PRODUCTION OF XENOGRAFT FROM ANIMAL BONES

Abstract

A method for producing xenogeneic bone graft, which is a material for use in bone tissue therapy, is provided. The method includes the steps of isolating the cancellous bones from cartilage and cortical bone by fragmenting them into pieces, washing the bone fragments with purified water to partially remove the organic phases and boiling them with purified water, contacting the bone fragments with a solvent for further removal/isolation of the organic phase, washing the bone fragments to remove the solvents used and other possible residues, subjecting them to a hydrothermal and/or solvothermal treatment with a solvent at a pressure of more than 1 atm and a temperature in the range of 100° C. to 300° C., after which washing them with purified water, and drying the cancellous bone fragments, for example, at a temperature in the range of 50° C. to 100° C. to dehydrate them.

Claims

1. A method for producing xenogeneic bone grafts, comprising subjecting cancellous bones to a hydrothermal and/or solvothermal treatment with a first solvent at a pressure of more than 1 atm and a temperature in a range of 100° C. to 300° C.

2. The method according to claim 1, wherein the first solvent is selected from the group consisting of organic solvents, inorganic solvents, ionic solutions, and mixtures of the organic solvents, the inorganic solvents, and the ionic solutions.

3. The method according to claim 2, wherein the first solvent is selected from purified water, sodium hypochlorite, chloroform, acetone, or mixtures of the purified water, the sodium hypochlorite, the chloroform, and the acetone.

4. The method according to claim 1, wherein the hydrothermal and/or solvothermal treatment is continued for a period of 6 hours to 24 hours.

5. The method according to claim 1, comprising the following steps in sequence, prior to the hydrothermal and/or solvothermal treatment: a) obtaining cancellous bone fragments by crushing an animal bone with a cancellous tissue; b) partially removing of organic phases in the cancellous bone fragments; c) further removing/isolating of the organic phases of the cancellous bone fragments; and d) washing the cancellous bone fragments.

6. The method according to claim 5, wherein in the step (b), the cancellous bone fragments are washed with a purified water with a first number of repetitions in a range of 10 to 15 times for a period of 1 hour to 2 hours and boiled with the purified water with a second number of repetitions in a range of 3 to 5 times for a period of 30 minutes to 60 minutes.

7. The method according to claim 5, wherein in the step (c), the cancellous bone fragments are contacted with a second solvent capable of dissolving organic components for a period of 7 days to 15 days.

8. The method according to claim 7, wherein the second solvent being able to dissolve the organic components is selected from organic solvents, inorganic solvents, ionic solutions, and mixtures thereof the organic solvents, the inorganic solvents, and the ionic solutions.

9. The method according to claim 5, wherein the washing in the step (d) is repeated with a purified water with a repetition number in a range of 10 to 15 times for a period of 1 hour to 2 hours.

10. The method according to claim 5, comprising the following steps in sequence after the hydrothermal and/or solvothermal treatment: e) washing the cancellous bone fragments; and f) drying the cancellous bone fragments.

11. The method according to claim 10, wherein the washing 9 in the step (e) is performed with a purified water for a period of 1 day to 7 days and repeated 3 or more times.

12. The method according to claim 10, wherein the drying in the step (f) is carried out at a temperature in a range of 50° C. to 100° C. for a period of 1 day to 2 days.

13. The method according to claim 2, wherein the hydrothermal and/or solvothermal treatment is continued for a period of 6 hours to 24 hours.

14. The method according to claim 3, wherein the hydrothermal and/or solvothermal treatment is continued for a period of 6 hours to 24 hours.

15. The method according to claim 6, wherein in the step (c), the cancellous bone fragments are contacted with a second solvent capable of dissolving organic components for a period of 7 days to 15 days.

16. The method according to claim 11, wherein the drying in the step (f) is carried out at a temperature in a range of 50° C. to 100° C. for a period of 1 day to 2 days.

17. The method according to claim 6, comprising the following steps in sequence after the hydrothermal and/or solvothermal treatment: e) washing the cancellous bone fragments; and f) drying the cancellous bone fragments.

18. The method according to claim 7, comprising the following steps in sequence after the hydrothermal and/or solvothermal treatment: e) washing the cancellous bone fragments; and f) drying the cancellous bone fragments.

19. The method according to claim 8, comprising the following steps in sequence after the hydrothermal and/or solvothermal treatment: e) washing the cancellous bone fragments; and f) drying the cancellous bone fragments.)

