User:Azam Rahimi/sandbox/Bioengineering Skin Constructs

INTRODUCTION
As the largest organ of the body in vertebrates, skin plays a crucial role in providing a barrier against the external environment. It protects the internal organs from mechanical disturbances, pathogenic microbial agents, radiation, and loss of body fluid, and also has functions of thermoregulation and immune defense .Skin has three anatomical layers from top to bottom, as shown in .The upper epidermal layer has a thickness of approximately0.1-0.2 mm and consists mainly of keratinocytes, which solely originate from the adjacent dermal capillary network. Keratinocytes progressively differentiate from cells in the basement membrane, lose their nucleus, finally form a layer of keratin, and are later shed. The underlying dermis layer directly integrates with the epidermis. Although the thickness of dermis is varied for different parts of the body, it is primarily composed of fibroblasts and extracellular matrix (ECM) such as collagen type I, elastin, and glycosaminoglycans (GAGs). With a rich and complex supply of nerves and blood, the dermis can perceive touch, temperature, and pain. Skin appendages, including hair follicles, sweat glands, and sebaceous glands, are lined with epidermal keratinocytes and maintain homeostasis by constant recycling of the cells located on basement membrane. The third layer, the hypodermis, is situated beneath thedermis and contains well-vascularized adipose tissue and loose connective tissue, which provides both thermoregulatory and mechanical functions Due to genetic disorders or the exposure of skin to theexternal environment, skin losses caused by injuries, burns,chronic wounds, or even surgical interventions often happen, and can be a huge threat to human life and health .Sometimes severe burns and scalds could lead to rapid, extensive, and deep wounds, where a substantial area of the dermis is damaged. Additionally, with the development of economy and society, life expectancy and affluence have increased remarkably, and chronic wounds associated with ageing and diabetes become very significant According to the structure of skin, wounds can be classified as epidermal, superficial partial-thickness, deep partialthickness, and full-thickness .Different treatments are required depending on the depth of skin injuries. For epidermal injuries such as sunburn or grazing with just erythema and minor pain, no particular surgical treatment is necessary.Superficial partial-thickness wounds can regenerate rapidly because of the large number of basal keratinocyte stem cells or epithelial stem cells derived from hair follicles and sweat gland remnants located in the deeper dermis. For defects extending into the deeper dermis with more damaged skin appendages, or even full-thickness injuries with complete destruction of epithelial-regenerative elements, wounds are closed by severe scarring and epithelialization from the wound edge. This repair process is driven by an evolutionarily optimized “self-protection” mechanism in order to achieve quick closure of the wounds to prevent infection and possible future wound breakdown, but at the expense of cosmetic and functional defects .Up to the present, the autograft is still the best way for the clinical treatment of full-thickness skin defects. However, it is greatly limited by the lack of donor sites and the risk of secondary surgery, especially for deep and large area burns .With the emergence and development of tissue engineering and regenerative medicine, various bioengineered skin constructs have been developed in the past decades which have reached many significant milestones of clinical therapies for full-thickness skin defects. This chapter first reviews traditional treatment methods, including autograft, allograft, and wound dressing for the skin injuries. The tissue engineering approach for fabricating bioengineered skin constructs is then particularly focusedon. Finally, some important challenges in the field of bioengineered skin constructs such as angiogenesis, scarring, and appendages are discussed.

Autograft
So far autologous split-thickness skin grafting is still the “gold standard” for clinical treatment of full-thickness skin defects .The graft contains all of the epidermis, and asuperficial part of the dermis which is harvested from the uninjured part of the body. This donor part will be redeveloped in a similar manner to that of the superficial partial thickness wound due to the remaining dermal components .When applied to the wound site, the graft easily survives, due to the nutrients supplied by its capillaries. Generally, the thicker the split-thickness skin graft is the less contraction the wound will suffer from, but it will take longer to heal the donor site. Moreover, over-removing of the healthy epidermis should be avoided, because death might result because of the ineffectivebarrier of skin and the onset of bacterial sepsis.

