Classifications

• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0068—General culture methods using substrates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers

Definitions

• the invention pertains to a temporary biologically active cover for extensive wound areas, the method of preparing thereof and the method of it's use.
• calf- ulcers For covering extensive skin defects such as burns, calf- ulcers (ulcus cruris) are used cultivated autologous kerati- nocytes.
• This method is based on the excision of a small piece of skin (3 cm 2 ), from which the skin cells (keratinocy ⁇ tes) are isolated. These cells are grown under the conditions for tissue cultures, enzymatically released from the bottom of the cultivation vessel, in the form of a continuous growth footed on a vaseline gauze and subsequently transferred to the skin defects.
• tissue cultures enzymatically released from the bottom of the cultivation vessel, in the form of a continuous growth footed on a vaseline gauze and subsequently transferred to the skin defects.
• Green et al. Proc. Natl. Acad. Sci. USA, 76, 5665-5668, 1979.
• the problem remains in the transfer itself which is technically very complicated and can be the cause of the failure.
• the object of the present invention is a temporary biologically active cover for extensive wound areas which comprises a support based on sparingly crosslinked hydro ⁇ philic polymers with cultivated keratinocytes.
• hydrophilic polymers according to the invention there are used hydrophi ⁇ lic polymers selected from the group comprising poly(2-hydro- xyethyl methacrylate) , copolymer of 2-hydroxyethyl methacry ⁇ late with diethylglycol methacrylate, copolymer of 2-hydroxy- ethyl methacrylate with diethylglycol methacrylate and sodium methacrylate, copolymer of 2-hydroxyethyl methacrylate with diethylglycol methacrylate and methacrylic acid containing up 1% of crosslinking agent.
• hydrogels are commonly designated as hydrogels.
• Another object of the invention is a method for prepa ⁇ ring the temporary biologically active cover which consists in the direct cultivation of keratinocytes on a preincubated hydrogel support.
• the use of the cover according to the invention is characterized in that the keratinocytes cultivated on the support are directly transferred to the skin defect so that the cover is put onto the surface of the defect by the side with the grown keratinocytes. The keratinocytes then migrate from the surface of the support and colonize the surface of the skin defect.
• the procedure takes place in such a way that from a patient there is taken a small dermoepidermal graft having a thickness of about 0.2-0.3 mm from which the keratinocytes are released by the treatment with a trypsine solution.
• a hydrogel support in disk form being 24 to 48 hours preincubated with a 10 to 25% bovine serum.
• On bottom of the cultivation vessel with the hydrogel support are seeded lethally irradiated (6000 rad) 3-T-3 mouse fibroblasts (feeding cells) in a density of 1,3 x 10 6 per 50 cm 2 promoting the adherence and the first phase of growth of keratinocytes (Green et al., 1979). After the adherence of the fibroblasts there were seeded the keratinocytes in a density of 2 x 10 6 per 50 cm 2 .
• the cells are cultivated at 37°C in the atmosphere of 3.3% C0 2 .
• the cultivation medium containing commonly 1) Eagl MEM with unessential aminoacids and sodium pyruvate (Institute of Molecular Genetics of ASC, Prague), 2) 0.3 mg/ l Glutamine (TJSOL, Prague), 3) 10% Bovine serum (Bioveta, Opava), 4) Hydrocortisone (0.5 ⁇ m/ml) (Hydrocortisone Spofa soluble, 5) Peniciline (200 units/ml), Streptomycine (10 " g/ml), 6) Insulin (5 ⁇ g/ml) (Actrapid MC NOVO, Denmark), 7) Choleratoxin (10 "1 ° M) (Sigma), 8) 10 ng/ml Epidermal growth factor (Sigma).
• the swollen disk was 24 hours preincubated with 25% (v/v) bovine serum.
• the keratinocytes for the cultivation were obtained by taking from the patient a fine dermoepidermal graft having the thickness of about 0.2-0.3 mm, from which the keratinocy ⁇ tes have been released by the treatment with a trypsine solution.
• the used cultivation medium consisted of the 1) Eagl MEM with unessential aminoacids and sodium pyruvate (Institute of Molecular Genetics of ASC, Prague), 2) 0.3 mg/ml Glutamine (USOL, Moscow), 3) 10% Bovine serum (Bioveta, Opava), 4) 0.5 ⁇ m/ml Hydrocortisone (Hydrocortisone Spofa soluble, 5) Peniciline 200 units/ml, Streptomycine 10 '4 g/ml, 6) 5 ⁇ g/ml Insulin (Actrapid MC NOVO, Denmark), 7) 10 "10 M Choleratoxin (Sigma), 8) 10 ng/ml Epidermal growth factor (Sigma).
• Example 2 There was used the procedure as in Example 1 with the difference, that as support in form of a swollen disk there was used the polymer poly(2-hydroxyethyl methacrylate)
• polyHEMA crosslinked with 1 wt. % of ethylene dimethacryla- te.
• Example 2 The same procedure as in Example 1 was carried out with the difference that as support there was used a swollen disk made from the copolymer 2-hydroxyethyl methacrylate (HEMA) (80 wt.%), diethylglycol methacrylate (DEGMA) (20 wt.%) and methacrylic acid (0.5 wt.%) crosslinked with ethylene dimethacrylate (0.5 wt.%) .
• HEMA 2-hydroxyethyl methacrylate
• DEGMA diethylglycol methacrylate
• methacrylic acid 0.5 wt.% crosslinked with ethylene dimethacrylate
• Example 4 There was used the same procedure as in Example 1 differing in that as support there was used the copolymer 2- hydroxyethyl methacrylate (HEMA) (70 wt. %), diethylglycol methacrylate (DEGMA) (30 wt.%) and methacrylic acid (0.5 wt.%) crosslinked with ethylene dimethacrylate (0.3 wt.%).
• HEMA copolymer 2- hydroxyethyl methacrylate
• DEGMA diethylglycol methacrylate
• methacrylic acid 0.5 wt.%) crosslinked with ethylene dimethacrylate (0.3 wt.%).

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• Medicinal Chemistry (AREA)
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• General Engineering & Computer Science (AREA)
• Dermatology (AREA)
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Cited By (3)

Citations (3)

• 1994
  • 1994-05-31 CZ CZ941314A patent/CZ281269B6/cs not_active IP Right Cessation
• 1995
  • 1995-05-30 WO PCT/CZ1995/000011 patent/WO1995032743A1/en active Application Filing

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