Slide 1:WELCOME


WELCOME TO :WELCOME TO ARTIFICIAL SKIN PRESENTED BY DR. DHIREN B. BHOI M. V. Sc. VETERINARY GYNAECOLOGY AND OBSTETRICS COLLEGE OF VETERINARY SCI. & ANIMAL HUSBANDRY ANAND


ARTIFICIAL SKIN :ARTIFICIAL SKIN INTRODUCTION Skin is a complex, largest organ and a protective barrier Injuries to skin (mainly burns) and its management Current focus on treatment to enable wound healing and reduce scarring to design and develop skin substitutes to heal skin by regeneration rather than by repair Use of skin substitutes


Slide 4:ARTIFICIAL SKIN a synthetic substitute for human skin a porous polymer film placed over the damaged skin consists of synthetic epidermis and a collagen based dermis (Yang et al., 2000) Artificial dermis acts as template for formation of new tissue Synthetic epidermis acts as temporary barrier replaced by epidermis cultured from patient’s own epithelial cells


Slide 5:Artificial skin lacks hair, sweat glands and pigmentation It has no Langerhans' cells, which are the cells that pass along bacteria and other microbes to the T cells for destruction by the immune system Contn….


Slide 7:NEED FOR ARTIFICIAL SKIN Drawbacks of conventional treatment In severe burns like 3rd degree burns, normal wound healing is slow and larger area is involved Natural skin has limited options to recover, hence need for synthetic skin Use of patient’s own skin - costly, hospitalization, anesthesia, pain, immobilization etc. Solution to skin grafting is artificial skin transplants


Slide 8:EVOLUTION OF BURN WOUND CARE Burn wounds were occluded with dressings Animal and reptile skin used as a "skin substitute" Pig skin became popularized in the 1960’s Human tissue used as a skin substitute (cadaver skin & human amnion)


Slide 9:First Cultivation of Human Epidermal Cells (1960’s) (autologous keratinocytes) Use of allogenic keratinocyte grafts Bilayered Artificial Skin Contn…


Slide 10:Historical landmarks of Artificial skin As early as the 6th century B.C., Hindu surgeons were involved in nose reconstruction, grafting skin flaps from the patient's nose In 1984 - Joseph Vacanti & Robert Langer constructed a biodegradable scaffolding on which skin cells could be grown using fibroblasts In 1975 - Rheinwald and Green cultivated human keratinocytes From 1989 - cryo-preservation of keratinocyte sheets O’Connor et al., - auto-transplantation of cultured keratinocytes in a patient with major burns


Slide 11:Yannas and Burke - described the use of bilaminate collagen-glycosaminoglycan matrix covered with a silicon surface In 1979 - Burke and Yannas used the artificial skin on their first patient Contn…


Slide 12:Ideal properties of Artificial Skin Rapid and sustained adherence to wound surface Impermeable to exogenous bacteria Control water evaporation and protein and electrolyte loss  Inner surface structure that permits cell migration, proliferation and in growth of new tissue Flexibility and pliability to permit conformation to irregular wound surface Elasticity to permit motion of underlying body tissue Provide an environment for accelerated wound healing Resistance to linear and shear stresses


Slide 13:Biodegradability Low cost Indefinite shelf life Minimal storage requirements Absence of antigenicity Tissue compatibility Absence of local and systemic toxicity Improves the cosmetic appearance of the scar


Slide 14:RAW MATERIALS The raw materials needed for the production of artificial skin fall into two categories 1. Biological components includes - Skin tissue from neonatal foreskins - Fibroblasts from the dermal layer of donated tissue - Keratinocytes from the foreskin - bovine collagen from the extensor tendon of young calves 2. Laboratory equipment includes - glass vials, tubing, roller bottles, grafting cartridges, molds, and freezers.


