Wound healing profile
View Patent ↗The present invention relates to compositions and methods for tissue regeneration, particularly for treating skin lesions such as wounds. In one aspect, the invention provides wound healing composition characterized by the higher expression levels of phenotypic marker genes such as apolipoprotein D, matrix metalloprotease (2), collagen 3a1 and smooth muscle actin than the housekeeping gene ribosomal protein L32. The compositions and methods of the invention are useful especially for assisting the process of wound healing, particularly chronic open lesions that are slow to heal or resistant to healing.
1. A wound healing composition comprising isolated living dermal fibroblast cells having a wound healing phenotype, characterized in that:
(i) the cells of the composition exhibit, at less than 14 days, a 2.08 to 48,200-fold higher level of expression of apolipoprotein D (ApoD); a 694 to 662,000-fold higher level of expression of matrix metalloprotease 2 (MMP2); a 33.7 to 43,300-fold higher level of expression of collagen 3a1 (Coll3a1); and a 20.2 to 121,000-fold higher level of expression of smooth muscle actin (SMA) relative to the expression level of Ribosomal protein L32 (RPL32); or
(ii) 75-99% of the cells of the composition have a banding pattern of polymerase chain reaction (PCR) products resulting from differential display identical or similar to that shown in FIG. 4 or FIG. 5 for nucleic acid expression in fibrin,
wherein the dermal fibroblast cells are comprised within a fibrin support matrix formed by thrombin-mediated polymerization of a fibrinogen and said dermal fibroblasts followed by incubation of said fibrin support matrix at about 37° C. for about 16-24 hours after formation of said matrix, and
wherein said composition has a shelf-life of at least 7 and up to 28 days, when stored at about 2° to 8° C.
2. The wound healing composition of claim 1 , in which the matrix is non-pyrogenic and/or sterile.
3. The wound healing composition of claim 1 , in which the matrix is solid or semi-solid.
4. The wound healing composition of claim 1 , in which the cells are mammalian.
5. The wound healing composition of claim 1 , in which the cells substantially exclude keratinocytes.
6. The wound healing composition of claim 1 , in which the cells exhibit, at less than 14 days, a 360-fold higher mean level of expression of ApoD; a 102,000-fold higher mean level of expression of MMP2; a 800 to 4370-fold higher mean level of expression of Coll3a1; or a 14,100-fold mean higher level of expression of SMA relative to the level of expression of RPL32.
7. The wound healing composition of claim 1 , in which the cells are suspended substantially uniformly within the matrix.
8. The wound healing composition of claim 1 , in which the matrix has a protein concentration in the range of about 3 to 12 mg.ml −1 .
9. The wound healing composition of claim 1 , in which the cells are human.
10. The wound healing composition of claim 1 , in which fibroblasts comprise between about 90% to 100% of the cells of said composition.
11. The wound healing composition of claim 1 , in which the cells are not proliferating or not senescent.
12. The wound healing composition of claim 1 , further comprising a protease inhibitor.
13. The wound healing composition of claim 12 , in which the protease inhibitor is aprotinin or tranexamic acid.
14. The wound healing composition of claim 1 , in which the composition has a thickness of approximately 8 mm or less.
15. The wound healing composition of claim 14 , in which the composition has a thickness of approximately 5 mm or less.
16. The wound healing composition of claim 1 , comprising about 450 to 2500 cells per mm 2 .
17. The wound healing composition of claim 1 , in which the composition is single-layered.
18. The wound healing composition of claim 1 , in which the composition is packaged in a container suitable for transporting the composition, storing the composition, or topically applying the composition to a skin surface.
19. The wound healing composition of claim 18 , in which the container comprises a flexible pouch consisting of two sheets of impermeable flexible material peripherally sealed to provide a means of containment for the composition, the pouch comprising a first internal surface to which the composition is adherent at a level of adhesion more than between the composition and a second internal surface of the pouch but less than that between the composition and the skin surface, such that in use the pouch may be opened by parting the sheets and the composition conveniently manipulated and directly applied to the skin surface without further requirement for the composition to be touched directly by any other means prior to application.
20. The wound healing composition of claim 1 , wherein said wound healing composition is a topical composition.
21. The wound healing composition of claim 1 , wherein 90-99% of the cells of the composition have a banding pattern of polymerase chain reaction (PCR) products resulting from differential display identical or similar to that shown in FIG. 4 or FIG. 5 for nucleic acid expression in fibrin.