WO2000032252A1 - Composes a base de peau - Google Patents

Composes a base de peau Download PDF

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Publication number
WO2000032252A1
WO2000032252A1 PCT/GB1999/003889 GB9903889W WO0032252A1 WO 2000032252 A1 WO2000032252 A1 WO 2000032252A1 GB 9903889 W GB9903889 W GB 9903889W WO 0032252 A1 WO0032252 A1 WO 0032252A1
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Prior art keywords
skin
ethylene oxide
tissue
collagen based
based tissue
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PCT/GB1999/003889
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English (en)
Inventor
Sheila Macneil
Kaushik Chakrabarty
Mary Babu
Eric Freelander
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University Of Sheffield
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Application filed by University Of Sheffield filed Critical University Of Sheffield
Priority to CA002350555A priority Critical patent/CA2350555A1/fr
Priority to AU12830/00A priority patent/AU1283000A/en
Priority to NZ511585A priority patent/NZ511585A/en
Priority to EP99956180A priority patent/EP1137449A1/fr
Publication of WO2000032252A1 publication Critical patent/WO2000032252A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/20—Gaseous substances, e.g. vapours
    • A61L2/206—Ethylene oxide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the invention relates to a method to prepare skin tissue for use in, particularly but not exclusively, the repair of wounds and/or damaged tissue and/or cosmetic reconstruction.
  • Skin is a highly complex organ covering the external surface of the body. Skin functions, amongst other things, to prevent water loss from the body and to act as a protective barrier against the action of physical, chemical or infectious agents. Skin has an elastic property and varies in thickness from 0.5 mm, on for example the eyelids, to 4 mm on for example the palms and soles.
  • Skin is composed of two layers.
  • the outer layer which is comparatively thin is called the epidermis. It is several cells thick and has an external layer of dead cells that are constantly shed from the surface and replaced from below by a basal layer of cells, called the stratum germinativum.
  • the epidermis is composed predominantly of keratinocytes which make up over 95% of the cell population, the rest include dendritic cells such as Langerhans and pigmented cells called melanocytes. It is essentially cellular and non vascular, there being relatively little extra cellular matrix except for the layer of collagen and other proteins beneath the basal layer of keratinocytes. Keratinocytes of the basal layer are constantly dividing, and daughter cells subsequently move outwards, during which they undergo a period of differentiation and are eventually sloughed off from the surface.
  • the inner layer of the skin is called the dermis and is composed of a network of collagenous extracellular material, elastic fibres, blood vessels and nerves. Contained within it are hair follicles and associated sebaceous glands, collectively known as the pilosebaceous unit.
  • the interface between the epidermis and dermis is extremely irregular and consists of a succession of papillae, or finger like projections.
  • Skin is therefore a highly complex organ the cells of which are periodically replaced. Although skin provides a hard wearing and resilient outer surface skin can be physically stressed with relative ease thus leading to irreparable damage. Damage of this sort can result from: high impact contusions, diseases or burns.
  • ambulatory ulcers are the most common and result from poor venous blood supply to the lower limbs.
  • Venous insufficiency results in severe necrosis leading to deep open wounds and scarring.
  • the scarring results in a condition referred to as lipodermatosclerosis which is a hardened and leathery skin overlying sites of fibrin cuffs.
  • lipodermatosclerosis is a hardened and leathery skin overlying sites of fibrin cuffs.
  • This is particularly evident in diabetes sufferers where foot ulcers can result in amputation of toes and/or feet.
  • There are a number of treatments of diabetes that alleviate many of these symptoms.
  • the ulcers can be deep and do not heal well. In these situations skin grafts may be appropriate to remedy the condition.
  • Gastric ulcers are a common ailment of the major industrialised countries. There is a strong correlation between the presence of Helicobacter pylori and the incidence of gastric ulcers and this correlation has changed how patients prone to gastric ulcers are treated. However in those individuals suffering from established gastric ulcers the only remedial action is to surgically repair the stomach wall. This may require replacing the tissue with an autograft from a region of the stomach that is unaffected.
  • first degree burns affect the outer layer of the skin, causing pain redness and swelling.
  • Second degree burns affect both the outer and underlying layer of skin causing pain, redness, swelling and blistering.
  • Third degree burns extend into deeper tissue causing brown or blackened skin that may result in irreparable damage; this requires surgery to remove dead skin and replacement, via grafting of skin, to repair the wound.
  • three types of graft are available to a surgeon:
  • an autograft in which a piece of tissue is removed from one area of a patient's body and placed in another location;
  • an allograft in which a section of tissue from one human, for example a cadaver, is grafted onto another human;
  • tissue is harvested from another animal species, for example a pig, and placed over the wound area.
  • Autografts can be problematic due to the availability of suitable tissue and the added trauma to the patient during the removal of the tissue from another part of the body to the wound area. Allografts are limited by immunological reactivity of the host and the availability of donor tissue. Xenografts are even more problematic due to the severe immunological reactivity.
