Gene modified tissue engineered skin

Adv Biochem Engin/Biotechnol (2006) 103: 241–274 DOI 10.1007/10_023 © Springer-Verlag Berlin Heidelberg 2006 Published online: 11 October 2006 Gene-Modified Tissue-Engineered Skin: The Next Generation of Skin Substitutes Stelios T. Andreadis Bioengineering Laboratory, Department of Chemical & Biological Engineering, University at Buffalo, The State University of New York (SUNY), Amherst, NY 14260, USA sandread@eng.buffalo.edu 1Introduction 242 2 Tissue Engineering of Skin 243 2.1 SkinStructureandPhysiology 243 2.2 Tissue-EngineeredSkin 244 2.2.1 BiomaterialDressings 244 2.2.2 Cell-basedSkinSubstitutes 245 2.3 LimitationsofCurrentTechnologies 246 3 Gene Therapy in Tissue Engineering of Skin 247 3.1 DeliveryVehicles 247 3.1.1 GeneDeliveryVehicles 247 3.1.2 Routes of Gene Delivery – Short- vs. Long-Term Gene Transfer . . . . . . 248 3.2 CandidateDiseaseConditionsforGeneTherapyoftheSkin 249 3.2.1 GeneticDiseases 249 3.2.2 WoundHealingandAngiogenesis 250 3.3 Gene-enhanced Tissue-Engineered Skin: ATransplantableBioreactorforTreatmentofSystemicDisorders 255 3.4 FutureDevelopmentsforEfficientGeneTransfer 258 3.4.1 GeneTransfertoEpidermalStemCells 258 3.4.2 RegulatableGeneTherapy 259 4 Gene-Modified Skin Substitutes as Biological Models of Tissue Development and Disease Pathophysiology 261 5 Summary 263 References 264 Abstract Tissue engineering combines the principles of cell biology, engineering and ma- terials science to develop three-dimensional tissues to replace or restore tissue function. Tissue engineered skin is one of most advanced tissue constructs, yet it lacks several important functions including those provided by hair follicles, sebaceous glands, sweat glands and dendritic cells. Although the complexity of skin may be difficult to recapit- ulate entirely, new or improved functions can be provided by genetic modification of the cells that make up the tissues. Gene therapy can also be used in wound healing to promote tissue regeneration or prevent healing abnormalities such as formation of scars 242 S.T. Andreadis and keloids. Finally, gene-enhanced skin substitutes have great potential as cell-based devices to deliver therapeutics locally or systemically. Although significant progress has been made in the development of gene transfer technologies, several challenges have to be met before clinical application of genetically modified skin tissue. Engineering challenges include methods for improved efficiency and targeted gene delivery; efficient gene transfer to the stem cells that constantly regenerate the dynamic epidermal tissue; and development of novel biomaterials for controlled gene delivery. In addition, advances in regulatable vectors to achieve spatially and temporally controlled gene expression by physiological or exogenous signals may facilitate pharmacological administration of therapeutics through genetically engineered skin. Gene modified skin substitutes are also employed as biological models to understand tissue development or disease progression in a realistic three-dimensional context. In summary, gene therapy has the potential to generate the next generation of skin substitutes with enhanced capacity for treatment of burns, chronic wounds and even systemic diseases. 1 Introduction Tissue Engineering applies the principles and methods of engineering and the life sciences toward the development of tissue substitutes to restore, maintain or improve tissue function [18, 71, 97]. The field of tissue engineering is motivated by the tremendous need for transplantation of human tissue. In particular, the large number of patients with severe burns (13 000 per year, with 1000 of these involving more than 60% of the body surface), diabetic ulcers (about 600 000 per year), venous ulcers (∼1millionperyear)andpres- sure sores (about 2 million per year), creates a pressing need for artificial skin substitutes [117]. In addition to providing an alternative to autologous transplantation, engineered tissues have great potential as realistic biological models to obtain fundamental understanding of the structure-function rela- tionships under normal and disease conditions and as toxicological models to facilitate drug development and testing. To engineer tissues in the laboratory, cells must grow on three-dimensional scaffolds that provide the right geometric configuration, mechanical support and bioactive signals that promote tissue growth and differentiation. The cells may come from the patient (autologous), another individual (allogeneic) or a different species (xenogeneic). Cell sourcing may be overcome by use of adult or embryonic stem