20. The method according to claim 9, comprising the following steps in sequence after the hydrothermal and/or solvothermal treatment: e) washing the cancellous bone fragments; and f) drying the cancellous bone fragments.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings, whose brief description is provided below are just given for better understanding of the present invention and as such, are not intended to determine the scope of the claimed subject matter, in the absence of the description.

[0015] FIG. 1 is a schematic diagram representing a process step of the present invention and the process steps that can be carried out before and after this process step.

[0016] FIG. 2 is a schematic sectional view of an exemplary autoclave reactor suitable for performing the hydrothermal/solvothermal treatment step of the present invention.

[0017] Reference numerals: 10: bolt; 20: filter pad; 30: holder; 40: coupling cover; 50: cover; 60: liner; 70: outer shell; 80: solvent(s); and 90: base.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0018] In the prior art, the problems of the methods used in the production of xenogeneic bone grafts (an increase in the crystallinity of the materials produced due to the heat treatment at high temperatures and consequently the lack of biodegradation in the desired period, the emergence of the TCP (tri calcium phosphate) phase due to the dehydroxylation of the hydroxyapatite in the structure of the bone during the heat treatment process and the degradation of the natural bone structure, a lower Ca/P ratio as compared to the natural bone resulting from the heat treatments, etc.) are overcome by the method of the present invention having a hydrothermal/solvothermal treatment step, in addition to other chemical and mechanical processes. With the hydrothermal/solvothermal treatment, a xenogeneic bone graft may be obtained, which preserves the structure of the natural hydroxyapatite crystal and has rapid biodegradation. The method of the present invention comprises the following process step: [0019] i) subjecting the cancellous bones to a hydrothermal/solvothermal treatment with a solvent at a pressure of more than 1 atm and a temperature in the range of 100° C. to 300° C.

[0020] Said process step may be regarded as hydrothermal when water is used as a solvent, and as solvothermal when another solvent is used. If one or more solvents are present in the process, it can be referred to as a hydrothermal and solvothermal treatment step. Suitable solvents for the hydrothermal/solvothermal treatment step may include various solvents such as water (preferably purified water), sodium hypochlorite, chloroform, acetone, and other organic, inorganic, and ionic solutions (salt solutions, etc.), or a mixture thereof. In other words, suitable solvents for the hydrothermal/solvothermal treatment step may be selected from the following: organic solvents, inorganic solvents, ionic solutions, and mixtures thereof. The hydrothermal/solvothermal treatment is preferably continued for a period of 6 hours to 24 hours. In the hydrothermal/solvothermal treatment step, the grain size does not change, because high temperatures that have to be reached in the prior art are avoided, and the natural crystalline structure, dimensions and porosity of the cancellous bone fragments can be preserved, without degradation.

[0021] By adapting the above-described step (i) instead of the heavy chemical processes and/or the process steps requiring high temperatures in the prior art methods, a method allowing production of xenogeneic grafts from animal bones with a cancellous structure may be thoroughly designed by a skilled person in the art.

[0022] Preferably, the present invention may comprise the following steps in sequence, prior to step (i): [0023] a) Obtaining cancellous bone fragments by crushing an animal bone with cancellous tissue (e.g., head of femoral bone-femur head). Accordingly, the cancellous part of the bone can be isolated from the cartilage and cortical bone, so the cancellous bone fragments, preferably in the order of 0.5-1.5 centimeters, can be obtained. [0024] b) Partial removal of the organic phases in the cancellous bone fragments (e.g., partial removal/isolation of fats, blood, and other unwanted organic phases). Accordingly, for example, the cancellous bone fragments may be washed with purified water 10-15 times for 1-2 hours and boiled 3-5 times with purified water for 30-60 minutes. [0025] c) Further removal/isolation of the organic phase of the cancellous bones. Accordingly, the cancellous bones may be contacted with a solvent capable of dissolving organic components (e.g., proteins, fats, DNA, cells, etc.); this step may be referred to as “chemical treatments step” and “decellularization”. In this step, they are contacted with the solvents that will dissolve the organic phase (fats, proteins, etc.) and remove it from the cancellous bones, such as sodium hypochlorite, chloroform, acetone, or mixtures thereof, additionally or also other organic, inorganic and ionic solutions (salt solutions, etc.) or mixtures thereof, for a period of e.g. 7-15 days, and it is preferably stirred in a continuous manner. [0026] d) Washing of the cancellous bone fragments. With the washing step, the cancellous bone fragments are removed from the solvent(s) mentioned in step (c) and other possible residues. Said washing process may be repeated 10-15 times for 1-2 hours, for example with purified water.