  • One approach is to provide human skin composites or reconstructed skin that have reduced immune reactivity leading to a decrease in tissue rejection by the patient.
  • research has been directed to the development of artificial skin composites which provide many of the features of natural skin.
  • the combination of cultured epithelium in combination with either an allograft dermis or synthetic dermis have been used to decrease rejection.
  • the allograft is free of the donor's host cells, thereby reducing immunogenecity, and is free from infectious agents that may compromise skin graft acceptance by the patient.
  • Currently methods of sterilisation and removal of cellular elements from allogeneic skin result in disruption of the collagen matrix leading to reduced colonisation of the graft by the hosts keratinocytes and early graft rejection.
  • a method for providing a skin composite comprising;
  • step (iii) thereof provides a skin composite that is unique in that it is both sterile and importantly, it retains its integrity or natural conformation.
  • said collagen based tissue is derived from skin, preferably human skin and more preferably still is an allograft.
  • collagen based tissue may originate from alternate sources, for example and not by way of limitation, gastric tissue, venous/arterial vessels, heart valves, bone, oral mucosa.
  • these alternative tissues may serve as autografts, allografts, or xenografts.
  • an allograft is the preferred source of collagen based tissue. This reduces the physical and psychological stress on the patient as graft material may be provided from a skin bank and abrogates the need to remove skin from a region of the patient's body as would be required if the graft was an autograft.
  • dermal samples derived from skin can be incubated long term in phosphate buffered saline or hyperosmotic saline solutions or proteolytic enzymes (e.g. trypsin, dispase) or combinations thereof. Samples are monitored periodically to assess the cellularity of the sample until such time as the dermis is reduced to a collagen matrix which is devoid of cellular elements. Acellular dermal samples can then be stored at -20°C without appreciable loss of structure. As mentioned, it is desirable to provide a collagen matrix that is essentially free of cellular material to reduce graft rejection due to immune recognition by the patient.
  • proteolytic enzymes e.g. trypsin, dispase
  • said dehydration of collagen based tissue is through incubation with a liquid dehydration agent.
  • liquid dehydration agent is glycerol.
  • said dehydration of collagen based tissue is through a sequential and incremental increase in glycerol concentration.
  • said glycerol concentration is increased from at least 50% glycerol to at least 98% glycerol.
  • said increase in glycerol concentration is in increments of at least 10% glycerol.
  • said dehydration agent is isopropanol
  • said dehydration of collagen based tissue is through the sequential and incremental increase in isopropanol concentration.
  • isopropanol concentration is increased from at least 50% isopropanol to at least 98% isopropanol.
  • said increase in isopropanol concentration is in increments of at least 10% isopropanol.
  • said sterilising agent is a fluid.
  • the sterilising agent may be provided as either a liquid or gas.
  • said sterilising agent is a liquid.
  • said sterilising agent is gaseous ethylene oxide.
  • said ethylene oxide is provided at at least 15% ethylene oxide, at least 85% carbon dioxide at an atmospheric pressure of at least 5.5 atmospheres at at least 55°C for at least 30 mins. Under these conditions a concentration of 1200 mg ethylene oxide per litre is maintained during the sterilising cycle.
  • the combination of dehydration in the presence of a sterilising agent has a twofold beneficial effect on the collagen based tissue. Firstly, the dehydration step maintains the integrity of the tissue and facilitates its long term storage. Secondly, the severe effects of for example ethylene oxide, on the collagen based tissue is ameliorated by the presence of, for example glycerol but retains its sterilising effect on infectious agents.
  • a skin composite produced by the aforedescribed method.
  • a therapeutic composition comprising the skin composite prepared by the method of the invention.
  • said therapeutic compostion is provided with storage means which facilitates and/or maintains the natural structural conformation of the skin composite.
  • a fifth aspect of the invention there is provided a method according to any previous aspect or embodiment of the invention for the preparation of collagen based tissues for use in cosmetic reconstructive surgery.
  • Figure 1 represents collagen IV in normal skin (b) and residual collagen IV in glycerol sterilised de-epidermised dermis(d), ethylene oxide sterilised de- epidermised dermis (f) and glycerol pretreated ethylene oxide sterilised de- epidermised dermis (h);
  • Figure 2 represents the appearance of a skin composite prepared by glycerol treatment
  • Figure 3 represents the appearance of a skin composite prepared by ethylene oxide treatment
  • Figure 4 represents the appearance of a skin composite prepared by glycerol and ethylene oxide treatment
  • Figure 5 represents the appearance of a skin composite prepared using keratinocytes expanded in culture and added either as a suspension or as an autograft sheet;
  • Table 1 represents the production of de-epidermised dermis using ethylene oxide and IM NaCl under various conditions;
  • Table 2 represents the influence of sterilisation protocols on the physical properties of dermis
  • Table 3 represents the scoring system used to assess skin composites
  • Table 4 represents a comparison of the in vitro appearance of skin composites prepared using ethylene oxide and glycerol/ ethylene oxide sterilised skin;
  • Table 5 represents the influence of sterilisation methodology on the appearance of composites grafted onto nude mice
  • Table 6 represents the influence of sterilisation methodology on the presence of collagen IV in composites on nude mice.