cells that have the capacity for self-renewal and can differentiate into multiple cell types, thus providing an unlimited supply of cells for tissue and cellular therapies. Application of stem cells in tissue engineering requires control of their differentiation into specific cell types, which in turn depends on fundamental understanding of the factors that affect stem cell self-renewal and lineage commitment [162, 173]. Gene-Modified Tissue-Engineered Skin: The Next Generation of Skin Substitutes 243 2 Tissue Engineering of Skin 2.1 Skin Structure and Physiology The skin has two distinct layers, the dermis (D) and the epidermis (E) (Fig. 1). The dermis is the inner thicker layer that provides mechanical strength and elasticity. The main cells of the dermis are fibroblasts that synthesize extracellular matrix, endothelial cells organized in small vessel capillaries (VC) and other cellular structures such as hair follicles, sebaceous glands and sweat glands. Individual cells and cellular structures are interspersed in a network of collagen and elastin fibers of varying diameter depending on the dis- tance from the epidermis. The dermal zone right underneath the epidermis (papillary dermis) contains small-diameter fibers and the lower dermal compartment (reticular dermis) contains collagen and elastin fibers of thicker diameter. Interestingly, the diameter of vessel capillaries follows a similar dis- tribution pattern. Epidermis is the outer layer that provides a barrier to infection and wa- ter loss. It is separated by the dermis with a basement membrane and is comprised of multiple layers of keratinocytes that form a stratified squa- mous epithelium. From the innermost to the outermost, these layers are the basal layer (BL), spinous layer (SL), granular layer (GL) and stratum corneum (SC) (Fig. 1). The epidermis undergoes continuous self-renewal Fig. 1 A Morphology of mouse skin tissue. Skin tissue was harvested from an athymic mouse and processed for histology. B Bioengineered skin was prepared by culture of neonatal human keratinocytes on acellular dermis at the air-liquid interface for 7 days. Paraffin embedded tissue sections were stained with hematoxylin and eosin following standard protocols (magnification 40×). BL: basal layer; SL: suprabasal layer; GL: granular layer; SC: stratum corneum; VC: vessel capillary 244 S.T. Andreadis through proliferation of the basal cells, the only cell compartment with the ability to proliferate. The epidermis contains cells with different growth potential and at different stages of differentiation: slowly dividing stem cells that continue to proliferate for the lifetime of the tissue; transit amplifying cells that divide fast but are limited to a finite number of cell divisions before their progeny must commit to differentiate; and cells that are committed to differentiation along a certain lineage, which will eventually reach full maturity and die. Stem and transit amplifying cells are located in the basal layer of the epidermis. Periodically the transit amplifying cells leave the basement membrane and move upwards as they undergo a process of terminal differentiation that results in the anucleate cells of the stratum corneum. In the last stages of differentiation, cells extrude lipids into the intercellular space to form the per- meability barrier and break down their nuclei and other organelles as they form the highly cross-linked protein envelope immediate beneath their cellular membranes. This envelope is connected to a network of keratin filaments that provide much of the physical strength of the epidermis. The cells of the stratum corneum are eventually sloughed off and replaced with new cells coming from the lower layers. The entire renewal process takes approximately 30 days. 2.2 Tissue-Engineered Skin Research in tissue-engineered skin has produced two types of skin substitutes: biomaterials that act as synthetic dermal and epidermal analogs and can serve as temporary skin dressings and engineered tissues that contain skin cells, which provide the basic functions of the skin and may actively stimulate tissue regeneration and wound healing. 