WO1995003073A1

WO1995003073A1 - Process for preparing an osseous ceramic material and bone regenerating and growth promoting composition

Info

Publication number: WO1995003073A1
Application number: PCT/BR1994/000024
Authority: WO
Inventors: Benedicto De Campos Vidal
Original assignee: Faissol Pinto, Olympio
Priority date: 1993-07-21
Filing date: 1994-07-21
Publication date: 1995-02-02
Also published as: AU7454694A

Abstract

The present invention provides an osseous ceramic material useful in a bone regenerating and growth promoting composition, as well as a process for the preparation thereof. The invention also provides a bone regenerating composition comprising said osseous ceramic material and collagen, which composition ensures an excellent bone regeneration without any inflammatory reaction or any type of rejection from an animal organism. According to the process of the invention, the osseous ceramic is prepared from naturally occurring bones, by a specific heating procedure which maintains both the hystological microstructural distribution of the in natura bones, as well as their mineral physical-chemical properties.

Description

"Process for preparing an osseous ceramic material and bone regenerating and growth pro¬ moting composition"

The present invention refers to a composition useful for promoting bone growth and regeneration which comprises collagen and a hidroxyapatite material from bones, the compo¬ sition presenting a molecular order and a microstructural reg¬ ister similar to natural bones. This characteristic enables the composition of the invention to provide excellent results in promoting the bone growth without causing any negative re¬ action in the organism.

BACKGROUND OF THE INVENTION

Compositions for osseous repair and regeneration comprising collagen complexes and osseous material are; already known from the prior art.

Patents US 4,472,840, US 4,394,370, EP 012,959 and US 4,440,750, for example, describe compositions useful for promoting bone growth which comprise collagen and products de¬ rived from bones. However, according to the teachings of these documents, the products derived from bones used in such com¬ positions should necessarily be submitted to a demineralization process in order to obtain good results.

Other documents known from the prior art disclose compositions which also provide bone growth or regeneration but should comprise collagen and hidroxyapatite in addition to other essential components to yield an effective bone repair without causing any kind of rejection. An example is patent US 4,911,641 which teaches a composition for promoting bone growth containing, as essential ingredients, hidroxyapatite materials of two different sizes, a source of collagen, tetracycline which is necessary, among other reasons, for its antibiotic effect and to guarantee the product integrity dur¬ ing storage, and fibronectin to adhere to and orient the new osseous cells.

Another example of such compositions is disclosed in US 4,780,450 and refers to a composition comprised of natural or synthetic hidroxyapatite in a mixture with collagen and a phosphophoryn calcium salt.

A further example which can be included in this group is described in patent US 4,794,046 and comprises an osseous ceramic material of hidroxyapatite prepared by pres- sure molding a laminated body consisting of a layer of net made of natural or synthetic fibers and a layer of natural or synthetic powdery hydroxyapatite.

Patent US 4,795,467 teaches a composition for bone repair containing hidroxyapatite and collagen, in which the hidoxyapatite should be from mineral origin because, according to this document, it would not be possible to obtain good re¬ sults if collagen protein was used together with bone minerals since, on the one hand, it would be very difficult to obtain appropriate physical characteristics for provide a matrix for the bone growth and, on the other hand, inflammatory manifes¬ tations might occur as a result of the immunogenic character of the implant, unless the proteic component is derived from a tissue from the same individual who would receive said im¬ plant. Still a further composition for osseous regeneration is know from US 4,623,553 and comprises collagen and sintered apatite. However, to obtain the desired results the collagen should be submitted to a partial cross-linking process with aldehydes prior to be admixed to the sintered apatite. It is, therefore, one object of the present in¬ vention to provide an osseous ceramic material obtained from bones which maintains all the microstructural and mineral physical-chemical characteristics of a bone in natura, that is to say which does not contain proteins and/or any other or- ganic product, whereby it permits the use of material obtained directly from bones in compositions for osseous growth and re¬ pair, without showing any one of the common inconveniences known from the prior art. A further object of the present invention is to pro¬ vide a composition essentially comprising an osseous material and collagen which ensure a bone regeneration without inflam¬ matory reactions or any rejection, as well as a process for the preparation thereof.

