2003 BIOTECHNOLOGY CONVENTION 2003 BIOTECHNOLOGY CONVENTION

Artificial Skin Technology Integra LifeScience Corporation: Ms. Murad, Dr. Amirrtha Genes R’ Us: Dr. Jacqueline, Dr. Naghmeh M.C.: Vuk Pavlovic

Skin Skin is the largest organ in the body

Facts Every year in the United States, 1.1 million burn injuries demand medical attention Ten thousand people die every year of burn-related infections

Solution INTEGRA® Dermal Regeneration Template Originally conceived and designed by Dr. John F. Burke and Dr. Ioannas Yannas INTEGRA ®... provides immediate wound closure provides immediate wound closure is an innovative bilayer matrix that provides a scaffold for dermal regenration is an innovative bilayer matrix that provides a scaffold for dermal regenration is a biodegradable template that induces organized regeneration of dermal tissue by the body is a biodegradable template that induces organized regeneration of dermal tissue by the body

And now for the biotechnology... Source: Design of an Artificial Skin. I, II, III, Journal of Biomedical Materials Research, Dagalakis et al.

Stages of Clinical Experience Stage 1: Physiochemical and Mechanical constraints Keep bacteria out Keep bacteria out Prevent tissue and organ water from evaporating Prevent tissue and organ water from evaporating Stage 2: Biochemical constraints Minimize formation of deep, disfiguring scars and contractures Minimize formation of deep, disfiguring scars and contractures

Preventing Infection Keep bacteria out Wound Bed Air Bubbles Damaged Skin Graft

Air Pocket Prevention Wetting the woundbed Surgically remove dead tissue Surgically remove dead tissue Bending rigidity of membrane must be small Bending rigidity of membrane must be small High peel strength High peel strength accidental shear force should not pull membrane off accidental shear force should not pull membrane off

Moderate Fluid Flux / Moisture Flux Rate Must be maintained Too high  graft dehydrates and air pockets form Too low  interface compromised Rate should be close to human physiological rate

Artificial Membrane No need to mimic all properties of skin Membrane only needs to have: Large tear strength Large tear strength Appropriate Elasticity Appropriate Elasticity Typical thickness should be 0.1 to 0.5 mm < 0.1 mm  Difficult to suture < 0.1 mm  Difficult to suture > 0.5 mm  Not flexible enough to wet woundbed effectively > 0.5 mm  Not flexible enough to wet woundbed effectively

How is Even Growth and Healing Ensured? Biodegradability of the membrane Pores in the membrane

How is Even Growth and Healing Ensured? Biodegradability of the membrane Must biodegrade in 25 days Must biodegrade in 25 days Faster degradation  membrane is reduced to a liquid-like state Faster degradation  membrane is reduced to a liquid-like state Slower degradation  membrane must be surgically removed Slower degradation  membrane must be surgically removed

How is Even Growth and Healing Ensured? Pores in the membrane Cell migration must occur  pores must be > 10  m Cell migration must occur  pores must be > 10  mMigration Depends on availability of gases and nutrients  depends on diffusion Depends on availability of gases and nutrients  depends on diffusion The dermal layer can regenerate within 25 days The dermal layer can regenerate within 25 days The epidermal layer cannot  a temporary synthetic is used until epithelial cells can be cultured The epidermal layer cannot  a temporary synthetic is used until epithelial cells can be cultured

Selection of Chemical Compounds For dermal layer  a cross-linked collagen-glycosaminoglycan coprecipitate is used For epidermal layer  Silastic is used

Membrane Compound Criteria Polypeptides and polysaccharides can degrade to non-toxic compounds can degrade to non-toxic compounds structurally similar to macromolecules in the woundbed structurally similar to macromolecules in the woundbed can exist in highly hydrated forms can exist in highly hydrated forms

Why Collagen? Hydrophilic polymer Biodegradability (which can be controlled by cross-linking) Weak antigen  low rejection Tough enough for surgical applications Physical properties are well understood  leads to design flexibility