2.2.1 Biomaterial Dressings Naturally derived and synthetic dressings, including Alloderm, Xenoderm and Integra [129], have been approved by the food and drug administration. Alloderm is acellular matrix from cadaver skin, which is processed to remove the cells of the epidermis and dermis. The processed acellular dermis is immunologically inert and retains an intact basement membrane [105]. When transplanted onto the wound bed, Alloderm is covered with a split thickness autograft to form a functional epidermis while the dermis is in- filtrated by cells of the host and populated by new blood vessels [40, 41]. Xenoderm is very similar to Alloderm but is obtained from porcine skin. Finally, Integra contains an artificial dermis composed of bovine collagen and chondroitin-6-sulfate and an artificial epidermis composed of a dis- Gene-Modified Tissue-Engineered Skin: The Next Generation of Skin Substitutes 245 posable silicone sheet [24, 169–172]. A few weeks after transplantation the collagen-glycosaminoglycan matrix is vascularized by the host and the silicone layer is removed and replaced by a split thickness autograft to form the epidermis. Although both Alloderm and Integra are immediately available, immunologically inert and have been used successfully in the treatment of burns and wounds, the lack of a functional epidermis necessitates a second surgery to implant a split thickness autograft from a neighboring site. 2.2.2 Cell-based Skin Substitutes Tissue-engineered skin substitutes with epidermal and dermal components have been designed to provide the lost cellular functions of the epidermis and dermis, respectively. The main types of skin substitutes employ three-dimensional biomaterials that provide the scaffolds for cell attachment, growth and differentiation to form functional tissues. To date three commer- cially available products have obtained FDA approval: Transcyte and Derma- graft, which are produced by Advanced Tissue Sciences, and Apligraf, which is produced by Organogenesis. Transcyte is a dermal substitute that is composed of allogeneic human fibroblasts cultured in a nylon mesh for 4–6 weeks to form a dense cellular tissue and secrete a plethora of growth factors and extracellular matrix molecules. This cellular construct is used for treatment of burns after render- ing the cells non-viable by a freeze-thaw process. Transcyte has been reported to provide considerable relief from pain, reduce scarring and prevent conver- sion of partial thickness burns into more serious full thickness injuries [107]. Dermagraft is also a dermal analog that is approved by the FDA for use in diabetic foot ulcers. It is composed of human fibroblasts cultured in a biodegradable polyglactin matrix, where they form a dense three- dimensional tissue containing extracellular matrix and growth factors. Der- magraft is used alone or as a base for the meshed autografts or possible epidermal cultures that provide barrier function. One of its main advantages is that it possesses considerable angiogenic activity, which is enhanced by the process of cryopreservation used to store the product [104, 127]. Apligraf is a living skin equivalent that contains both dermal and epidermal components and is approved by the FDA for treatment of venous ulcers. The dermal component consists of human fibroblasts embedded in type I collagen and cultured for a few weeks until the cells contract the matrix. At this point epidermal keratinocytes are added to overlay the matrix and cultured to the air-liquid interface to promote complete differentiation and stratification [15, 16, 124, 165]. Apligraf resembles human skin histologically and biochemically and possesses limited barrier function [124, 165]. Surpris- ingly, transplanted skin equivalents are not rejected by the host possibly due 246 S.T. Andreadis to the absence of endothelial cells, suggesting that engineered tissues from allogeneic cells are appropriate for transplantation in humans [23]. This is an important consideration as it suggests that tissue-engineered products can be produced from a limited number of donors and stored to provide immediately available, off-the-shelf tissues for transplantation. Other tissue-engineered skin equivalents are also available or under development in industrial or academic laboratories for clinical applications or toxicological testing [9, 77, 132, 160]. Several studies have used human acellular dermis as a matrix for culture of epidermal keratinocytes [111–113]. Acellu- lar dermis retains the biochemical components of the basement membrane (e.g., collagen IV, VII and laminin), the microtopology of human dermis (rete-ridge pattern) and dermal porosity that promotes ingrowth of fibroblasts and blood vessels along the pathway of preexisting vascular conduits. When keratinocytes are seeded on the basement membrane side of the dermis and raised to the air-liquid interface they differentiate to form a fully stratified epidermis with basal, suprabasal, granular and cornified cell layers exhibiting barrier function [6, 65]. In contrast to skin equivalents with collagen gels, the dermis retains the mechanical strength