Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
  • C07C311/30—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups
  • C07C311/37—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
  • C07C311/38—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton
  • C07C311/39—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound nitrogen atoms, not being part of nitro or nitroso groups having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring having sulfur atoms of sulfonamide groups and amino groups bound to carbon atoms of six-membered rings of the same carbon skeleton having the nitrogen atom of at least one of the sulfonamide groups bound to hydrogen atoms or to an acyclic carbon atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
  • A61F2/02—Prostheses implantable into the body
  • A61F2/10—Hair or skin implants
  • A61F2/105—Skin implants, e.g. 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
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/24—Collagen
• • 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
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
  • C12N5/0698—Skin equivalents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
  • A61F2310/00005—The prosthesis being constructed from a particular material
  • A61F2310/00365—Proteins; Polypeptides; Degradation products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • 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
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
  • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
• • 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
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1323—Adult fibroblasts

Definitions

• This invention is in the field of biology and par- ticularly relates to a material which can be used to cover wounds to skin.
• This invention relates to the discovery that a living tissue can be formed.
• This living tissue is produced by forming a hydrated collagen lattice, in vitro, and plating fibroblast cells into the lattice.
• a convenient technique for simultaneously forming the lattice and plating cells therein involves neutralizing an acidic collagen solution maintained in a culture dish with nutrient medium containing fibroblast cells. Upon neutralization, collagen fibrils precipitate from the solution to form the lattice with fibroblast cells homogeneously dispersed therethrough. The cells and collagen lattice are then maintained under conditions which allow the cells to attach to the collagen lattice and to contract it to a fraction of its original size * thereby providing the living tissue.
• a skin-equivalent can be produced from this liv- ing tissue by plating keratinocyte cells upon the liv ⁇ ing tissue and providing for their growth.
• This skin- equivalent is uniquely different from previously de ⁇ scribed artificial skins because its basic organization is like that of skin and its living constituent cells can be donated by a potential graft recipient.
• FIG. 1 is a plot of data illustrating the contrac ⁇ tion of a hydrated collagen.lattice
• FIG. 2 is a plot of data illustrating the contrac- tion of hydrated collagen lattices having different collagen contents
• FIG. 3 is a plot of data illustrating the contrac ⁇ tion of hydrated collagen lattices containing different numbers of fibroblast cells;
• FIG. 4 is a plot of data indicating the contractile capacity in hydrated collagen lattices of cells of dif ⁇ ferent population doubling levels;
• FIG. 5 is a plot of data illustrating the effect of 10.0 jug/ml of the inhibitor cytochalasin B on the capacity of cells to contract a hydrated collagen lattice;
• FIG ⁇ . 6 is a plot of datai ' Huslrrating the effect 5 of 0.36 jug/ml of the inhibitor colcemid on the capacity of cells to contract a hydrated collagen lattice;
• FIG. 7 is a plot of data illustrating the effect of cytosine arabinoside on the capacity of cells to contract a hydrated ' collagen lattice.
• Hydrated collagen lattices have been prepared employing collagen derived from rat tail tendon and calf skin collagen. Other sources of collagen would be suitable, of course. Solutions of collagen were pre-
• Fibroblast cells actually used in the experiments described herein were human foreskin fibroblasts and guinea pig dermal fibroblasts. Fibroblasts from other sources can also be used, and it is believed, in fact, that fibroblasts from any vertebrate animal would be suitable for contractin -hydrated-co-llage -lattices.
• fibroblast cells into hydrated collagen lattices caused-the lattices to contract as trapped water was squeezed out. If the surface on which the lattice was formed was non-wettable, e.g., a hydro- phobic plate, the resulting tissue was of regular geometry. On tissue culture plates, some cells migrated from the lattice to the plate surface and contraction of the lattice was not always regular. When a non- wettable surface * such as a bacteriological Petri plate was used, the lattice remained nearly a perfect disc as its radius was decreased by- the cells.
• a non- wettable surface * such as a bacteriological Petri plate
• Fibroblast cells were found homogeneously dispersed throughout collagen lattices and not merely upon the lattices' surface. This simulates, then, the dermal layer of humans and other mammals.
• conditioned medium prepared by growing 1 x 10 human foreskin fibroblast cells for five days in nutrient medium caused no con- traction when no cells were present.
• the initial diameter of a lattice was determined by the plate on which it was formed. Thus, maximal contraction was an arbitrary measure. Although most contracted hydrated collagen lattices were formed as sheets, other shapes could be formed. Tube*s-->---f.or- exampie, " can be formed, by forming-the con ⁇ tracted lattice in an annular mold, or a glove of skin can be performed from an appropriate mold. Human foreskin keratinocytes, ' obtained in a biopsy, were deposited on the contracted hydrated collagen ma ⁇ trix. The same could have been done, of course, with keratinocytes cultivated in vitro.
• Plating of keratino ⁇ cytes can be-done at the time the matrix-gel forms.,., at any time during the period of contraction of the lattice, or any time after contraction has been com ⁇ pleted.
• the cells formed a confluent layer on the lattice surface and the process of kera- tinization began leading to the formation of a corneal layer which would prevent loss of tissue fluids.
• the living tissue described herein is suitable for the treatment of a wound to the skin of a human being or other mammal. It is particularly suitable for massive burns wherein it functions as a skin-equiva ⁇ lent. Contraction of hydrated collagen lattices by fibroblast cells can also be used as an assay for meas ⁇ uring fibroblast function, including contractile power.
