MAMMAL-DERIVED MATRIX AS A SAFE BONE REPLACEMENT

Abstract

The invention provides acellular matrix from mammalian teeth for preparing a safe graft or bone replacement in regenerative medicine.

Claims

1. A bone graft or bone replacement comprising acellular powder of crushed mammalian teeth or bones or parts thereof, extracted with an alcoholic hydroxide and heated to a temperature between 110 and 190° C.

2. The bone graft or bone replacement of claim 1 reacting positively with an anti-RGD antibody.

3. (canceled)

4. The bone graft or bone replacement of claim 1, for use as a xenograft.

5. A method of manufacturing the bone graft or a bone replacement of claim 1, comprising steps i) providing a mammalian tooth or bone or a part thereof ii) immersing said mammalian tooth or bone or a part thereof in alcoholic solution of an alkali hydroxide; iii) washing said tooth or bone or a part thereof after immersing in said alcoholic hydroxide with a saline solution; iv) heating and drying said washed tooth or bone or a part thereof at a temperature of between 110 and 190° C.; and v) crushing said tooth or bone or a part thereof, wherein the step of crushing is performed after any one of steps i) to iv); thereby providing an acellular powder of crushed mammalian teeth or parts thereof.

6. The method of claim 5, comprising steps i) providing a mammalian tooth or bone or a part thereof, washing and crushing said tooth or bone or a part thereof; ii) immersing said crushed mammalian tooth or bone or a part thereof in alcoholic solution of an alkali hydroxide for a period of from 0.25 to 5 hours; iii) washing said crushed material after said immersing of step ii) with a saline solution; and iv) heating said crushed and washed material of step iii) at a temperature of between 120 and 180° C. for a period of from 0.1 to 3 hour.

7. The method of claim 5, wherein said part of the tooth comprises dentine.

8. The method of claim 5, further comprising a step of removing soft tissue or enamel after step i) and performing steps ii) to iv) without these components.

9. The method of claim 5, further comprising irradiation.

10. The method of claim 5, wherein said alcohol comprises ethanol.

11. The method of claim 5, wherein said alkali hydroxide comprises sodium hydroxide.

12. The method of claim 5, wherein said alcohol is aqueous ethanol, 50-90 vol. %, and said hydroxide is sodium hydroxide in a concentration of up to saturation in said aqueous ethanol at ambient temperature.

13. The method of claim 5, wherein said immersing is performed for from 0.3 to 3 hours, and said step of heating for from 0.2 to 1 hour at 150°.

14. The bone graft or bone replacement of claim 1 for use in replacing a missing bone in dental surgery, in treating fractures, or after resection of bone tumors.

15. A matrix comprising mammalian teeth or bone or parts thereof treated with an alcoholic hydroxide and heated to a temperature of between 110 and 190° C. for use in the preparation of a bone xenograft.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0013] It was shown that bone morphogenesis is induced by special peptides, found, for example, in demineralized bone or teeth (see, for example, U.S. Pat. No. 6,677,306). Accordingly, many works attempted to provide bone grafts derived from demineralized bones or teeth, or from materials treated with hydrochloric acid (see already cited EP 2 462 899 and U.S. Pat. No. 9,610,383). It has now been found that a powder obtained from mammalian teeth or parts thereof, comprising dentine and rid of cells, can advantageously serve as a safe bone graft if said teeth or parts thereof are treated with alcoholic hydroxide and heated to a temperature of 110° C. or more, such as 150° C.; it may seem surprising, as the instant conditions do not demineralize the teeth and, moreover, the conditions would supposedly destroy and/or remove the special growth peptides. The inventors believe, without wishing to be limited by any particular theory, that the instant teeth-derived material comprises residual dentine, which supports the bone regeneration. Particularly, the inventors believe that the teeth-derived material contains hydroxyapatite and residual collagen, which collagen, even if being modified by alcoholic hydroxide and by thermal treatment, exhibits osteoinductive effects and together with the hydroxyapatite also osteoconductive effects, the effects resulting from combined structural properties of the modified collagen protein and the inorganic matrix. In one embodiment of the invention, the teeth-derived material is particularly active when providing a positive reaction with antibodies to the Arg-Gly-Asp (RGD) sequence; the RGD sequence is known to be comprised in receptors for cell adhesion molecules, as well as in collagen I which is a part of dentine. In another aspect of the invention, the method of the invention is employed with mammalian bones or parts thereof, providing a superior bone graft or a bone replacement.

[0014] Based on the above findings, the present invention is directed to a composition for treating bone fractures, or for replacing a missing bone in dental surgery, or for providing bone after resection of bone tumors. The composition has no toxicity, low immunogenicity, and is free of infectious agents. Particularly, the composition is absolutely free of any infectious or toxic agents, including eukaryotic microorganisms, spores, bacteria, viruses, proteins, lipopolysaccharides, or nucleic acids.

[0015] The composition of the invention comprises teeth-derived dentine or bone-derived base treated with alcoholic hydroxide and heated to a temperature of from 110 to 190° C., for example at about 150° C. In one embodiment, the teeth-derived or bone-derived material is heated at two steps at two different temperatures. In a preferred embodiment, the invention provides a composition to be used as a bone graft, derived from mammalian teeth or bones or their parts, for example from teeth rid of enamel and cellular components, exposed to an alkali hydroxide, such as sodium or potassium hydroxide in alcoholic solution, the solution comprising a lower alcohol, optionally mixed with another organic solvent or water. Said alcohol comprises, for example, ethanol. In one embodiment, the composition of the invention positively reacts with anti-RGD antibodies.

[0016] The invention will be further described and illustrated in the following examples.

EXAMPLE

[0017] All steps were done in clean hood until blisters packaging stage. The process included grinding the teeth, for example pig teeth, and screening on 300 μm, and re-grinding the greater particles. The teeth were immersed for 40 minutes in sodium hydroxide dissolved in 70 vol % ethanol, 70 wt % NaOH+30 wt % ethanol (70 vol %), while occasionally stirring to release non-dentin material and let it float and removing said floating material with a peristaltic pump. The alcoholic hydroxide was removed, and the crushed teeth were washed with distilled water ten times and allowed to be immersed in PBS for five minutes. The PBS was removed, and the immersion was repeated until the pH of the wash liquid was between 7.5 and 8.5. The crushed and washed teeth were placed on a stainless steel tray, a layer not higher than 1 cm, and heated in an oven for 10 minutes at 180° C. and for 90 minutes at 130° C. If the loss on drying was higher than 5%, the heating at 130° C. was prolonged by 30 minutes.

[0018] Desired portions were packed into glass or plastic vials or other medical class materials. If desired, portions were transferred to gamma radiation. No microbial growth was observed in the final package. Bio compatibility testing and the cytotoxicity in vitro tests were performed, and the product complied with the requirements.

[0019] While this invention has been described in terms of some specific examples, many modifications and variations are possible. It is therefore understood that within the scope of the appended claims, the invention may be realized otherwise than as specifically described.