and elasticity of human skin, and therefore it is easy to handle during transplantation. Others have used fibrin as a biomaterial for growth of tissue engineered skin. Fibrin is particularly attractive because it is a natural biomaterial that acts as a scaffold for tissue regeneration during wound healing and has been widely used as an adhesive in plastic and reconstructive surgery. Fibrin was found to maintain the stem cell phenotype and the proliferative potential of epidermal keratinocytes, while improving the “take-rate” of epidermal grafts onto massive full thickness burns [126]. Others used fibrin as a scaffold for fibroblast growth to re-create the dermal component of the skin before addition of epidermal keratinocytes [110, 131]. In combination with novel methods that have been developed to incorporate peptides and growth factors [65, 123, 135–137] fibrin formulations may be ideal for cell, growth factor and gene delivery to accelerate the healing response. 2.3 Limitations of Current Technologies Although substantial progress has been made, several drawbacks must be overcome to increase the clinical success of tissue-engineered skin substitutes. Current skin substitutes lack several skin cells including mast cells, Langerhans cells and adnexal structures. Although some of these structures may not be necessary for patient survival, they are important for restora- tion of normal skin functions such as sensation and sweating. Despite suc- cessful attempts to add other cell types into tissue-engineered skin e.g., melanocytes [19, 103, 154], engineering the full complexity of the skin tissue may be much more challenging. Gene-Modified Tissue-Engineered Skin: The Next Generation of Skin Substitutes 247 Part of the challenge may be addressed by appropriate design of bioactive scaffolds that provide the appropriate molecular signals and mechanical en- vironment to guide cellular infiltration and function. Alternatively, cellular function may be directed by molecular engineering at the most fundamental level, the genome. Gene delivery can be applied in tissue engineering in order to impart new functions or enhance existing cellular activities in tissue substitutes. This is achieved by genetic modification of cells that will be part of the implant or gene transfer to the site of injury to facilitate in situ tissue regeneration. Cells can be genetically engineered to express a variety of molecules including growth factors that induce cell growth/differentiation or cytokines that prevent an immunologic reaction to the implant. Therefore, gene delivery has the potential to improve the quality of skin substitutes by altering the genetic basis of the cells that make up the tissues. 3 Gene Therapy in Tissue Engineering of Skin 3.1 Delivery Vehicles The skin is an attractive target for gene therapy because it is easily accessible and shows great potential as an ectopic site for protein delivery in vivo. The cells that are primarily used to recreate skin substitutes are epidermal keratinocytes and/or dermal fibroblasts, which can be genetically modified with viral or non-viral vectors [42, 118]. Genetically modified cells are then used to engineer three-dimensional skin equivalents, which when transplanted in vivo can act as in vivo “bioreactors” to produce and deliver the desired therapeutic proteins either locally or systemically. Local delivery of proteins may be used for treatment of genetic diseases of the skin or wound healing of burns or injuries, while systemic delivery may be used for correction of systemic diseases like hemophilia or diabetes. 3.1.1 Gene Delivery Vehicles Gene delivery vehicles can be broadly classified in two categories: viral and non-viral. The genome of recombinant viruses has been modified by deletion of some or all viral genes and replacement with foreign therapeutic or marker genes. Recombinant viruses that are currently used in gene therapy include retrovirus, lentivirus, adenovirus and adeno-associated virus. Since viruses have evolved to infect cells, they display significantly higher gene transfer efficiency than non-viral systems. Recombinant retrovirus is the most commonly 248 S.T. Andreadis Table 1 Physicochemical and biological properties of the most common gene transfer technologies ∗ Properties RT LT AV AAV Plasmid DNA Titer 10 5 –10 7 10 5 –10 7 10 7 10 12 10 7 Integrates into Yes Yes No Yes No host genome? Persistence of Years Years Months Years Weeks gene expression Stability No No Yes Yes Yes Maximum 7–8 7–8 36 4–5 Unlimited transgene size Immunogenicity No No Yes Yes No Gene transfer No Yes Yes Yes Yes to non-dividing cells P ote n t i a l f o