Allograft
A cadaveric allograft is sometimes utilized temporarily to cover the wound until a permanent skin graft is available When used in a fresh form, the cadaver skin plays a role in relieving pain and providing a temporary durable cover for the first few weeks post-injury. In this case, the immune response in a badly burnt patient should be pathologically suppressed. However, with the vascularizatio of the allograft, the highly immunogenic epithelial cells will trigger the immune response of the host and it will be rejected. As a result, it is preferable to use glycerolized or lyophilized allograft whose cellular components are destroyed with diminished immunological reaction. Allografts have been used for decades, and some of them, such as Karoskin® (Karocell Tissue Engineering AB, Sweden), are commercially available .However, the application of allografts is limited by some issues such as lack of supply, high cost, and variability in quality, as well as many safety risks, e.g., viruses, transmissible spongiform encephalopathies. Sweat gland duct Basement membrane Touch receptor Subcutaneous layer Capillary Dermis Hair shaft Sweat gland pore Epidermis.

Wound Dressing
In 1960s, Winter found that the healing rate of a rapidly closed wound is higher than that of an open wound .It has also been proposed that a moist environment benefitswound healing .In order to provide a quick cover for the wound and create a moist micro-environment, a variety of wound dressings in the form of films, sponges, hydrogels, etc., have been developed, and some of them are commercially available. An ideal dressing should have the properties of hemostasis, adhesion, suitable moisture permeability, excellent protection against bacteria, formability, operability, etc. The wound dressing can generally be divided into three types: passive dressing; interactive dressing; and bioactive dressing. Traditional dressings, such as gauze and mesh fabric, are representative of passive dressings. Nylon, a well-known suture material, is also widely used for wound cover in the form of nylon mesh .Interactive dressings comprise a polymeric film and a sponge which can prevent the invasion of bacteria and water loss, but allow permeability of moist gas to promote wound healing. The Opsite® (Smith & Nephew, UK) is composed of a semi-permeable polyurethane film with good adhesion properties. It can relieve pain, accelerate wound healing, and effectively prevent infection of the wound. Another famous polyurethane-based, commercially available, wound dressing is Tegaderm® (3 M Health Care). The product is cost-effective for small-sized split-thickness skin defects. Bioactive dressings usually contain bioactive components for wound healing, such as proteoglycans, collagen,alginate, and chitosan, etc,.

TISSUE ENGINEERING APPROACH FOR SKIN REPAIR
Millions of patients suffer from skin diseases and defects every year, and acute and chronic skin wounds are the main challenges in skin repair. The key factor in skin defect healing is to promote wound closure to reduce the complications caused by disorders of the internal environment or invasion of microbes. Without timely transplantation, patients can easily suffer from loss of body fluid, acute infection, and even sepsis which may threaten their lives. Although the traditional autografting treatment is widely used in clinics, it has the limitation of availabilty. Besides, the repeating harvest of split-thickness skin may delay the process of reepithelialization .Tissue engineering is an interdisciplinary field of cell biology, biomaterials, and engineering to improve or replace the functions of damaged tissues or organs .Based on the principle of tissue engineering, bioengineered skin constructs consisting of cells, biomaterials, and bioactive factors have been developed in past decades which provide a novel approach for repair of full-thickness skin defects. In general, tissue-engineered skin must comply with three major requirements, i.e., safety for patients, clinical efficiency, and convenience of handling.

