CATALYST FOR THE REGENERATION OF TISSUES AND RELATED METHOD FOR MAKING IT

Abstract

A biological catalyst for the regeneration of tissues, obtainable directly from an adipose starting material, includes adipose material in liquid solution in a quantity of between 1.0 and 1.5 grams per millilitre of adipose starting material, in which a quantity of between 2 and 30 milligrams of proteins is present, said proteins comprising at least: from 0 to 60 picograms of PDGF, from 300 to 1300 picograms of VEGF, from 10 to 100 picograms of TGFb1, from 3000 to 7500 picograms of FGFb, from 400 to 4000 picograms of IL-1 RA. The biological catalyst is obtained from a method including at least the steps of collecting an adipose starting material; centrifuging the adipose material, to separate the collected material at least into an oily fraction, an aqueous fraction and a cellular fraction; removing the surface oily fraction; collecting the aqueous fraction and the cellular fraction, in which the above-mentioned proteins are found.

Claims

1. A biological catalyst for the regeneration of tissues, obtainable from an adipose starting material, wherein it comprises adipose material in liquid solution in a quantity of between 1.0 and 1.5 grams per millilitre of adipose starting material, in which a quantity of between 2 and 30 milligrams of proteins is present, said proteins comprising at least: from 400 to 4000 picograms of IL-1 RA, an IL-1b receptor inhibitor.

2. The biological catalyst according to claim 1, wherein said proteins comprise at least: from 1 to 40 picograms of IL-4 and from 0.5 to 5 picograms of IL-10.

3. The biological catalyst according to claim 1, wherein said proteins comprise at most: from 5 to 500 picograms of IL-6, from 1 to 10 picograms of TNFa, from 0.5 to 10 picograms of IL-1b.

4. A biological catalyst for the regeneration of tissues, obtainable directly from an adipose starting material, wherein it comprises adipose material (1) in liquid solution in a quantity of between 1.0 and 1.5 grams per millilitre of adipose starting material, in which a quantity of between 2 and 30 milligrams of proteins is present, said proteins comprising at least: from 0 to 60 picograms of PDGF, from 300 to 1300 picograms of VEGF, from 10 to 100 picograms of TGFb1, from 3000 to 7500 picograms of FGFb.

5. A method for obtaining a biological catalyst for the regeneration of tissues, wherein it comprises at least the following steps: collecting an adipose starting material; centrifuging said adipose material at least once, to separate the collected material at least into an oily fraction, an aqueous fraction and a cellular fraction; removing the surface oily fraction; collecting the aqueous fraction separated from the cellular fraction.

6. The method according to claim 5, wherein the cellular fraction is treated according to the following steps: breaking down the cellular fraction; centrifuging; removing the surface oily fraction; collecting the supernatant aqueous solution; adding the supernatant aqueous solution to the aqueous fraction.

7. The method according to claim 5, wherein it comprises at least one subsequent step of filtering the aqueous fraction.

8. The method according to claim 5, wherein it comprises a subsequent step of freezing the aqueous fraction.

9. The method according to claim 5, wherein it comprises a further step of dehydrating the aqueous fraction, in such a way as to obtain the biological catalyst in freeze-dried form.

10. The biological catalyst according to claim 1, wherein it comprises freeze-dried adipose material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Further advantages and features of the invention are more apparent in the detailed description which follows, with reference to the accompanying drawings, in which:

[0027] FIG. 1 is a schematic illustration of the basic steps of the method used to obtain the invention;

[0028] FIGS. 2 and 3 show the results of in vitro tests of the invention;

[0029] FIG. 4 shows the results of in vivo tests of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

[0030] The invention consists of a biological catalyst for the regeneration of tissues, directly obtainable (that is to say, without any biological manipulation, in English enzyme free) from an adipose starting material (1). In fact, it was observed that the adipose material of many mammals contains, chiefly in the form of proteins, most of the active factors released by stem cells, and therefore also traceable in the supernatant of stem cell cultures.

[0031] By means of a method described below, a biological catalyst 10 is obtained, comprising adipose material in a liquid solution in a quantity of between 1.0 and 1.5 grams per millilitre of adipose starting material 1, containing a 2 to 30 milligram quantity of proteins. Those proteins comprise at least: from 0 to 60 picograms of PDGF, from 300 to 1300 picograms of VEGF, from 10 to 100 picograms of TGFb1, from 3000 to 7500 picograms of FGFb.