r g e n e Ye s Ye s N o Ye s N o transfer to stem cells ∗ RT = Retrovirus; AV = Adenovirus; AAV = Adeno-associated virus; LT = Lentivirus used vehicle for gene transfer to epidermal keratinocytes and skin substitutes. Lentivirus, adenovirus and non-viral gene transfer technologies have also been used but to a much lesser extent. Non-viral methods include delivery of DNA using physical and chemical means. Physical methods such as particle acceleration (gene gun) facilitate entry into target cells and may be useful in direct gene transfer to tissues which are difficult to penetrate such as skin. Although delivery of DNA com- plexed with lipids or polymers has met with some success with other cell types, epidermal cells have been difficult to transfect efficiently. On the other hand, development of biomaterials for DNA delivery in vivo has met with significant success especially in the area of tissue regeneration and wound healing. A comparison of the main characteristics of viral and non-viral technologies is given in Table 1. For a more detailed discussion on other viral and non-viral technologies see [5]. 3.1.2 Routes of Gene Delivery – Short- vs. Long-Term Gene Transfer Use of gene delivery technologies that result in temporary or permanent genetic modification depends on the requirements of the disease or condition to be treated. In tissue engineering, cells are isolated from the patient or an allogeneic source, genetically modified, expanded in culture and combined with biomaterials to recreate three-dimensional tissues that can be used to restore Gene-Modified Tissue-Engineered Skin: The Next Generation of Skin Substitutes 249 the lost function. Genetic modification may be used to suppress immune re- jection of the transplant or to generate cell-based devices for protein delivery into the systemic circulation. In these cases, permanent genetic modification is required to provide long-lasting effects. Consequently, the most suitable vectors are recombinant viruses that can mediate permanent gene transfer such as retrovirus, lentivirus and adeno-associated virus. On the other hand, genetic modification of the engineered tissue may be used to increase the rate of graft survival by promoting angiogenesis. Alter- natively, genes can be delivered in vivo using viral or non-viral technologies to promote wound healing. These applications may require transient gene expression until the transplant integrates with the surrounding tissue or until wound healing is complete. Therefore, adenoviruses or non-viral gene transfer technologies may be more appropriate. 3.2 Candidate Disease Conditions for Gene Therapy of the Skin 3.2.1 Genetic Diseases Identification of genes that are responsible for genetic diseases opens the pos- sibility for treatment by gene therapy approaches. Attempts to correct genetic defects using gene therapy include different forms of epidermolysis bullosa, lamellar ichthyosis and even psoriasis. Epidermolysis bullosa (EB) is a skin blistering disease that is caused by mutations in several genes expressed in the basal cells of the epidermis leading to loss of attachment to the basement membrane. There are three forms of EB that are caused by mutations in different genes. The simplex form of EB is caused by mutations in the keratin genes K5/K14; junctional EB is due to mutations of genes encoding for α3, β3andγ 2 chains of laminin, integrin α6β4 or bullous pemphigoid antigen; and dystrophic EB is caused by mutations in type VII collagen [80, 156, 157]. Another genetic disease, ichthyosis is a scaling disorder caused by mutations in genes that regulate the assembly of the cornified envelope. One form of the disease, namely lamellar ichthyosis is the result of mutations in the transglutaminase-1 gene, while another form, X-linked ichthyosis is caused by steroid sulfatase deficiency. Although the etiopathogenesis of these disor- ders is known, there is no available conventional treatment offering a unique opportunity to develop models for corrective gene delivery. Several studies have attempted to correct these complex genetic defects using gene therapy. When the γ 2orβ3 integrin subunits were introduced to epidermal keratinocytes from patients with junctional EB using recombinant retrovirus, the modified cells restored expression of laminin-5 and reversed the disease phenotype as evidenced by enhanced cell-substrate adhesion and reduced motility [44, 61, 159]. A clinical trial is under way to test a retroviral- [...]