize. On the other hand, angiogenesis is shown to be promoted in human umbilical vein endothelial cells (HUVEC) in vitro on a fibrin-based matrix covalently bonded with ligand L1Ig6.

Overall, despite the difficulties in meeting the clinical and commercial expectations, skin replacement therapy has developed significantly and benefited to patients. Continued researches will overcome challenges and may lead to advanced regenerative tissue-engineered replacement therapies.

I choose this paper because skin substitutes is always an interesting subject and currently a growing area in bioengineering. Thus, this review paper may update our knowledge regarding bioengineered skin, and perhaps may interest some of you to specifically study more about this.