Why a Glycosaminoglycan (GAG) Coprecipitate? Coprecipitate is more flexible than cross- linked collagen Biodegradation can still be controlled Significantly more porous Weak antigen Degrades into non-toxic compounds

Drawback to GAG precipitate GAG can be eluted out Cross-linking can prevent this Cross-linking can prevent this

Why Silastic? Silastic is a synthetic medical grade flexible silicone sheet StrongFlexible Controls water flux like real skin

Preparation of Collagen From bovine hide Collagen is depolymerized by liming Sliced to thin pieces Washed with acid for 4 hours Water removed through filter Pulverized to tiny pieces Freeze-dried yielding loose, shredded mats

Preparation of GAG From shark cartilage GAG used as received from Sigma Chemical company

Preparation of Membrane Freeze-dried collagen flakes pulverized to minute particles dispersed in acetic acid dispersed in acetic acid ACID (A)

Preparation of Membrane Freeze-dried collagen flakes pulverized to minute particles dispersed in acetic acid dispersed in acetic acid GAG dispersed in acetic acid GAG added drop-wise to collagen while stirred ACID (B)

Preparation of Membrane Freeze-dried collagen flakes pulverized to minute particles dispersed in acetic acid dispersed in acetic acid GAG dispersed in acetic acid GAG added drop-wise to collagen while stirred Dispersion stirred for additional 5 minutes Collagen GAG

Preparation of Membrane Precipitate is then dehydrated by one of 3 different methods 1.Casting  solvent evaporation at room temperature 2.Filtration in Buechner funnel fitted with filter (assisted by aspiration) 3.Direct freeze-drying at –60 o C The various methods affect pore size

Preparation of Membrane The formed membrane is then cross- linked Cast films and filtered membranes are placed in glutaraldehyde bath Excess aldehyde is removed Freeze-dried membranes are cross- linked differently

Preparation of Membrane First, the membrane is treated in a vacuum oven so it doesn’t collapse in an aqueous solution Then, the membrane is bathed in glutaraldehyde Cross-linked membrane is freeze-dried and stored Cross-linking of Freeze-Dried Membrane

Collagen-GAG Membrane Formation Summary

Integra This membrane is the dermal layer of artificial skin The epidermal layer is made of Silastic The two fused together make Integra Silastic Membrane Integra

Biological Properties of Artificial Skin Facilitate rapid migration of fibroblasts and microvascular cells (neodermis synthesis) Absent inflammatory and foreign body reaction Synthesis of normal neodermal tissue Controlled rate of biodegradation Low/no antigenicity Nontoxic metabolites Prevent scar formation and contracture Infection prevention

Mechanical Properties of Artificial Skin Epidermal portion impermeable to bacteria Moderate fluid flux Tear strength Bending rigidity Control of pore structure in dermal portion Modulus of elasticity High peel strength Stable handling and suturing characteristics

The Healing Process Source: Integra LifeSciences Corporation

The Healing Process

Advantages of Artificial Skin Wound Closure Availability in large quantities Long shelf life DurabilityPermeability

Advantages of Artificial Skin Good Adherence Bacterial defense Ease of application and removal Cosmetic Acceptability Feels more like natural skin than autografts or cultured human cells.

Advantages of Artificial Skin

Complications and Unusual Cellular Responses Loss of Integra due to a haematoma under graft Wrinkling of Integra over the arm or lower leg Sometimes associated with collection of sterile fluid under the Integra Sometimes associated with collection of sterile fluid under the Integra Silastic sometimes loosens at edges 2 to 3 weeks after placement In areas of wear or motion caused by joint movement In areas of wear or motion caused by joint movement Infection can occur in the graft bed under Integra Fewer lymphocytes formed than expected during the healing process

Complications and Unusual Cellular Responses

The Future of Integra The dermal matrix of Integra seeded with autologous human keratinocytes In vitro seeded Integra (scanning electron microscopy)

Artificial Skin Technology Questions and Answers...