  • Table 7 represents a comparison of the in vitro appearance of skin composites prepared using freshly isolated and cultured keratinocytes using ethylene oxide sterilised skin.
  • PBS Phosphate buffered saline
  • Glycerol and isopropanol were from BDH Laboratory Supplies, Lutterworth, Leicestershire, UK. MTT, cholera toxin, epidermal growth factor (EGF), adenine, insulin, sodium chloride, transferrin, triiodothyronine and primary antibody to laminin, transferrin, triiodothyronine, ethylenediaminetetra acetic acid (EDTA), trypan blue, anti-human laminin antibody, anti-human collagen IV antibody were from Sigma Chemical Co, Poole, Dorset. ABC immunostaining kits was bought from Vector Laboratories, Peterborough, UK. Primary antibody to collagen IV was obtained from DAKO Laboratories, High Wycombe.
  • Stainless steel clips, Ligaclips were from Ethicon, Ltd, Edinburgh, UK.
  • Stainless steel grids and rings were supplied by the Department of Medical Physics, Royal Hallamshire Hospital, Sheffield, UK. All pre-sterilised plastic containers for use in tissue culture were from Costar, High Wycombe Buckinghamshire, UK. All tissue was handled in class II laminar flow hoods, provided by Walker Safety Cabinets, Glossop, Derbyshire, UK.
  • Nude mice were obtained and investigated in the Research Department of Northwick Park Hospital, Middlesex, UK. Silicone wound chambers of 1cm diameter used on the nude mice were purchased from Renner GMBH, Dannstadt, Germany.
  • Keratinocytes expanded in culture Primary keratinocytes in suspension were seeded onto a pre-coated feeder layer of lethally irradiated 3T3 cells (L3T3). These were cultured and refed every 3 days until approximately 80% confluent, usually by 7-9 days. Residual i.3T3 were removed with 0.02% EDTA before the secondary keratinocytes were detached using trypsin. The suspension of cells was centrifuged at 200g for 5 minutes and the pellet of cells re-suspended in a known volume of keratinocyte medium (KM). A cell count was then performed and these cells were now ready for use as secondary suspensions of keratinocytes.
  • L3T3 lethally irradiated 3T3 cells
  • Keratinocyte Sheets 2 x 10 6 primary keratinocytes in suspension were seeded onto a petri dish that had been pre-coated with a feeder layer i3T3 cells (2 x 10 6 per petri dish) and cultured as previously described until a confluent multilayer sheet of cells.
  • the cultured epithelial sheet was then detached by incubating for approximately 20-30 minutes in a 0.25% dispase solution.
  • a tegapore backing dressing was applied to the sheet of cells and secured to the dressing using stainless steel ligaclips, so that the basal cells were facing up, on the backing dressing.
  • the keratinocyte sheets were now ready for application.
  • confluent fibroblasts were enzymatically detached using 0.1 % w/v trypsin for 5-10 minutes.
  • the detached cells were aspirated and the trypsin was neutralised by the addition of FCS or FM.
  • the suspension of cells was centrifuged at 200g for 5 minutes.
  • the pellet of cells was re-suspended in a known volume of FM solution.
  • a viable cell count was performed cells were now ready for use in composites, or for further passaging. Only cells between passage 4 and 9 were used in the composites.
  • the 2 sterilisation techniques used for the preparation of dermis were glycerol and ethylene oxide.
  • STSG was placed sequentially in an excess of 50%, then 85% glycerol in PBS for 24 hours each at room temperature. Finally the STSG was transferred into 98% glycerol, where it was stored at room temperature, for 6 weeks. This methodology enabled the slow dehydration of skin. Previous work from this laboratory showed that 3 weeks in 98% glycerol at room temperature was sufficient to inactivate intracellular viruses in normal human fibroblasts ( Marshall et al 1995)(19). The skin samples were ready to go on to the next stage , that of acellularisation.
  • Skin was transferred from theatre to the laboratories in normal saline.
  • the first step was to wash the skin in distilled water to eradicate the saline and thereby prevent any future chlorhydrin production during ethylene oxide sterilisation.
  • STSG were washed in 6 changes of and then immersed in fresh distilled water in a sealed container and placed in a shaking water bath for a minimum of 10 minutes. This was repeated a minimum of 3-4 times more. Excess water was allowed to drip off and then the skin was placed in a 90mm petri dish base with fenestrated aluminium foil on top and frozen to -20°C at l°C/minute, before being freeze-dried overnight.
  • Freeze-dried skin was then double bagged before ethylene oxide sterilisation, using a standard SterivitTM process, using a mixture of 15% ethylene oxide and 85% carbon dioxide at a pressure of 5.5 atmospheres at 55°C.