SUMMARY OF THE INVENTION

The osseous ceramic material of the invention is prepared by a process which comprises grinding the bones, cleaning the bone particles to remove fat and any other or¬ ganic material and heating the clean bone particles, wherein the heating step comprises an initial step of gradual heating up to a temperature of about 200°C during approximately 1 hour, followed by a plurality of additional heating steps, these additional steps being separated from each other by in¬ tervals of constant temperature, until a final temperature of about 1200°C, whereby the osseous ceramic thus obtained pre¬ sents a hystological microstructural distribution and mineral physical-chemical properties substantially similar to those of the bones from which it was obtained.

The invention also provides a composition for pro- moting the osseous regeneration containing the above osseous ceramic material and collagen. The organism cells to which such composition is applied recognize therein the biological microstructural registers in terms of molecular order and this fact is responsible for the stimulation of the bone growth. DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a process for pre¬ paring a hidroxyapatite ceramic material, hereafter named "osseous ceramic", which process essentially comprises the following steps: a) triturating or grinding the bones; b) cleaning and treating the bone particles obtained in step (a) in order to remove fat and any other residue of organic matter; c) heating the fat-free bone particles up to a temperature of 1200°C.

The bones useful as raw material for the osseous ce¬ ramic of the present invention are preferably long bones, such as bovine bones, and long-bone ends which contain spongeous bone material.

In step (a), the bones should be ground into parti¬ cles of an appropriate size to allow the separation of the ends which contain spongeous matter and which may also be used for preparing the ceramic. The resultant particles are then treated for removing fat and all organic matter using proce¬ dures well known from the art. Such procedures include, for example, washing the bone particles with inert soap and water and treating the washed bones with solutions of hypochlorite, hydrogen peroxyde and sodium hydroxide for removing fat. Then the bones are usually treated with solvents such as ethanol and acetone, and finally dried.

In a next step, after the bone particles are com¬ pletely clean and free from fat the heating step is carried out. In the first hour, the temperature is elevated to about 200°C, the heating velocity being controlled in such a way that the temperature of 200°C is achieved only in the end of the first hour. At this moment, the temperature is increased to 1200°C, wherein this additional heating step comprises in fact a plurality of heating steps being separated from each other by intervals of time, in which the temperature is kept constant. Preferably, in each of said additional heating steps the temperature is increased of 200°C and each of the men¬ tioned intervals is of about 2 hours. According to a preferred embodiment of the in¬ vention, in the end of the first heating hour, that is to say when the temperature reaches 200°C, the gradual heating is continued so that the temperature is 400°C in the end of the second heating hour. The material is then maintained at this temperature for 2 hours and, after these 2 hours at 400°C, the temperature is increased to approximately 600°C and this tem¬ perature is kept constant for about 2 hours. Then the temper¬ ature is elevated to 800°C and maintained constant for more 2 hours. At this stage the temperature is increased to 1000°C where the material is kept for a period of 1 hour, after which the temperature is finally increased to 1200°C. It is essen¬ tial that the temperature does not go beyond 1200°C since higher temperatures would destroy the osseous molecular ar- rangement.

Preferably, the ceramic material remains at 1200°C for 3 hours and, then its allowed to cool very slowly. After cooling the ceramic material, it is possible to effect the me¬ chanic grinding thereof in order to obtain an appropriate granulometry to allow the ceramic to be used in osseous repair compositions.

It has been found that in the ceramic material produced according to the process of the invention, the micro- anatomy of the molecular order and distribution of the crys- tals (minerals) is maintained. For this reason, the osseous ceramic of this invention is particularly useful for preparing compositions for promoting bone growth and regeneration. Such compositions comprises a complex of the osseous ceramic and collagen, especially Type I collagen. It is known that, the crystalline portion of bones presents precise relations of order and surface complementarity, which up to now had not been achieved in syn¬ thetic products. In the osseous ceramic-collagen complex of the invention, both the lamelar structures and the osteoplastic spaces of the bones are maintained, which spaces are filled by the collagen. When the osseous ceramic-collagen complex is applied or grafted to the bone, the collagen acts as a "carrier" for the cellular growing, whereby the new cells which are responsible for the osseous regeneration penetrate into the above referred spaces.