• the living tissues can be tailored -for other applications.
• collagen lattices contracted by fibroblasts could be employed as carriers for other cell types, such as pancreatic islet cells, in the pro ⁇ duction of a tissue having pancreas function, particu ⁇ larly one produced from cells obtained from the poten ⁇ tial recipient.
• tubes or other shapes might be pre- pared from lattices containing fibroblasts, heart muscle cells, or other cells, to form living protheses.
• Such protheses may be even further treated, such as by irradiation or with chemicals, to convert them to non ⁇ living but biocompatible protheses.
• the invention can be further specifically illus ⁇ trated by the following examples.
• Protein lattices were prepared in 60 mm Falcon bac ⁇ teriological dishes to which fibroblasts adhere poorly. Each dish contained: 1.0 ml 5X McCoy's 5a medium, 1.0 ml Fetal Calf Serum, 0.25 ml 0.1M NaOH, 1.5 ml collagen solution, and 1.0 ml fibroblasts suspended in IX McCoy's medium. The dishes were first filled with the above volume of McCoy's medium, serum and NaOH and then set aside until the fibroblast suspension was prepared. Speed was important in simultaneous addition of collagen solution and fibroblasts since the gel. started setting immediately. Dishes were placed in an incubator at 37°C in a 5% COumble atomosphere.
• fibroblasts were com ⁇ pletely set after 10 minutes.
• the fibroblasts employed were human foreskin fibro ⁇ blasts, strain 1519, obtained from the Human Genetic Cell Repository at the Institute for Medical Research in Camden, New Jersey. These cells were grown and maintained in McCoy's 5a- modified medium with 20%. serum,., penicillin, and streptomycin. The cultures were free of mycoplasma.
• the M.I.T. Cell Culture Center prepared and froze cells of every tenth population doubling level (PDL) .
• the effect of the number of cells on contraction of hydrated collagen lattices was determined as follows. A number of. hydrated collagen lattices containing 720 jag/ml of Protein "R" were prepared according to the procedure of Example 1. Human foreskin fibroblasts, strain 1519,.-were-employed and the-medium-in each of the cultures was changed in. the third, seventh and tenth days. Controls were employed to which no cells were added. In addition, four series of experiments were run in which varying numbers of cells were added. The data obtained are plotted in Fig. 3 in which each point represents the average of the contraction of three or four lattices. Deviations are not shown because they were all very.small ( ⁇ t 1.0 mm).
• the contractile- capacity in hydrated collagen lattices of cells of different population doubling level (PDL) was determined as follows. Cultures were formed of hydrated collagen lattices pre ⁇ pared according to the .procedures of Example 1 and containing 720 jug/ml Protein "R". Medium was changed on the third, seventh and tenth days.
• Control cultures contained no cells.
• a series of experiments with cells of different PDL levels were carried out. The data collected are plotted in FIG. 4 in which each point represents the average of three or four lattice contractions. Deviations were ⁇ 1.0 mm. As can be seen, cells of the 35th PDL performed as well as those of the 19th PDL, but cells of the 50th PDL were unable to contract lattices at a commensurate rate.
• the effect of the inhibitor cytochaiasin B on the capacity of cells to contract a hydrated collagen lattice was determined as follows. Hydrated collagen lattices were prepared according to Example 1 which contained a
• cytosine arabinoside The effect of 1.0 ug/ml of cytosine arabinoside on protein lattice contraction by cells of different PDL level was tested as follows. Cultures containing hydrated cdllagen lattices containing Protein "C" in a concentration 570 ug/ml were- prepared. Human foreskin fibroblasts, strain 1519, of the 19th PDL or 47th PDL were added -to-both " controls containing no cytosine ara ⁇ binoside and test cultures containing sytosine arabinosid Data obtained are plotted in FIG. 7, where it can be seen that the 47th PDL cells outperformed the lower PDL cells even though they were fewer in number. In these experiments cytosine arabinoside was used to block DNA synthesis and thereby keep the number of cells in the lattice constant.
• a hydrated collagen lattice was prepared according to the procedures of Example 1 which contained 500 j ⁇ g/ml of Protein "C".
• Human foreskin fibroblasts obtained in a biopsy were removed from a culture plate with a solution of EDTA and trypsin. The suspension of single cells was centrifuged to pellet the cells, after which the cells were resuspended in culture-medium and then deposited on top of the hydrated collagen matrix seven days after the fibroblast cells had been introduced. Within three days, the keratinocyte cells had attached to the lattice substrate and the process of keratiniza- tion began leading to the formation of an impervious cornium. Histological observations were made by""means of electron microscopy.
• Skin biopsies were taken from guinea pigs and ' dermis was separated from epidermis surgically. Dermis was dissociated enzymatically into constituent cells which were plated onto tissue culture dishes and allowed to undergo proliferation.. Cells from each experimental animal were grown in separate dishes so that their identity was preserved. Tissues were made up in ' vitro by forming contracted-hydrated collagen lattices accord- int to the procedures of Example 1 except employing fibroblasts from the guinea pigs.
• lattices after contraction, were plated according to Example 9 with epidermal cells or keratinocytes taken from second biopsies so that keratinocytes as well as fibroblasts in each graft were from the animal which was to become the recipient of the graft.
• This invention has industrial applicability in the preparation of living tissue, such as skin tissue.

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• Health & Medical Sciences (AREA)
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• Animal Behavior & Ethology (AREA)
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• 1979
  • 1979-12-26 JP JP55500310A patent/JPS6133593B2/ja not_active Expired
  • 1979-12-26 DE DE8080900201T patent/DE2967509D1/de not_active Expired
  • 1979-12-26 JP JP80500310A patent/JPS55501130A/ja active Pending
  • 1979-12-26 WO PCT/US1979/001120 patent/WO1980001350A1/en not_active Ceased
• 1980
  • 1980-07-14 EP EP80900201A patent/EP0020753B1/en not_active Expired

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