  • ethylene oxide sterilisation using a protocol which did not exceed 37°C. This produced similar reticular damage to that observed with our own conditions.
  • STSG were placed in a large volume of sterilised 50% glycerol in PBS followed by 85% glycerol in PBS, each for 4 hours at room temperature. Finally the STSG was transferred into 98% glycerol, and stored at room temperature for 40 hours.
  • the skin was then firmly padded dry to remove any excess glycerol and placed in autoclave bags and seal with autoclave tape. These were then sent to CSSD for vacuum sealing of bags and ethylene oxide sterilisation as already discussed above. Once returned from CSSD, skin can be kept at room temperature until required for use.
  • STSG were placed in a large volume of sterilised 50% isopropanol in PBS followed by 75% isopropanol in PBS, each for 4 hours at room temperature. Finally the STSG was transferred into 98% isopropanol , and stored at room temperature for 40 hours.
  • Glycerolised sterilized skin was rehydrated for at least 4 hours with several changes of sterile PBS containing 625 ⁇ g/ml amphotericin B, l OOOiu/ml penicillin and lOOO ⁇ g/ml streptomycin.
  • the graft was then placed in an excess of PBS for 4-14 days at 37°C to produce a dermal/epidermal split using the modified technique of Krejci et al (1991)(20).
  • Such separation techniques have shown collagen IV and laminin retention on the dermal surface (Woodley et al 1983 and Moore et al 1993)( 21,22).
  • Grafts were transferred into sterile 90mm petri dishes, the epidermis was gently peeled from the dermis and discarded. The remaining dermis was washed in PBS with 625 ⁇ g/ml amphotericin B, lOOOiu/ml penicillin and lOOO ⁇ g/ml streptomycin for the rest of the acellularisation duration.
  • Freeze-dried sterilised skin was removed from the vacuum sealed packets and hydrated in a large volume of sterile PBS for 24 hours at 37°C to eradicate any residual ethylene glycol. Skin was then immersed in IM sodium chloride solution for 6-8 hours at 37°C and the epidermis was removed using sterile forceps. Skin was then rehydrated in an excess of KM for 48 hours at 37°C.
  • the split thickness skin contained anucleate epithelium and no obvious fibroblasts in the dermis. After de- epidermisation the dermis was completely acellular and no further processing was required. For glycerolised skin, however, it was necessary to remove cells as below:
  • the de-epidermised dermis was washed in sterile PBS with 0.625 ⁇ g/ml amphotericin B, lOOOiu/ml penicillin and lOOO ⁇ g/ml streptomycin and then immersed in PBS with 0.625 ⁇ g/ml amphotericin B, lOOOiu/ml penicillin and lOOO ⁇ g/ml streptomycin at 37°C. Twice weekly changes of PBS with 0.625 ⁇ g/ml amphotericin B, lOOOiu/ml penicillin and lOOO ⁇ g/ml streptomycin were performed.
  • biopsies of the dermis were taken for confirmation of acellularity by H&E stain. Once grafts were free of cells, a biopsy was taken for culture to confirm sterility. Once this was confirmed to be negative, the DED was ready for use in composite manufacture.
  • the stress-strain characteristics and tensile strength were measured by the method of Vogel (1971). Dumb-bell shaped skin strips of 12mm length and 4mm width were punched out in the same orientation from all the dermal preparations. These strips were immersed and stored in phosphate buffer solution (pH 7.2) at 4°C prior to testing. At room temperature, each wet strip was tested uniaxially in tension on an Instron Tensile Tester (Model 1112) in a liquid cell with the deformation applied along the strip length. The thickness of the samples was measured using a screw gauge. The broadened ends were gripped between the Instron grips and the stress-strain curves were recorded at an extension rate of 0.5cm/min. The tensile strength (kg/cm ) was obtained by dividing the alternate load by the area of original cross section (thickness X width).
  • DED de-epidermised dermis
  • DED De-Epidermised Dermis
  • a suspension of 1 x 10 6 primary keratinocytes (or 3 x 10 5 in the case of secondary keratinocytes unless otherwise stated) in KM was applied in the stainless steel ring and the remainder of the well was filled with KCM. 24 hours later the KM within the ring was aspirated and the keratinocytes were re-fed fresh KM. The following day, 48 hours after application of keratinocytes, the KM was aspirated from inside the rings and from the wells and the skin equivalents were raised onto stainless steel grids and fresh KM was added to the level of the base of the skin equivalent so that they were at an air liquid interface.
  • Skin composites were cultured for the required period, usually 10 days. Medium was changed every 2 - 3 days and at the end of the time period, they were assessed for their histological morphology and basement membrane and some of these were assessed for their in- vivo performance on nude mice.