In the preparation of the composition containing osseous ceramic and collagen, it is possible to use collagen both in the form of a solution or in the form of a gel. The compacting degree and the biomechanic characteristics of the composition depends on the specific objective aimed by the specialist skilled in the art and are a function of the polymerization degree obtained during the process of reagregation of the collagen fibers used in the composition. An example of collagen useful for the present in¬ vention may be prepared from bovine tendons. The tendons are washed and made free from fat by using, for example, sodium hypochlorite and hydrogen peroxyde.

The resultant material is then solubilized in a acidic solution such as an acetic acid or hydrochloric acid solution, together with a proteolytic enzyme, for example a protease, which dissolves the residual collagen. A collagen in the form of a solution is then obtained.

If a collagen in the form of a gel is desired, a first extractive step is carried out after the above solution has been obtained, during which step the tropocollagen mole¬ cules - the collagen units in which the fibers are not aggre¬ gated yet - are separated. These tropocollagen molecules are solubilized and a transparent solution of Type I collagen is obtained.

The fibers thus reconstituted are submitted to an aggregation step and the resulting fibers present a molecular and aggregation order similar to natural collagen and this characteristic is very important for the future cellular re- cognizance and adherence. The step of reconstituting the fi¬ bers is initiated by the addition of a chemical agent, preferably NaCl, which increases the pH of the collagen sol¬ ution and permits the reassociation of the fibers. This step is carried out at low temperatures, preferably in a range of 8 to 10°C, and the fibers remain in the solution for 20 to 30 hours in order to achieve an optimum stage of aggregation and molecular order, which stage is detectable by the anisotropic properties - intrinsic textural birefringence and linear pleochroism. The above aggregation step is followed by a dialysis process to remove the chemical agent used to aggregate the fi¬ bers and to make it possible to continue said aggregation at the same time of carrying out the hydration of the fibers. The required time for the dialysis, as well as the size of the tubes used in this process, depend upon the type of the de¬ sired gel. If a tube with a small diameter is used for a small period of time, for example 72 hours, the resultant reassoci- ated fibers form an almost transparent, high viscosity but non-rubbery gel. On the other hand, if the same tubes are used for a longer time, for example for 8 days, a rubbery fibrous gel is obtained, which can even be "cut". Although such a gel has stronger mechanic characteristics than the for¬ mer mentioned gel, it can also be handled, added to and/or complexed with other substances or products. In both cases, if larger tubes are employed it is usually necessary to wait more 2 or 3 days for achieving results similar to those obtained with tubes of smaller diameter.

In addition to the above cited gels, it is also pos- sible to obtain a highly polymerized collagen gel by effecting a gamma-ray polymerization of the reconstituted and dialyzed collagen fibers. The final product is in the form of injectable crystalline granules.

As previously mentioned, the composition of the present invention comprises a ceramic product of hidroxyapatite prepared according to the process defined above and collagen in the form of solution or gel.

When a collagen solution is employed, the osseous material, which may be in the form of fragments or powder, is added to the collagen solution preferably immediately after the ceramic has been prepared. If ceramic fragments are used they usually have a volume of 0.5 to 3 cm . In this case, the mixture is submitted to vacuum so that the tropocollagen sol¬ ution can penetrate into the voids which were previously occu- pied by the organic matter of the bones. The ingredients are further mixed, always under vacuum, until an excess of collagen solution is detected. In such conditions, the whole mineral surface of the osseous ceramic interacts with the collagen forming a highly organized complex, reconstituting a biological structure which will be recognized by the animal organism.

In the event a powdery osseous ceramic is used, its preferred that the particle size varies in a range of 2.5 to 1000 um and that they are added to the collagen solution under gentle stirring. The osseous ceramic-collagen complex settles when the stirring is interrupted.

The inventor has found that the cells (osteoblasts) of the regions to which a composition prepared in accordance to the above cited procedure is applied recognize the collagen inserted in the osseous ceramic, adhere thereto and begin to multiply, promoting the repair of osseous wounds and losses.