[0032] The acronyms used identify proteins which are particularly active in the repair of tissue damage, and in particular they stand for: PDGF (Platelet derived growth factor) a protein which regulates growth and differentiation of cells of mesodermal origin; VEGF (Vascular endothelial growth factor) a protein which regulates the growth of endothelial cells and angiogenesis; TGFb1 (Transforming growth factor b1) regulates growth, differentiation, adhesion, migration and other cellular functions; FGFb (Fibroblast growth factor b) regulates growth and differentiation of cells of mesenchymal origin.

[0033] The in vitro and in vivo tests demonstrate the effectiveness of this product, confirming the stimulating action of the invention on various cellular components of tissues. In fact, as shown in FIG. 2, use of the invention at a 10% concentration (Adipose Derived 10%) increased the proliferation capacity of mesenchymal stromal cells derived from human adipose tissue (AD-MSCs) and of dermal fibroblasts (SFs), relative to the controls in which the same cells have traditionally been supported using fetal bovine serum at a 10% concentration (FBS10%), or in the absence of supplements (no supplements).

[0034] A further test of the effectiveness of this product is shown in FIG. 3, which illustrates how use of the invention, at a concentration of 6 and 10%, and containing a quantity of FGFb respectively of 0.3 ng/ml and 0.5 ng/ml, increased the proliferation capacity of mesenchymal stromal cells derived from human adipose tissue (AD-MSCs) and of dermal fibroblasts (SFs) relative to the same cells in the presence of recombinant FGFb (rFGFb) used at the concentration of 0.5 ng/ml.

[0035] FIG. 4 shows how experiments conducted on mice 6, which were given subcutaneous grafts of the invention on an amorphous support 5, after an eight week interval resulted in a considerable cellular and vascular proliferation with abundant formation of collagen fibres.

[0036] Moreover, the above-mentioned proteins also comprise many molecules characterised by a strong anti-inflammatory action, accompanied by a rather small quantity of pro-inflammatory molecules.

[0037] In particular, they contain at least: from 400 to 4000 picograms of IL-1 RA, a strong inhibitor of the receptor of the inflammatory molecule IL-1b, from 1 to 40 picograms of IL-4 and from 0.5 to 5 of IL-10, which are interleukins with a strong anti-inflammatory action.

[0038] On the other hand, the same proteins only contain: from 0.5 to 10 picograms of IL-1b, the inflammatory protein opposed by IL-1RA, from 1 to 10 picograms of TNFa, also known as tumour necrosis factor, and from 5 to 500 picograms of IL-6, an interleukin with pro-inflammatory action.

[0039] It is obvious that topical administration of the invention with such high quantities of IL-1 RA allows a local availability of this cytokine at concentrations such that it performs its biological action at cellular level and effectively opposes the inflammatory diseases which the patient treated suffers from.

[0040] The method for obtaining the biological catalyst for the regeneration of tissues described above, schematically illustrated in FIG. 1, comprises first collecting an adipose starting material 1: that starting material could even be the result, for example, of liposuction performed on the same patient who is to be treated with the invention.

[0041] The adipose material 1 collected is centrifuged at least once, for separating the collected material at least into an oily fraction 2, an aqueous fraction 3 and a cellular fraction 4; since the active factors useful for the regeneration of tissues are mainly in the aqueous fraction 3 and to a certain degree in the cellular fraction 4, the surface oily fraction 2 is removed, whilst the aqueous fraction is separated from the cellular fraction 4.

[0042] Because the aqueous fraction 3 contains the most active ingredients, it is already suitable for use as a biological catalyst 10 for the regeneration of tissues. However, a better result can be achieved, by further processing the cellular fraction 4: in this case, first the cellular fraction 4 is broken up, and then it is centrifuged at least once and the surface oily fraction is subsequently removed. At this point, the supernatant aqueous solution is collected and added to the aqueous fraction 3 previously obtained.

[0043] In any case, the aqueous fraction which is obtained is subsequently filtered, to eliminate any tissue and cellular debris, as well as to remove a possible microbial contamination, and preferably frozen.

[0044] Before or after freezing, it is possible to proceed with dehydration of the aqueous fraction 3, in such a way as to obtain the biological catalyst in freeze-dried form 10: in fact, in this form the stability of the active ingredients is extended, preservation is facilitated and, at the moment of use, it can be diluted within a wide range depending on the needs of the patient.