Skin equivalent culture

Abstract

Disclosed is a method of preparing a collagenous construct comprising (i) casting viable collagen-producing human dermal fibroblasts in a support matrix comprising fibrin onto a support material, (ii) incubating in situ said support matrix with said viable collagen-producing human dermal fibroblasts in a collagen-inducing medium thereby (a) inducing or enhancing collagen production by said viable collagen-producing human dermal fibroblasts to form a collagenous construct, and (b) degrading said fibrin, (iii) rendering said collagenous construct free of said viable collagen-producing human dermal fibroblasts, and (iv) cross-linking said collagenous construct with a chemical or exposure to ultraviolet light.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 12 / 902,710 filed Oct. 12, 2010, which is a continuation of U.S. application Ser. No. 11 / 886,113, now U.S. Pat. No. 8,114,670, filed Jun. 24, 2008, which is a national phase application under 35 U.S.C. §371 of International Application No. PCT / GB2006 / 000890, filed Mar. 14, 2006, which claims the benefit of Great Britain Application No. 0505202.2, filed Mar. 14, 2005. The contents of all of these referenced applications are incorporated into the present application by reference.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates to methods of forming soft connective tissue compositions such as skin equivalents, compositions made by the methods and their uses.[0004]2. Description of Related Art[0005]Bioengineered skin substitutes have emerged over the past two decades, with the initial intention to replace autograft, allograft, and xenog...

AI Technical Summary

Benefits of technology

[0015]The freeze-dried construct may be re-populated or seeded with epidermal keratinocytes, for example, and allowing cornification to occur, so as to provide further strength and rigidity to the connective tissue equivalent.

[0079]In the description of ICX-SKN, we have illustrated examples of incubating constructs intended to form “living skin equivalents” for 49 days to produce a collagen matrix with sufficient structural integrity through the formation of crosslinks between and within collagen molecules in order that it will act substantially as a dermal replacement in a “living skin equivalent”. In addition, ascorbic acid, and other ingredients, are added to the growth media in order that the necessary post-translational modifications to the collagen occur within the long maturation period that we describe. Furthermore, we describe the application of physical (mechanical, ultrasound, etc.) stress to our constructs in order to produce yet more collagen and attendant structure to further enhance their properties. Freeze-drying the constructs, as we have described, further adds stability and strength to the constructs of our invention.

Problems solved by technology

This results in a concave rather than the desired convex shape, and loss in height of the construct.

Whilst it is accepted that collagen will be produced in the period of incubation (10 days) as described by Hewitt et al., without the addition of substantial means of synthesizing or obtaining ascorbic acid (vitamin C) it is doubtful that the collagen will obtain its final structure that is crucial to its function in skin, tendon, etc.

Furthermore, within the 10 day incubation described in Hewitt et al., it is unlikely that sufficient structure will form in and between collagen fibers for the resulting construct to be useful as a “living skin equivalent”.

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