  • composites were made using different combinations of cells at different seeding densities and different DED. These combinations included DED with no cells, keratinocytes alone and keratinocytes and fibroblasts together. If cells were not to be added at a particular step, then 0.5ml of the appropriate medium was added in the same way into the ring to avoid any variation in experimental conditions.
  • Anaesthetised nude mice were placed on a sterile towel. Graft sites were prepared on the anterolateral back with routine aseptic preparation using 70% alcohol and this was then washed off with sterile normal saline.
  • the skin equivalent was trimmed and applied as a lay-on graft.
  • the skin equivalent was then dressed with an overlying non- adherent dressing and saline soaked damp gauze, which was cut to size to fill the chamber.
  • 2.5cm Elastoplast tm was wrapped around the chambers and the trunk of the mouse tightly enough to secure the positions and not compromise the breathing of the mouse.
  • the mice were then placed in individual cages for 2 weeks, after which they were sacrificed by overdose of intraperitoneal barbiturate. Once the mouse was dead, the wound chambers were removed and the wound and its underlying base were excised en bloc. The specimen was cut into pieces for frozen section and light microscopy.
  • Table 1 documents removal of epidermis using IM sodium chloride at 4°C and 37°C.
  • Table 1 documents removal of epidermis from fresh skin and from ethylene oxide sterilized skin varying the temperature and duration of exposure to IM sodium chloride. Confirmation of the presence of basement membrane antigens was achieved by immunostaining for collagen IV.
  • the results summarized in Table 1 show that at 37°C we were able to remove the epidermis reliably within 8 hours both from fresh skin and from ethylene oxide skin whereas it requires 72 hours at 4°C to obtain equivalent removal of the epidermis from fresh skin at 4°C. In the case of skin at 4°C, we only lost basement membrane after de-epidermisation at 48 hours.
  • Figure 1 illustrates the morphology of (a) normal skin, of (b) glycerol sterilized de-epidermised dermis, of (c) ethylene oxide sterilized de- epidermised dermis and of (d) glycerol pre-treated ethylene oxide sterilized de-epidermised dermis.
  • glycerol sterilization gives a dermis, which is indistinguishable from that of normal skin at the light microscopy level.
  • ethylene oxide sterilization often leads to some degree of damage to the reticular dermis.
  • glycerol pre-treatment of ethylene oxide sterilized dermis gave a dermal architecture which was indistinguishable from that of normal skin.
  • Ethylene oxide sterilized and glycerol pre-treated ethylene oxide sterilized dermal preparations were routinely produced in 5 days and, by this time, were totally acellular.
  • Figure 2 illustrates the presence of residual collagen IV in (a) normal skin, (b) glycerol sterilized de-epidermised dermis, (c) ethylene oxide sterilized de- epidermised dermis and (d) glycerol pre-treated ethylene oxide sterilized de- epidermised dermis.
  • Figure 2e also illustrates that allodermis prepared by ethylene oxide sterilization (whether pre-treated with glycerol or not) retain the pliability of normal skin following 2 days soaking in phosphate buffered saline or in medium.
  • This photograph shows a piece of de- epidermised acellular dermis draped over a needle to illustrate its pliability.
  • this scoring system takes into account the nature of the dermis (whether it had normal architecture or not), the extent of fibroblast penetration into the dermis for those composites where fibroblasts were added, the nature of the keratinocyte layer, the nature of the keratin layer and the quality of the dermal epidermal junction. A maximal score of 15 was possible where both cell types were included in the composites with features resembling normal skin.
  • Table 4 compares the performance of composites based on ethylene oxide and glycerol/ethylene oxide sterilized skin. Four experiments are compared, each using skin from a separate donor dermis. (Table 5 compares the in vivo performance of the composites prepared using glycerol, ethylene oxide and glycerol/ethylene oxide sterilized composites on the nude mice.)
  • Figure 3 illustrates that glycerol sterilized dermis gave rise to composites which showed a poor epidermal attachment in the absence of fibroblasts once composites had undergone the shearing forces of cryosectioning. It is unlikely that such composites will provide secure epidermal attachment on a wound bed when subjected to any manipulation.
  • composites prepared with fibroblasts performed relatively well on the nude mice ( Figure 3 c) but showed poor epidermal attachment on the mice in the absence of fibroblasts ( Figure 3d).
  • Figures 4 and 5 show that the composites prepared using ethylene oxide sterilization and sodium chloride removal of the epidermis performed much better than the glycerol based composites.
  • Epidermal cells remained attached following cryosectioning both in vitro and ex vivo.
  • Clearly composites could be prepared in the absence of fibroblasts and yet maintain secure epidermal attachment for at least 7 days in culture and after 2 weeks of grafting on the mouse.
  • Table 4 for the semi-quantitative scoring of the ethylene oxide and glycerol/ethylene oxide composites in vitro, it was clear that inclusion of the glycerol dehydration step prior to ethylene oxide significantly improved the quality of the dermis.