Slide 15:The Manufacturing Process I. Mesh scaffolding method Frozen fibroblasts are thawed and expanded Cells are transferred to a culture system combined with nutrient-rich media mixed with biodegradable mesh scaffolding (lactic acid and glycolic acid) in a cassette cells adhere to the mesh and grow new dermal skin layer is formed cryoprotectant is added cassettes stored, labeled and frozen until use


Slide 16:II. Collagen method Cold collagen & nutrient media added to fibroblast At room temperature, collagen warms and forms gel fibroblasts entrapped in gel and new skin cells grow After 2 weeks, thawed keratinocytes are seeded on to new dermal skin this is allowed to grow for several days new epidermal layer is formed new skin thus formed is stored is sterile containers until use


Slide 17:PREPARATION OF SKIN SUBSTITUTE


Slide 18:COMMERCIALLY AVAILABLE SKIN SUBSTITUTES With advancing technology, a host of both permanent and temporary biologically active skin substitutes are available to replace allograft and xenografts.- I. Naturally occurring tissues - Cutaneous allografts - Cutaneous xenografts - Amniotic membranes II. Skin substitutes - Synthetic bilaminate - Collagen based composites       Biobrane       TransCyte       Integra


Slide 19:III. Collagen based dermal analogs - De-epithelized allograft - Alloderm IV. Culture-derived tissue - Bilayer human tissue (Apligraf) - Cultured autologous keratinocytes - Fibroblast seeded dermal analogs - Collagen-glycosaminoglycan matrix - Polyglycolic or acid mesh (Dermagraft) - Epithelial seeded dermal analog


Slide 20:BIOBRANE Biobrane is a bilayer synthetic skin substitute Outer epidermal analog constructed of a thin silicone film with barrier functions Small pores present in silicone to allow for exudates removal and permeability to topical antibiotics Inner dermal analog composed of nylon filament weave upon which is bonded type I collagen peptides


Slide 21:BIOBRANE


Slide 22:BIOBRANE APPLIED TO WOUNDS


Slide 23:PROCESS OF HEALING A superficial partial thickness burn The zone of necrosis is confined to the upper dermis & is usually separated by a layer of edema from the viable wound surface


Slide 24:Viable wound bed showing fibrin and collagen


Slide 25:Bilayer BIOBRANE placed on clean wound


Slide 26:Biobrane adhered to surface by nylon-collagen mesh. Preservation of thin water layer on surface to allow epithelial migration along inner layer


Slide 27:Biobrane peeled back from surface to demonstrate rapid migration of new epithelium along nylon-collagen mesh


Slide 28:Biobrane removed with re-epithelialization


Slide 29:BIOBRANE REMOVAL APPLICATION OF BIOBRANE


Slide 30:TRANSCYTE Trancyte is a bilayer skin substitute Outer epidermal analog is a thin nonporous silicone film with barrier functions Inner dermal analog is layered with human fibroblast products mainly collagen type 1, fibronectin and Glycosaminoglycan Subsequent cryo-preservation destroys fibroblasts but preserves activity of fibroblast-derived products Thin water layer at surface is maintained for epidermal cell migration It is removed after re-epithelialization TransCyte is stored and sealed in a cassette & is thawed just prior to use


Slide 31:TRANSCYTE


Slide 32:TRANCYTE IN PLACE


Slide 33:TRANSCYTE


Slide 34:ALLODERM AlloDerm is an acellular dermal matrix designed to serve as a biologic scaffold for normal tissue remodeling It is a donated human tissue processed to remove all epidermal and dermal cells while preserving the remaining biological dermal matrix It directs normal revascularization and cell repopulation as blood vessels, collagens, proteoglycans and elastin are preserved This extracelullar matrix contains the blood vessel channels which serve as conduits for revascularization Collagens, proteoglycans and elastin provide structure and information for cell repopulation The preserved proteoglycans and proteins direct the patient's own cells to initiate revascularization and cell repopulation


Slide 35:NORMAL DERMIS ALLODERM


Slide 36:Day 1: Biologic Scaffold


Slide 37:Day 7-10 Host fibroblast cells and blood vessels respond to the transplantation of the AlloDerm matrix Initiation of the revascularization and normal tissue remodeling process


Slide 38:Day 45 Replacement and revascularization of the transplant continues as normal connective tissue


Slide 39:Day 90 AlloDerm repopulated with the patient's own cells Fibroblasts continue to lay down autologous collagen


Slide 40:INTEGRA INTEGRA is a bilayer membrane system for skin replacement The dermal replacement layer - porous matrix of fibers of cross-linked bovine tendon collagen and a glycosaminoglycan (chondroitin-6-sulfate) The temporary epidermal substitute layer - synthetic polysiloxane polymer (silicone) and functions to control moisture loss from the wound The collagen dermal replacement layer serves as a matrix for the infiltration of fibroblasts, macrophages, lymphocytes, and capillaries derived from the wound bed As healing progresses an endogenous collagen matrix is deposited by fibroblasts