Scaffolds
Besides different types of cells, the three-dimensional network (namely ECM) composed of biomacromolecules, such as collagen and glycosaminoglycans, plays a key role in the development and regeneration of a tissue or organ with certain structures and functions. Therefore, the first priority in tissue engineering is to construct a scaffold with well-designed components and microstructure to mimic the functions of ECM to support the infiltration, proliferation, and differentiation of seeded cells .In principle, the scaffold must meet the following specific requirements .First, high porosity and adequate pore size are necessary to promote cell infiltration and diffusion of both nutrients and wastes throughout the whole structure. Second, the scaffold must enable or even actively support cell adhesion, proliferation, and differentiation to build up new tissue. Third, the scaffold should preferably be absorbed by the surrounding tissues without extra surgical removal, at a rate coinciding as much as possible with that of tissue regeneration. That is to say, the scaffold should be able to provide structural integrity within the body and gradually break down while cells are fabricating their own natural matrix structure, until eventually the newly formed tissue takes over the mechanical load. Therefore, the strategy in scaffold design is basically to mimic the components and microstructure of the target tissue or organ, to provide a temporary support for the growth of cells and regulate the process of repair and regeneration. The skin scaffold is generally made up of ECM components such as collagen and chondroitin sulfate. Collagen is the principle ECM component of connective tissues, and is mostly found in the form of elongated fibrils in tendon, ligament, and dermis. It is composed of a triple helix with two identical chains (α1), with an additional chain that differs slightly in its chemical composition (α2). Due to its structural and functional similarity to native ECM, as well as biodegradability, collagen with a supplement of polysaccharide was used early in the fabrication of wound dressings and skin scaffolds, including some famous commercially available products. For example, theIntegra® is engineered primarily with crosslinked bovine collagen and chondroitin-6-sulfate (8 wt%).fabricated a porous dermis scaffold using collagen and glycosaminoglycans which were crosslinked with 1- ethyl-3,3-(dimethyl amine propyl) carbodiimide (EDC). The scaffold could promote wound healing significantly after implantation onto nude mice. In order to balance some disadvantages of collagen, and even most natural materials, such as the weaker mechanical properties, and severe shrinkage, many crosslinking treatments have been developed to increase the structural stability of collagen. We previously fabricated a collagen/chitosan scaffold with good biocompatibility to promote fibroblast growth and dermis regeneration. The chitosan is used because of its advantages of biocompatibility, biodegradability, hemostatic activity, and antibacterial properties. Furthermore, the chitosan could function as a crosslinking bridge under the treatment of glutaraldehyde, as shown in An EDC/N-hydroxysuccinimide (NHS)- based crosslinking method with the assistance of lysine is effective in enhancing the biostability of collagen scaffolds, and can be further exploited for the crosslinking of collagen/chitosan scaffolds for skin repair. Mao et al. also constructed a chitosan-gelatin scaffold with a bilayer structure. Other natural materials such as gelatin, hyaluronan, and fibrin glues are also widely used to construct bioengineered skin substitutes. For example, Lin et al. designed a gelatin/ chondroitin sulfate/hyaluronan scaffold with a bilayered structure, and wound healing with a high survival rate after implantation was achieved. Lee et al. fabricated a bio-artificial skin composed of gelatin and (1 → 3), (1 → 6)-β-glucan, and compared its healing efficiency with the acellular dermal matrix. A relatively higher reepithelialization rate of the wound treated with the artificial skin was observed. In addition, natural polymer-based scaffolds can be further functionalized via some chemical compounds and specific biological ligands to obtain better properties .The acellular dermal matrix (ADM) maintains most components and structure of natural ECM. It is primarily composed of the extracellular matrix of the dermis by removal of the cells, which is more capable of providing the micro-environment for ingrowth of cells from the wound site. Currently, AlloDerm®, made by the LifeCell Corporation, is the most widely employed of the commercially available products. However, the main drawbacks of ADM lie in the limited sources, possible immune response elicited by the allogenic collagen, and the risk of virus infection. The most commonly used synthetic polymers are polyesters such as polylactic acid (PLA), polyglycolic acid (PGA), and poly (lactic-co-glycolic acid) (PLGA), which have been approved by the US Food and Drug Administration (FDA). Chen et al. combined collagen with knitted PLGA to fabricate a hybrid scaffold which can promote the growth offibroblasts and the regeneration of dermis .Venugopal et al. fabricated a porous polycaprolactone(PCL)/collagen membrane by electrospinning. Its well-defined nanostructure can promote the growth and adhesion Schematic representation of collagen crosslinked with glutaraldehyde in the presence of chitosan. The Advanced Tissue Science Company cultured fibroblasts in a PLA scaffold to form an artificial active dermis, (TransCyte®), which has been approved by the FDA for healing of third degree burns. In general, compared with the natural polymers, the synthetic polymers are less expensive and have better batch consistency. They can be tailored using different fabrication techniques to provide various physical properties. To overcome the drawbacks of poorer cellular recognition, structural modification should be considered to improve biocompatibility.