  • Table 5 summarizes the in vivo performance of composites prepared using glycerol, ethylene oxide and glycerol/ethylene oxide sterilized skin focussing on the quality of the collagen IV basement membrane antigen layer in these composites both in the presence and absence of fibroblasts.
  • the quality of the collagen IV in the glycerol composites was relatively poor irrespective of the presence of fibroblasts, whereas a good layer of collagen IV was seen in composites prepared with ethylene oxide or glycerol/ethylene oxide.
  • the ethylene oxide sterilization protocol is clearly better than the glycerol based sterilization protocol for composite performance in vivo. Collagen IV staining was equally good in ethylene oxide sterilized composites and in those pre-treated with glycerol dehydration prior to ethylene oxide sterilization.
  • keratinocytes expanded in culture could also be used to produce composites, which performed well (as will be discussed in the following section of the application). Scaling up and speeding up of production of composites for clinical use To address the issue of speeding up composite production, we then examined the use of keratinocytes expanded in culture in composite production. Our preliminary data in this area show that keratinocytes initially expanded in culture will give reasonable composite performance both in vitro ( data not shown ) and in vivo ( please see table 5) even in the absence of fibroblasts.
  • EOS Ethylene oxide sterilized skin
  • the aim of this study was to progress the development of a human skin replacement based on a sterile human allodermis with cultured autologous keratinocytes for clinical use.
  • Many studies of epidermal/dermal composites, based either on human allodermis or artificial dermis show that composites can be produced to an experimental level and can perform well on nude mice ( please see refs 1-12).
  • glycerol sterilisation of skin is almost certainly based on the hygroscopic properties of glycerol which allow it to slowly dehydrate the skin providing the basis of its bacteriacidal and viricidal activity ( Marshall et al 1995)(19).
  • exposure to 98% glycerol for 3 weeks at room temperature is sufficient to prevent infectivity of herpes simplex and polioviruses.
  • glycerol has been shown to be effective in the elimination of viruses, bacteria and fungi ( Marshall et al 1995, Basile et al 1982, Van Baare et al 1994)( 19,24,25) it is not a standard method of sterilisation.
  • Dermis produced by this method maintains good histological morphology and remains pliable with good handling properties and retains expression of some basement membrane antigens.
  • ethylene oxide sterilisation of dermis was explored and showed that successful composites could be produced using ethylene oxide sterilised skin.
  • Ethylene oxide sterilization is a universally recognised method of sterilization used for surgical equipment ( Brigden et al 1980)(28). It is readily available and is a widely used sterilization process, being present in the majority of hospitals.
  • keratinocytes expanded in culture can be used to produce successful composites on nude mice and, further, that there are two options to the expansion either using the application of cultured epithelial autografts onto the sterilised dermal preparations or using passaged keratinocyte suspensions.
  • Cultured keratinocytes have been used in composites on nude mice (please see refs 3-6 and 8-12) and have been used in conjunction with a human allodermis ( Medalie et al 1996 and Demanches et al 1992)(11,12). Work is now continuing in this laboratory to scale up and speed up the production of such composites based on this information.
  • Kearney JN Franklin UC, Aguirregocoa V, Holland KT, (1989). Evaluation of ethylene oxide sterilisation of tissue implants. J Hosp Infection, 13: 71-80.

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Abstract

La présente invention concerne un procédé de préparation de tissus, généralement mais pas exclusivement de la peau, destinés à être utilisés pour la cicatrisation de blessures et/ou de tissus lésés et/ou pour la reconstruction cosmétique.