Slide 41:Upon adequate vascularization of the dermal layer and availability of donor autograft tissue, the temporary silicone layer is removed A thin, meshed layer of epidermal autograft is placed over the "neodermis"(usually 14-21 days after application) Cells from the epidermal autograft grow and form a confluent stratum corneum, thereby closing the wound reconstituting a functional dermis and epidermis After final healing of the wound, the neodermis tissue histologically and functionally is similar to normal dermis used for child limb injuries (Violas et al., 2005) Contn…


Slide 42:HEALING WITH INTEGRA


Slide 43:DERMAGRAFT is an example of a synthetic matrix combined with allogenic fibroblasts and has good resistance to tearing (Bello et al., 2001) Dermal fibroblasts are seeded onto biocompatible Vicryl scaffold to form a living tissue The scaffold Vicryl is a blend of polylactic and polyglycolic acids (synthetic absorbable surgical sutures) Vicryl is inert, non-antigenic, non-pyrogenic and elicit only a mild tissue reaction during absorption Dermagraft is a total skin replacement for full thickness burns and chronic wounds like diabetic foot ulcers


Slide 44:Dermagraft cassettes ready for patient use DERMAGRAFT


Slide 45:APLIGRAF is supplied as a living, bi-layered skin substitute The lower dermal layer combines bovine type 1 collagen and human fibroblasts (dermal cells), which produce additional matrix proteins The upper epidermal layer is formed by promoting human keratinocytes (epidermal cells) first to multiply and then to differentiate to replicate the architecture of the human epidermis APLIGRAF does not contain melanocytes, Langerhans' cells, macrophages, and lymphocytes, or other structures such as blood vessels, hair follicles or sweat glands approved by the FDA to treat patients exhibiting venous leg ulcers & for diabetic foot ulcer treatment


Slide 47:SOME CASE STUDIES FOR APLIGRAF APPLICATION VENOUS LEG ULCER 1 2 3 4


Slide 48:APLIGRAF APPLIED AFTER SKIN CANCER REMOVAL


Traditional Skin Graft Artificial skin :Skin graft from the patient applied to wound Grafted dermis does not regenerate, resulting in scars that contract Larger donor sites are needed to compensate for graft shrinkage Harvested donor sites are painful, itchy and red Stiffness of graft area Two-layer template composed of a porous matrix inner layer and a silicone outer layer applied to the wound Dermis is regenerated and grows Regenerated dermis maintains shape and strength Thin epidermal graft does not create lasting donor site wound Pliable skin Traditional Skin Graft Artificial skin


Current Research and Challenges :Current Research and Challenges A novel absorbable scaffold composed of chitosan and gelatin has been fabricated (Mao et al., 2003) Liu et al. (2006) has incorporated Hyaluronic acid into the Cs-gel complex which increase the water uptake ability & biocompatibility of the scaffold An artificial skin with cross linked silicon sheet on surface of APD called SAPD was produced and tested on rats by Wang et al. (2005) DNA biomarkers were measured to evaluate the safety of tissue engineered skin (Rodriguez et al., 2004)


Slide 53:A new fish derived membrane as artificial skin has been developed by scientists at CIFT, Kochi (Polymerised product from collagen of fish air bladder and chitosan of prawn shell) The artificial human skin may be safely frozen to -80° C or stored in liquid nitrogen in order to have a skin tissue bank Challenges: Cost related concerns - The price of human skin equivalent is about $ 1,000 for a 7 inch square circle Inadequate physician education (Mendes et al., 2006) Contn…


Slide 54:Future Directions More effective cell preservation techniques could enhance shelf life and minimize issues related to storage Simplified thawing and rinsing of cryopreserved products would make such products more user-friendly A more complete understanding of the mechanism of therapeutic action of bioengineered skin could lead to even more efficacious products eg) genetic modification of the cells to overproduce specific cytokines like growth factors might be feasible and productive


Slide 55:Efforts by manufacturers to further reduce the cost of cellular skin substitute wound therapy could change the role of this approach dramatically Lower cost could also allow for multiple applications and possibly increase the efficacy of the course of treatment Contn…


Slide 56:THANK YOU