Cells
Seed cells are the essential element for tissue regeneration.The sources of cells for bio-engineered skin mainly include autologous cells, allogeneic cells, and xenogenic cells. The allogeneic and xenogenic cells are relatively abundant, but the existing immune response and differences between cell types have limited their application. Recent progress in making use of autologous cells falls into two categories depending on the ability of cell differentiation. The somatic cells are biological cells forming the body of an organism other than germ cells, gametocytes, or undifferentiated stem cells. Many kinds of the somatic cells, such as fibroblasts, keratinocytes, epidermal cells, melanocytes, and hair follicle cells have been used to reconstruct skin substitutes. Fibroblasts are known to play a key role in skin wound healing, and they are the most dominant cell type in dermis. Vascular endothelial cells contribute to blood vessel regenesis. Berthod et al. added laminin into a collagen/chitosan scaffold for the co-culture of fibroblasts and keratinocytes, laminin is well-known for promoting neurite extension, and more regenerated nerves were detected 120 days postimplantation on nude mouse back. Neil et al. co-cultured melanocytes with keratinocytes to deal with the lack of pigment during wound healing. Wu et al. seeded dermal sheath cells and epithelial cells in collagen gels, which formed hair follicle-like structures after being applied in nude mice. With self-renewing and multipotent differentiation abilities, stem cells have recently been widely used in tissue engineering. While embryonic stem cells are topipotent, adult stem cells tend to be multipotent and are able to function well as a source of cells for tissue repair. Various types of adult stem cells can be found from skin, such as epidermal stem cells, sebaceous stem cells, hair follicle stem cells, sweat gland stem cells, melanocyte stem cells, mesenchymal stem cells (MSCs), neural stem cells, and endothelial stem cells. Ruszczak et al. outlined an approach to reconstruct an in vitro skin which can be transplanted directly to the wound bed and can permanently replace the missing tissue. They cultured epidermal stem cells to generate an epidermal sheet and maintain the stem cell population. The epidermal sheet is then placed on top of a dermal substitute comprising devitalized dermis or bioengineered dermal substitutes seeded with dermal fibroblasts. The hair follicle bulge is the main source of multipotent hair follicle stem cells. Kobayashi and Nishimura dissected dermal papillae cells and reconstituted them with fragments of hair follicle. Viable hair follicles formed after transplantation in nude mouse. Bone marrow-derived mesenchymal stem cells (BMSCs) possess multi-lineage potential to undergo osteogenic, chondrogenic, and adipogenic differentiation. It was recently reported that the BMSCs could be induced into hair dermal papilla-like cells and accelerate the wound healing rate. Sheng et al. managed to reconstruct sweat gland by combining BMSCs and sweat gland cells, which may lead to the road of using stem cells to reconstruct skin appendages to fulfill the complete function of regenerated skin.It is worth mentioning that embryonic stem cells (ES) derived from the inner cell mass of the embryonic blastocyst are totipotent, and able to be maintained indefinitely SEM images of PLGA/collagen hybrid mesh (a), and fibroblasts cultured in the mesh for 5 days (b). Tissue Engineering Skin and Oral Mucosa in vitro without loss of differentiation potential, and further can be differentiated into many different types of cells. The ES cell provides an attractive and viable proposition for cell-replacement therapy, especially for the regeneration of complex organs such as skin, although human ES cells have only recently become available and there are still many controversial ethical and technical problems that need to be overcome before the full potential of this type of cell can be applied. ES cells look quite promising in regenerative medicine and tissue engineering, and there is no doubt that well-functionalized skin substitutes comparable to normal skin can be achieved in the future.