PCT/GB1999/003889 1998-11-27 1999-11-23 Composes a base de peau WO2000032252A1 (fr)

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CA002350555A CA2350555A1 (fr) 1998-11-27 1999-11-23 Composes a base de peau
AU12830/00A AU1283000A (en) 1998-11-27 1999-11-23 Skin composites
NZ511585A NZ511585A (en) 1998-11-27 1999-11-23 Skin composites
EP99956180A EP1137449A1 (fr) 1998-11-27 1999-11-23 Composes a base de peau

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GBGB9825938.5A GB9825938D0 (en) 1998-11-27 1998-11-27 Skin composites
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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1184040A1 (fr) * 2000-08-23 2002-03-06 Surface Care GmbH Matrice de peau pour la couverture et regénération de parties de peau lésées et son procédé de fabrication
WO2002024244A2 (fr) * 2000-09-20 2002-03-28 Regeneration Technologies, Inc. Procede de preparation et de traitement de tissus de greffe
WO2008154628A2 (fr) * 2007-06-12 2008-12-18 Musculoskeletal Transplant Foundation Procédé pour la stérilisation d'un tissu mou acellulaire sous vide
WO2009085199A2 (fr) * 2007-12-21 2009-07-09 Edwards Lifesciences Corporation Protection de tissu bioprosthétique pour en réduire la calcification
US8007992B2 (en) * 2006-10-27 2011-08-30 Edwards Lifesciences Corporation Method of treating glutaraldehyde-fixed pericardial tissue with a non-aqueous mixture of glycerol and a C1-C3 alcohol
EP1804045B1 (fr) * 2005-12-30 2014-03-12 QIAGEN GmbH Procédé et kit pour traitement d'un échantillon biologique
US8846390B2 (en) 2010-03-23 2014-09-30 Edwards Lifesciences Corporation Methods of conditioning sheet bioprosthetic tissue
US8906601B2 (en) 2010-06-17 2014-12-09 Edwardss Lifesciences Corporation Methods for stabilizing a bioprosthetic tissue by chemical modification of antigenic carbohydrates
US9101691B2 (en) 2007-06-11 2015-08-11 Edwards Lifesciences Corporation Methods for pre-stressing and capping bioprosthetic tissue
US9351829B2 (en) 2010-11-17 2016-05-31 Edwards Lifesciences Corporation Double cross-linkage process to enhance post-implantation bioprosthetic tissue durability
US9615922B2 (en) 2013-09-30 2017-04-11 Edwards Lifesciences Corporation Method and apparatus for preparing a contoured biological tissue
CN106561635A (zh) * 2016-11-07 2017-04-19 上海纽脉太惟医疗科技有限公司 一种用于干燥保存生物组织的方法
US10238771B2 (en) 2012-11-08 2019-03-26 Edwards Lifesciences Corporation Methods for treating bioprosthetic tissue using a nucleophile/electrophile in a catalytic system
US10959839B2 (en) 2013-10-08 2021-03-30 Edwards Lifesciences Corporation Method for directing cellular migration patterns on a biological tissue
US11517428B2 (en) 2018-11-01 2022-12-06 Edwards Lifesciences Corporation Transcatheter pulmonic regenerative valve
US12023416B2 (en) 2019-11-25 2024-07-02 Edwards Lifesciences Corporation Collagen fibers and articles formed therefrom

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DE4425776A1 (de) * 1994-07-13 1996-01-25 Britta Schimmack Verfahren zur Herstellung eines verbesserten Kollagentransplantats mit aufgelockerter Struktur
WO1998007452A1 (fr) * 1996-08-21 1998-02-26 Sulzer Vascutek Limited Procede de sterilisation de matieres destinees a l'implantation

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US5263983A (en) * 1988-03-09 1993-11-23 Terumo Kabushiki Kaisha Medical material and prosthetic skin in which cells can invade
DE4425776A1 (de) * 1994-07-13 1996-01-25 Britta Schimmack Verfahren zur Herstellung eines verbesserten Kollagentransplantats mit aufgelockerter Struktur
WO1998007452A1 (fr) * 1996-08-21 1998-02-26 Sulzer Vascutek Limited Procede de sterilisation de matieres destinees a l'implantation

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CHAKRABARTY K.H., DAWSON R.A., HARRIS P.: "Development of autologous human dermal-epidermal composites based on sterilized human allodermis for clinical use", BRITISH JOURNAL OF DERMATOLOGY, vol. 141, 1999, pages 811 - 823, XP000878676 *
GHOSH M.M., BOYCE S., LAYTON C., FREEDLANDER E., MACNEIL S.: "A comparison of Methodologies for the preparation of Human Epidermal-Dermal composites", ANNALS OF PLASTIC SURGERY, vol. 39, no. 4, 1997, pages 390 - 404, XP000878553 *

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EP1184040A1 (fr) * 2000-08-23 2002-03-06 Surface Care GmbH Matrice de peau pour la couverture et regénération de parties de peau lésées et son procédé de fabrication
WO2002024244A2 (fr) * 2000-09-20 2002-03-28 Regeneration Technologies, Inc. Procede de preparation et de traitement de tissus de greffe
WO2002024244A3 (fr) * 2000-09-20 2003-05-30 Regeneration Tech Inc Procede de preparation et de traitement de tissus de greffe
EP1804045B1 (fr) * 2005-12-30 2014-03-12 QIAGEN GmbH Procédé et kit pour traitement d'un échantillon biologique
