Chemistry: molecular biology and microbiology – Spore forming or isolating process
Patent
1991-09-12
1993-12-28
Lacey, David L.
Chemistry: molecular biology and microbiology
Spore forming or isolating process
4352401, 530356, 424934, C12N 506, A61K 3512, A61K 3712
Patent
active
052739007
DESCRIPTION:
BRIEF SUMMARY
o the biologically active molecules are reacted with the biotinylated dermal component to incorporate the active molecules into the dermal component. The so-modified dermal component is then combined with the epidermal cells to form the composite skin replacement of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
The details of typical embodiments of the present invention will be described in connection with accompanying drawings in which:
FIGS. 1A and 1B are diagrammatic representations of the method used to prepare the composite skin replacement (1A) and the grafting of the skin replacement onto a wound (1B).
FIG. 2 is a photograph of a composite skin replacement prepared according to the method of the invention.
FIGS. 3A-3E are photomicrographs of composite skin replacements in vitro (3A-3D), and of split thickness skin (3E). 3A: composite skin replacement after 5 days incubation in culture medium containing 20%(v/v) FBS. 3B: composite skin replacement after 5 days incubation in serum-free culture medium. 3C,3D: mitotic cells in composite grafts. 3E: split thickness skin graft.
FIGS. 4A and 4B are transmission electron micrographs of a composite skin replacement after 11 days in culture. 4A: bottom to top showing pores (P) in the collagen substrate (CS). Numbers on the right identify 10 HK layers. 4B: Uppermost HK strata with cornified cell envelopes (CCE), and uncornified plasma membrane (PM), scale bars=10 .mu.m.
FIGS. 5A and 5B are transmission electron micrographs of HK cells in culture. 5A shows desmosomes that interconnect HK cells in culture, 5B shows enlargement of desmosomal junctions between cells. Scale bar=10 .mu.m.
FIGS. 6A-6C are transmission electron micrographs showing biological attachments between HK cells and the collagen substrate. 6A: HK cell attached to the CS by hemidesmosomes (HD) and formation of extracellular matrix (ecm). (V)=vesicles in the plasma membrane (pm). 6B: HD, pm and ecm. 6C: Arrows indicate banded collagen substrate. A,B, scale bars=1 .mu.m; C, scale bar=10 .mu.m.
FIG. 7 is a photomicrograph of a full thickness skin defect treated six weeks before, using the composite skin replacement of the invention. Arrows indicate reticulated spaces formed by as yet non-resorbed dermal membrane. Scale bar=100 .mu.m.
FIG. 8 is an exploded isometric view of the apparatus for preparing the dermal membrane of the invention.
FIG. 9 is an isometric view of the assembled apparatus.
FIG. 10 is a section view taken along line 10--10 of FIG. 9.
FIG. 11 is a partial exploded section view taken along line 10--10 of FIG. 9.
FIG. 12 is a schematic diagram depicting the various methods for attaching biologically active compounds to a substrate as described in Examples V and VI, infra.
FIG. 13 illustrates spot test results for binding of horseradish peroxidase (HRP) covalently bound to avidin with biotinylated collagen and non-biotinylated collagen on nitrocellulose paper as described in Example IV, infra.
FIG. 14 is photographs of biotinylated HRP conjugated to biotinylated collagen.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes a method for preparing a permanent composite skin replacement consisting of a biological epidermal component and an acellular, biosynthetic (biological material prepared by synthetic procedures), porous, resorbable dermal membrane component. Preferably, the epidermal component includes cultured human epithelial cells such as normal human keratinocytes (HK) which are cultured on the dermal membrane and attach to the membrane to allow subsequent grafting of the composite formed to a wound. For use in the composite, the epidermal cells may be derived from epidermal tissue which is genetically identical to the tissue of the composite recipient (autogeneic cells) or may be derived from isospecific (same species) but genetically dissimilar (allogeneic) tissue.
The dermal membrane component may be prepared using the apparatus of the invention. The dermal membrane is preferably prepared from collagen, for example bovine collagen and a mucopolysac
REFERENCES:
patent: 3472228 (1969-10-01), Tanner, Jr.
patent: 3526927 (1970-09-01), Villain
patent: 3782387 (1974-01-01), Falabella
patent: 4060081 (1977-11-01), Yannas et al.
patent: 4140537 (1979-02-01), Luck et al.
patent: 4233360 (1980-11-01), Luck et al.
patent: 4252759 (1981-02-01), Yannas et al.
patent: 4280954 (1981-07-01), Yannas et al.
patent: 4326523 (1982-04-01), Wolfrom et al.
patent: 4350629 (1982-09-01), Yannas et al.
patent: 4418691 (1983-12-01), Yannas et al.