Bioactive Factors
Wound healing and skin repair are the result of the synergistic effects of different types of cells whose proliferation, migration, differentiation, and ECM secretion are well-regulated by bioactive factors such as cell growth factors and cytokines. Therefore, bioactive factors strongly affect vascularization, collagen secretion, and re-epithelialization during skin regeneration. Combining bioactive factors with scaffolds is a promising way to promote the efficiency and quality of wound healing. Cell growth factors are proteins or steroid hormones regulating a variety of cellular processes, such as cell proliferation and differentiation. They typically act as signaling molecules between cells and ECM to promote cell differentiation and maturation, and have been widely used in tissue engineering skin constructs. Some growth factors are promising mediators of wound healing, such as epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), insulin-like growth factor-I (IGF-I), transforming growth factors α and β (TGF-α and TGF-β), and hepatocyte growth factor (HGF). By combining the growth factors with micro-vehicles using chemical or physical methods, bioactive skin scaffolds can be constructed. Richardson et al. incorporated VEGF and PDGF in a porous PLGA scaffold, allowing for a controlled dose and rate of delivery. This strategy pioneers the research of a vehicle delivering multiple angiogenic factors with distinct kinetics. Perets et al. incorporated bFGF-loaded microspheres within three-dimensional porous alginate scaffolds, which enhanced vascularization in vivo. Shah et al. confirmed that high expression of TGF-β3 contributes to a scarless wound healing result. Tabata et al. combined FGF, HGF, and VEGF with collagen gels, which promoted the regeneration of hair follicles after implantation. Mao et al. combined FGF on a surface via layer-by-layer assembly to fabricate bioactive films on which fibroblasts proliferated better and secreted more Potential for genetic engineering Full-thickness skin wounds Dermal substitute Single-cell suspension Epidermal stem cell Epidermis Scaffold (Seed with dermal fibroblasts) Ex vivo expansion conditions to maintain ‘holoclones’ (stem-cell population) Principle of tissue-engineered skin using epidermal stem cells. Regarding hair follicle regeneration, HGF, a mitogen, motogen, and morphogen for many different organs, is expressed by isolated and cultured human hair follicles, and is involved in the cycle of hair growth. HGF has been shown to stimulate follicle growth and DNA synthesis in human hair, as well as mouse vibrissae, up-regulate DNA synthesis in hair bulb-derived keratinocytes, and modulate cyclic hair growth in mice. Another factor involved in hair growth is IGF. The use of IGF-I (10 ng/mL) combined with IGF-II (100 ng/mL) showed a greater ability than insulin to prevent the catagen stage in hair follicles. As summarized above, with the ability to modulate and direct cells efficiently, cell growth factors have been extensively used as bioactive factors to combine with tissue engineering scaffolds. Recently, emerging gene techniques show several advantages over cell growth factors. Functional genes can be incorporated into scaffolds as bioactive factors, and locally express encoded growth factors at the wound site. Specific examples of the application of gene therapy in skin tissue engineering will be introduced further below.

CURRENT PROGRESS
In order to obtain the necessary number of keratinocytes for therapeutic needs, isolation of keratinocytes from a donor and subsequent in vitro culture is the key step in the design and production of epidermal substitutes. The approaches to production of epidermal substitutes may be quite different. The cell culture techniques, the stage of cell differentiation and epithelial organization, the methods of cell delivery to the patient, and the use of additional substrates to enhance cell culture and delivery together determine what epidermal substitutes can be obtained. It could be imagined that the very nature of the confluent, layered, cell culture system to a large extent determines an unpredictable clinical outcome. An aerosol of cell suspension is one way to apply subconfluent keratinocytes to the wound bed. Culturing a monolayer of subconfluent keratinocytes on delivery membranes is an alternative approach. The membranes can be either mechanically peeled from the culture vessel, or can be applied with the cultured cells directly to the wound site. Cao et al. cultured epidermal cells on a chitosan/gelatin membrane to construct a tissue engineered epidermis. This artificial epidermal membrane could survive in vivo, and has good clinical application in promoting wound healing of the skin graft donor site and inhibiting hypertrophic scar formation.Epicel®(Genzyme Biosurgery, USA)is one of earliest commercialized epidermal substitutes which are manufactured using the patient’s own keratinocytes. It takes nearly 15 days for the keratinocytes to grow confluently to form cultured epithelial autografts (CEAs). ,