[0027] In addition to the above steps, the present invention may comprise the following steps in sequence, after step (i): [0028] e) Washing of the cancellous bone fragments. Thus, any residues that may still present on the cancellous bone fragments are removed. Said washing process may be carried out, for example, with water, e.g., purified water. The washing process may be repeated for a period of e.g., 1 to 7 days, and e.g., 3 or more times. [0029] f) Dehydration of the cancellous bone fragments by drying. Said drying process can be carried out, for example, at a temperature in the range of 50° C. to 100° C., and for a period of time in the range of for example 1 to 2 days.

[0030] FIG. 1 is a diagram depicting the possible steps of the method according to the invention. In step 1, the original version of the head of an exemplary animal femoral bone is illustrated. Step 2 represents the isolation of the cancellous bone from the tendon, cartilage, and cortical bone by fragmenting the head of the femoral bone into small pieces. Step 3 represents the washing of the cancellous bone fragments several times in purified water and boiling them with purified water, in order to partially defat same. In step 4, the decellularization of the cancellous bone fragments by means of a chemical process and the removal of the organic residues such as fat, etc. is represented.

[0031] Step 5 represents further removal of the chemical residue and dissolved organic materials by washing the cancellous bone fragments with purified water. Step 6 is directed to the hydrothermal/solvothermal treatment step of the method according to the present invention, wherein it is represented that the bone fragments are treated at a temperature in the range of 100° C. to 300° C. and under a pressure above 1 atm, preferably for a period of 6 hours to 24 hours. In step 7, the washing process (e.g., with purified water) is represented to remove any organic excess that is likely to form residue on the cancellous bone fragments after the hydrothermal/solvothermal treatment step. In step 8, it is represented to obtain xenogeneic bone grafts with preserved natural crystalline structure and porosity by drying the obtained purified cancellous bone fragments.

[0032] FIG. 2 is a schematic sectional view of an exemplary autoclave reactor suitable for performing the hydrothermal/solvothermal treatment step of the present invention. A known commercially available device can be used as a reactor in the hydrothermal/solvothermal step. For example, as of the date of application, it is available from TOPTION under the name of “Teflon lined hydrothermal autoclave reactor” (see FIG. 2, source: https://www.toptiontech.com/info/how-to-use-hydrothermal-autoclave-reactor-9587371.html). Said reactor may comprise a liner (60) surrounded by an outer shell (70) and a base (90), and acting as a reservoir through which one or more solvents (80) are brought into contact with the cancellous bone fragments. The liner (60) may be made of PTFE, for example. There may be a coupling cover (40) to cover an inlet opening located on the liner (60), and a holder (30) that grips it. The coupling cover (40) may preferably be made of the same material as the liner (60) so that it does not corrode the liner. In order to tightly couple the holder (30) with the outer shell (70) against the high pressures that can be reached in the hydrothermal/solvothermal treatment step, the reactor may include a cover (50) that surrounds the holder (30) and the outer shell (70) from the outside, and that is attached to the holder (30), for example by means of a bolt (10), and to the outer shell by means of a threaded perimeter. A filter pad (20) may be located between the bolt (10) and the holder (30). In order to be resistant to high pressures, the holder (30), the cover (50), the outer shell (70) and the base (90) may be made of steel.

[0033] The hydrothermal/solvothermal method may also be performed at temperatures above 300° C. if the reactor is not PTFE-lined, but higher temperatures are not required as the temperatures in the range of 100° C. to 300° C. yield excellent results. Therefore, there is no obstacle to the use of the reactors that can be easily cleaned by virtue of being PTFE-lined. Thus, the method makes it easy to clean the reactor after the hydrothermal/solvothermal treatment step.

[0034] In the method, chemical processes applied on the cancellous bone fragments to remove proteins and other organic components are both less in number and less damaging. In addition, the reactor temperatures in the hydrothermal/solvothermal treatment step (step i) forming the basis of the method are lower than those required by the prior art methods. Thus, white cancellous bone fragments (xenogeneic bone grafts) can be obtained, which preserve its natural inorganic structure and are free of organic components. The resulting cancellous bone fragments have a lower crystallinity (relatively small grain size) than those provided in the prior art.

[0035] The block and particle forms of the bone graft material to be produced using the cancellous bone fragments provided by the method of the present invention are suitable for use in dentistry and various orthopedic applications.

[0036] The xenogeneic bone graft material produced with the hydrothermal/solvothermal treatment step of the inventive method is industrially producible by suppling appropriate equipment and materials and thus is industrially applicable. Thanks to the inventive improvement, the deficiencies of the prior art have been eliminated and the aforementioned problems have been solved.