DECELLULARIZED BONE BIOMATERIAL ENRICHED WITH A HYDROGEL CONTAINING DECELLULARIZED EXTRACELLULAR BONE MATRIX

Abstract

It is a biomaterial developed from decellularized animal bone tissue and coated with a bone extracellular matrix in the form of a gel, which is capable of conferring efficient mechanical and biological support, and which can also be enriched with cells, nanocomposites or drugs, when used as a bone graft, bioreactor, or vehicle in treatments, research and development of other biomaterials; That is, it was developed from decellularized, lyophilized, porous and rigid, manipulable, safe and non-immunogenic bone material, coated and enriched with stimulating substances specific to bone tissue, presented/used in particulate or block form, thus possessing ability to promote the development of in vitro mature or progenitor cell lines when used as a bioreactor, and demonstrates a high integration capacity and a faster rate of fracture healing and filling of bone defects when used as an in vivo graft; The biomaterial also allows the promotion of cellular development, from the maintenance of the integrity of the organic extracellular matrix of the bone tissue, being able to improve healing time, reduce costs and contribute scientifically to basic research demonstrating the biotechnological importance, the investigative and applicability of decellularized organic matrices in biomaterials.

Claims

1. A bone preparation comprising a bone extracellular matrix hydrogel and a decellularized bone biomaterial obtained from an animal or human bone from an adult, young, neonate or fetal subject, wherein said bone biomaterial is decellularized by a chemical and enzymatic decellularization process, after several stages of cleaning, sorting, grinding, immersion, washing, drying, freeze-drying and freezing.

2-11. (canceled)

Description

[0037] The decellularized bone biomaterial enriched with bone extracellular matrix hydrogel can be better understood through the detailed description in accordance with the following attached figures wherein:

[0038] FIG. 01 shows pictures of fragments of bone tissue before (left) and after immersion in solution (right), during the decellularization process of the decellularized bone biomaterial enriched with bone extracellular matrix hydrogel.

[0039] FIG. 02 shows pictures of lyophilized particles (left) and hydrogel (right) of decellularized bone matrix, of decellularized bone biomaterial enriched with bone extracellular matrix hydrogel.

[0040] FIG. 03 shows steromicroscopy images of the decellularized bone biomaterial with magnification of 8 (a), 12.5 (b) and 20 (c), of the decellularized bone biomaterial enriched with bone extracellular matrix hydrogel.

[0041] FIG. 04 shows scanning microscopy images of the decellularized bone biomaterial at the magnifications of 50 (A), 100 (b), 200 (c) and 350 (d), of decellularized bone biomaterial enriched with bone extracellular matrix hydrogel.

[0042] In accordance with the above figures it can be seen that the decellularized bone biomaterial enriched with decellularized bone extracellular matrix hydrogel corresponds to a natural biomaterial, developed exclusively from decellularized, lyophilized, porous and rigid, manipulable, safe and non-immunogenic, coated and enriched with stimulating substances specific to bone tissue bones, presented/used in particulate or bulk form, with potential to be exploited by industrial production and basic science in Health Sciences (Medicine, Veterinary Medicine and Dentistry) and Biotechnology. It has the ability to promote the development of in vitro mature or progenitor cell lines when used as a bioreactor, and demonstrates high integration capacity and greater speed of fracture healing and filling of bone defects when used as an in vivo graft.

[0043] For this, the collected animal bones are of regulated origin/certified (Federal Inspection Service of the Ministry of Agriculture, Livestock and SupplySIF/MAPA/Brazil), collected from slaughterhouse and sent to the laboratory. After cleansing and dissection of the collected bones, specific areas of the bone tissue will be selected and treated in order to preserve to the maximum the physical, biological and morphofunctional characteristics of the organic extracellular matrix and tissue present. The selected tissue will be treated by immersion in detergent solution (Triton 1-3%, Sodium Dodecyl Sulfate-SDS 0.1-2.5%, or other) under agitation of 200-500 rpm for 24-96 hours until that the material presents a quantity of sample DNA of less than 50 ng. After decellularization, the resulting solid matrix will be repeatedly washed with buffer solution (PBS pH 7.4-7.8 or other), oven dried at controlled temperature between 25-50 C. for at least 12-48 hours, then lyophilized, sterilized in ethylene oxide and preserved for gel coating.

[0044] The decellularized bone extracellular matrix gel will be produced from the same material collected. After selection of the bone tissue, the material frozen in liquid nitrogen will be ground into small fragments or until it becomes powder. Demineralization in acid solution (0.1-2.5 N HCl or other) is then performed under stirring at 200-500 rpm at room temperature for 24-96 hours and thoroughly washed with distilled water. After drying the material will be degreased in chloroform/methanol solution, under stirring at 200-500 rpm, at room temperature, for 1-3 hours, and washed with distilled water insistently. After drying, the material will be decellularized by immersion in enzymatic solution (0.01%-0.5% Trypsin and 0.01-0.2% EDTA or other) under agitation of 200-500 rpm at 37 C. for 12-48 hours, until the material has less than 50 ng sample DNA. After that time, 1% antibiotic and antifungal solution (Streptomycin/Penicillin, Gentamicin, or other) will be added under agitation of 200-500 rpm at 4 C. for 12-48 hours.

[0045] After this period, the contents will be tested in culture against contamination, lyophilized and kept in freezer 80 C. From the sterile lyophilized content, enzymatic digestion with acidic solution (HCl 0.01-0.1 N) of Pepsin 0.5-2.5 mg/mL is performed under magnetic stirring at room temperature for 48 hours-120 hours. Thereafter, the material called digested matrix is kept in a freezer 80 C. From the digested matrix, neutralization is performed by 0.05-0.5N NaOH solution and buffer solution (PBS pH 7.47.8 or other) at 4 C. For formation of the hydrogel the material is placed at 37 C. for at least 1-6 hours.

[0046] With the decellularized materials, solid matrix and gel produced, the biomaterial will be produced by immersing the solid matrix into the gel resulting from the decellularized matrix itself, so that the hydrogel fills the pores present therein and is capable of coating the entire material. Then the lyophilization and preservation of the biomaterial in freezer 80 C. can be carried out until the moment of use.

[0047] Given the description of the technology above, and given the preferred embodiments and possible implementations after the patent filing, follow in a way that does not tend to limit it, and there may be constructive variations that are equivalent without, however, escaping the protection scope of the invention.