US8796028B2 (en) 2005-12-30 2014-08-05 Qiagen Gmbh Method for treating a biological sample with a composition containing at least one polyol and excluding water
EP1969341B1 (fr) * 2005-12-30 2019-05-29 Qiagen GmbH Procede de traitement d'un echantillon biologique
US8007992B2 (en) * 2006-10-27 2011-08-30 Edwards Lifesciences Corporation Method of treating glutaraldehyde-fixed pericardial tissue with a non-aqueous mixture of glycerol and a C1-C3 alcohol
US9918832B2 (en) 2006-10-27 2018-03-20 Edwards Lifesciences Corporation Biological tissue for surgical implantation
US10434218B2 (en) 2007-06-11 2019-10-08 Edwards Lifesciences Corporation Pre-stressing and capping bioprosthetic tissue
US11305036B2 (en) 2007-06-11 2022-04-19 Edwards Lifesciences Corporation Bioprosthetic tissue having a reduced propensity for in vivo calcification
US9101691B2 (en) 2007-06-11 2015-08-11 Edwards Lifesciences Corporation Methods for pre-stressing and capping bioprosthetic tissue
WO2008154628A3 (fr) * 2007-06-12 2009-02-12 Musculoskeletal Transplant Procédé pour la stérilisation d'un tissu mou acellulaire sous vide
WO2008154628A2 (fr) * 2007-06-12 2008-12-18 Musculoskeletal Transplant Foundation Procédé pour la stérilisation d'un tissu mou acellulaire sous vide
CN101965205B (zh) * 2007-12-21 2014-01-01 爱德华兹生命科学公司 加帽生物假体组织以减少钙化
EP2647393A1 (fr) * 2007-12-21 2013-10-09 Edwards Lifesciences Corporation Protection de tissu bioprosthétique pour en réduire la calcification
WO2009085199A2 (fr) * 2007-12-21 2009-07-09 Edwards Lifesciences Corporation Protection de tissu bioprosthétique pour en réduire la calcification
US10966822B2 (en) 2007-12-21 2021-04-06 Edwards Lifesciences Corporation Heart valve with reduced calcification
US9029418B2 (en) 2007-12-21 2015-05-12 Edwards Lifesciences Corporation Capping bioprosthetic tissue to reduce calcification
CN103705980A (zh) * 2007-12-21 2014-04-09 爱德华兹生命科学公司 加帽生物假体组织以减少钙化
WO2009085199A3 (fr) * 2007-12-21 2009-12-17 Edwards Lifesciences Corporation Protection de tissu bioprosthétique pour en réduire la calcification
US8748490B2 (en) 2007-12-21 2014-06-10 Edwards Lifesciences Corporation Capping bioprosthetic tissue to reduce calcification
CN101965205A (zh) * 2007-12-21 2011-02-02 爱德华兹生命科学公司 加帽生物假体组织以减少钙化
EP3453409A1 (fr) * 2007-12-21 2019-03-13 Edwards Lifesciences Corporation Capsulage de tissu bioprosthétique pour réduire la calcification
US10188511B2 (en) 2007-12-21 2019-01-29 Edwards Lifesciences Corporation Bioprosthetic tissue with reduced calcification
US9492230B2 (en) 2010-03-23 2016-11-15 Edwards Lifesciences Corporation Methods of conditioning sheet bioprosthetic tissue
US8846390B2 (en) 2010-03-23 2014-09-30 Edwards Lifesciences Corporation Methods of conditioning sheet bioprosthetic tissue
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US9498287B2 (en) 2010-03-23 2016-11-22 Edwards Lifesciences Corporation Methods of conditioning sheet bioprosthetic tissue
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US11213385B2 (en) 2010-03-23 2022-01-04 Edwards Lifesciences Corporation Methods of conditioning sheet bioprosthetic tissue
US8906601B2 (en) 2010-06-17 2014-12-09 Edwardss Lifesciences Corporation Methods for stabilizing a bioprosthetic tissue by chemical modification of antigenic carbohydrates
US9351829B2 (en) 2010-11-17 2016-05-31 Edwards Lifesciences Corporation Double cross-linkage process to enhance post-implantation bioprosthetic tissue durability
US10238771B2 (en) 2012-11-08 2019-03-26 Edwards Lifesciences Corporation Methods for treating bioprosthetic tissue using a nucleophile/electrophile in a catalytic system
US11590260B2 (en) 2012-11-08 2023-02-28 Edwards Lifesciences Corporation Methods for treating bioprosthetic tissue using a nucleophile/electrophile in a catalytic system
US9615922B2 (en) 2013-09-30 2017-04-11 Edwards Lifesciences Corporation Method and apparatus for preparing a contoured biological tissue
US10350064B2 (en) 2013-09-30 2019-07-16 Edwards Lifesciences Corporation Method and apparatus for preparing a contoured biological tissue
US10959839B2 (en) 2013-10-08 2021-03-30 Edwards Lifesciences Corporation Method for directing cellular migration patterns on a biological tissue
CN106561635A (zh) * 2016-11-07 2017-04-19 上海纽脉太惟医疗科技有限公司 一种用于干燥保存生物组织的方法
US11517428B2 (en) 2018-11-01 2022-12-06 Edwards Lifesciences Corporation Transcatheter pulmonic regenerative valve
US12023416B2 (en) 2019-11-25 2024-07-02 Edwards Lifesciences Corporation Collagen fibers and articles formed therefrom
US12023417B2 (en) 2020-07-20 2024-07-02 Edwards Lifesciences Corporation Method for pre-stretching implantable biocompatible materials, and materials, and devices produced thereby

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GB9825938D0 (en) 1999-01-20
NZ511585A (en) 2003-09-26
AU1283000A (en) 2000-06-19
CA2350555A1 (fr) 2000-06-08

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