... the body surface are in need of skin replacement to prevent infection and dehydration Therefore, tissue- engineered skin composed of genetically modified cells to promote healing and/or prevent scar formation would be the most appropri- Gene- Modified Tissue- Engineered Skin: The Next Generation of Skin Substitutes 251 ate treatment In this case the cells need to be cultured, genetically modified in vitro and... Therefore, further development of these genetic switches to control the temporal and spatial expression of the transgene quantitatively and reversibly [90] is necessary to provide physiological control of transgene expression of primary cells and engineered tissues Gene- Modified Tissue- Engineered Skin: The Next Generation of Skin Substitutes 261 Fig 2 Schematic of controlled gene expression A In the absence... hormone gene by transplantable human epidermal cells Science 237:1476– 1479 117 Morgan JR, Yarmush ML (1997) Bioengineered skin substitutes Science & Medicine July/August:6–15 Gene- Modified Tissue- Engineered Skin: The Next Generation of Skin Substitutes 271 118 Morgan JR, Yarmush ML (1998) Gene therapy in tissue engineering, p 278–310 In: Patrick CWJ, Mikos AG, McIntire LV (eds) Frontiers in Tissue Engineering... controlled delivery of genes and proteins thus minimizing the number of animal experiments 5 Summary Although tissue- engineered skin is the most advanced tissue engineering product, it lacks several functions provided by the natural tissue Gene therapy can be used to create the next generation of skin equivalents by imparting new properties and enhancing cellular function Gene- enhanced skin substitutes may... lentiviral gene transfer to stem cells is needed to provide a clinically acceptable means of stem cell gene therapy Gene transfer to stem cells can be achieved by isolation and expansion of stem cells that are subsequently genetically modified or by targeting stem cells in cultures containing stem and differentiated cells Early reports Gene- Modified Tissue- Engineered Skin: The Next Generation of Skin Substitutes... potential clinical applications of gene therapy and tissue engineering, genetically modified engineered tissues can be employed as model systems for studying tissue development, physiology and disease pathogenesis They may also be useful as toxicological models for development of new drugs and gene therapeutics Gene transfer can be used to alter distinct genes in a biosynthetic pathway or express adhesion... and functional similarities to human tissues Furthermore, while deletion of some genes may be lethal for animal embryos, it may still be possible to study their effects using “transgenic” tissue equivalents Several studies have used genetically modified tissues to understand the effects of gene expression on tissue development We prepared skin equivalents that were genetically modified to express keratinocyte... immune system and neighboring tissues At later times, pro-inflammatory cytokines and some growth-related genes were significantly reduced but enzymes that participate in lipid synthesis increased suggesting that the epi- Gene- Modified Tissue- Engineered Skin: The Next Generation of Skin Substitutes 263 dermal cells attempted to restore the lost barrier Finally, we identified novel genes that were expressed... cutaneous gene therapy and for biological studies that require efficient and permanent genetic modification 3.4.2 Regulatable Gene Therapy The majority of gene transfer vehicles provide constitutive (always on) gene expression that may be advantageous for treatment of genetic diseases However, tissue- engineering applications may require physiologically regulated gene expression by a subset of cells in the regenerated... (2002) Retroviral gene transfer to epidermal keratinocytes correlates with integrin expression and is significantly enhanced on fibronectin Hum Gene Ther 13:1821–1831 12 Bajaj B, Lei P, Andreadis ST (2001) High efficiencies of gene transfer with immobilized recombinant retrovirus: kinetics and optimization Biotechnol Prog 17:587– 596 Gene- Modified Tissue- Engineered Skin: The Next Generation of Skin Substitutes . commitment [162, 173]. Gene-Modified Tissue-Engineered Skin: The Next Generation of Skin Substitutes 243 2 Tissue Engineering of Skin 2.1 Skin Structure and Physiology The skin has two distinct layers,. 14260, USA sandread@eng.buffalo.edu 1Introduction 242 2 Tissue Engineering of Skin 243 2.1 SkinStructureandPhysiology 243 2.2 Tissue-EngineeredSkin 244 2.2.1 BiomaterialDressings 244 2.2.2 Cell-basedSkinSubstitutes 245 2.3 LimitationsofCurrentTechnologies. entire renewal process takes approximately 30 days. 2.2 Tissue-Engineered Skin Research in tissue-engineered skin has produced two types of skin substitutes: biomaterials that act as synthetic