patent: 4448718 (1984-05-01), Yannas et al.
patent: 4458678 (1984-07-01), Yannas et al.
patent: 4485096 (1984-11-01), Bell
patent: 4488911 (1984-12-01), Luck et al.
patent: 4505266 (1985-03-01), Yannas et al.
patent: 4507255 (1985-03-01), Shizawa
patent: 4511653 (1985-04-01), Play et al.
patent: 4522753 (1985-06-01), Yannas et al.
patent: 4546500 (1985-10-01), Bell
patent: 4600461 (1986-07-01), Guy
patent: 4600533 (1986-07-01), Chu
patent: 4604346 (1986-08-01), Bell et al.
patent: 4605413 (1986-08-01), Urry et al.
patent: 4621029 (1986-11-01), Kawaguchi
patent: 4655980 (1987-04-01), Chu
patent: 4670014 (1987-06-01), Huc et al.
patent: 4673649 (1987-06-01), Boyce et al.
patent: 4689399 (1987-08-01), Chu
patent: 4703108 (1987-10-01), Silver et al.
Boyce and Hansbrough, "Biologic attachment, growth and differentiation of cultured human epidermal keratinocytes on a graftable collagen and chondroitin-6-substrate", Surgery, 103:421-431 (Apr. 1988).
Boyce et al., "Athymic (Nude) Mice as a Model for Evaluation of Wound Contraction After Grafting of Materials for Human Skin Replacement" (Abstr.), Proc. Amer. Burn. Assocn. 19:78 (1987).
Boyce and Hansbrough, "Studies of Growth Requirements of Cultured Human Epidermal Cells of a Collagen-Glycosaminoglycan (GAG) Membrane", American Burn Association Sixteenth Annual Meeting (1984) Exhibit 3.
G. Gregory Gallico, III, M.D., et al., "Permanent Coverage of Large Burn Wounds With Autologous Cultured Human Epithelim," Medical Intelligence vol. 311, pp. 448-451 (1984).
John F. Hansbrough, M.D., Steven T. Boyce, B.A., "What Criteria Should be Used for Designing Artificial Skin Replacements and How Well do the Current Grafting Materials Meet These Criteria?" The Journal of Trauma, vol. 24, No. 9 Supplement, pp. S31-S35 (Sep. 1984).
Steven T. Boyce, Richard G. Ham, "Cultivation, Frozen Storage, and Clonal Growth of Normal Human Epidermal Keratinocytes in Serum-Free Media," Journal of Tissue Culture Methods, vol. 9, No. 2, pp. 83-93 (1985).
Steven T. Boyce, B. A., and Richard G. Ham., Ph.D., "Calcium-Regulated Differentiation of Normal Human Epidermal Keratinocytes in Chemically Defined Clonal Culture and Serum-Free Serial Culture" The Journal of Investigative Dermatology, vol. 81, No. 1 Supplement, pp. 33s-40s (1983).
Cuono, et al., "Use of Cultured Epideral Autographs and Dermal Allografts As Skin Replacement After Burn Injury" The Lancet, pp. 1123-1124, May 17, 1986.
Heck, et al., "Composite Skin Graft: Frozen Dermal Allografts Support the Engraftment and Expansion of Autologous Epidermis" The Journal of Trauma vol. 25, No. 2, pp. 106-112 (1985).
Doillon, et al., "Collagen-based Wound Dressings: Control of the Pore Structure and Morphology" Journal of Biomedical Materials Research, vol. 20, 1219-1228 (1986).
Tavis, et al., "A New Composite Skin Prosthesis" Burns, vol. 7, pp. 123-130 (1979).
Burke, et al., "Successful Use of a Physiologically Acceptable Artificial Skin in the Treatment of Extensive Burn Injury" Ann. Surg., vol. 194, No. 4, pp. 413-428 (1981).
Yannas, et al., "Wound Tissue Can Utilize a Polymeric Template to Synthesize a Functional Extension of Skin" Science, vol. 215 Jan. 1982.
Boyce, et al., "Reduced Wound Contraction After Excision and Grafting of Full Thickness Burns with a Collagen-Chondroiton-6-Sulfate Dermal Skin Replacement And Coverage With Biobrane" (Abstract) Program of the Amer. Burn Assoc., (1986).
Bell et al., "The Reconstitution of Living Skin" The Journal of Investigative Dermatology, vol. 81, No. 1 Supplements, pp. 2s-1s (1
Elliott George C.
Lacey David L.
The Regents of the University of California
LandOfFree
Method and apparatus for preparing composite skin replacement does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method and apparatus for preparing composite skin replacement, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and apparatus for preparing composite skin replacement will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1542154