If desired, the osseous ceramic-collagen composition may be further submitted to a dialysis process, always main- taining an excess of collagen, followed by a radiation treat¬ ment, for example with gamma rays until 2000 R. The object of such treatment is , on the one hand, to promote higher polymerization and aggregation of the collagen and, on the other hand, to sterilize the final product. In another embodiment of the invention, the composi¬ tion is prepared by using collagen in the form of a gel, which can be a fibrous gel or a rubbery gel. According to this em¬ bodiment, powdery osseous ceramic is preferably used with par¬ ticle sizes varying from 250 to 1000 um, said ceramic powder being added to a layer of the collagen gel usually in a ratio of 1:1 to 1:2, based on the weight of the collagen. The re¬ sultant composition is mechanically mixed and can be further submitted to a sterilization step by the use of gamma rays. Finally a thick mass is obtained which can be easily cut and manipulated for use in the treatment of bone failures or cavi¬ ties. If a rubbery collagen is employed, the final product presents excellent mechanic characteristics appropriate for osseous failures of large proportions.

It is observed that a composition according to the present invention comprises an osseous ceramic and collagen as its essential components. However, such a composition may also contain optional ingredients such as antibiotics, hormones, vitamins, hydration agents, absorbents, chemotherapeutic agents, etc. The invention can be better illustrated by the fol¬ lowing examples, which however should not be considered as limitative thereof.

Example 1

Preparation of the osseous ceramic

Long bovine bones were cut into 2 cm pieces, which were washed with tap water and common odorless detergent and further washed with filtered water.

The material was introduced in a hypochlorite sol- ution for 12 hours and then washed again with fresh water, im¬ mersed in a 2% hydrogen peroxyde solution where it was allowed to rest for 12 hours. The bones fragments were then boiled in a 2% solution of NaOH for 3 hours and washed with water.

The obtained white, fat-free material was dried and immersed in a mixture of 3 parts of 100% ethanol and 1 part of acetone for 24 hours, during which time the material was stirred from time to time.

After being dried, the final material was placed in an oven where the temperature were gradually increased as fol- lows:

1st hour - increased to 200°C;

2nd hour - increased to 400°C, the material remaining at this temperature for 2 hours; 4th hour - increased to 600°C, remaining at this temperature for more 2 hours;

6th hour - the temperature reaches 800°C, at which temperature the material remains also for 2 hours; 8th hour - increased to 1000°C, the temperature being kept constant for 1 hour.

Finally, after 9 hours, the temperature was in¬ creased to 1200°C and kept constant for 3 hours. The oven was then turned out and the material was allowed to cool. When completely cold, the material was submitted to a gentle me¬ chanic grinding which yield particles from 200 to 1000 um.

Example 2 Preparation of a collagen solution

Bovine tendons were cleaned, whereby the residues of blood and fat were removed, and treated with a 1% solution of sodium hypochlorite for 10 hours. They were then washed with 5 distilled water, immersed in hydrogen peroxyde 1-2% for about 5 hours and washed with water. The temperature was maintained between 8 and 10°C during the whole cleaning procedure.

The material thus obtained was placed in acetic acid solution 0.5 M together with a protease in a ratio of 1 mg of 10 the enzyme to each gram of collagen fragment. A collagen sol¬ ution (A) was thus produced.

Example 3

Preparation of a collagen gel

Fibrous gel

15 Firstly a collagen solution was prepared as de¬ scribed in Example 2.

After filtration of the collagen solution, NaCl 20% was added thereto so that a final concentration of 10% of NaCl is achieved. Upon the addition of NaCl, collagen fibers were

20 formed in the starting solution, which was maintained at a temperature of 8 to 10°C for a period ranging from 4 to 5 hours in which the aggregation of fibers were continued. After this time, the original bath was replaced by a fresh solution of NaCl 5% where the fibers remained for a further period of

25 20 hours at a temperature of 8-10°C.

In the next step, the formed fibers were submitted to a dialyisis process against distilled water, at a temper¬ ature about 8-10°C by employing small dialysis tubes. The process was carried out for 72 hours, with the distilled water

30 being changed at each period of 12 hours in a ratio of 1 liter of water to 100 g of collagen.

At the end of the dialysis process a fibrous collagen gel was obtained.

Rubbery gel The same procedure of the preceding example was re¬ peated, with exception of the dialyisis step which was carried out at 10°C for a period of 8 days.

Example 4

Preparation of the composition of osseous ceramic and collagen in the form of solution

Powdery osseous ceramic prepared according to the above process and comprising particles ranging from 250 to 1000 um was gradually added to an excess of the solution of tropocollagen in acetic acid 0.5M of Example 2, under gentle stirring.

When the stirring was interrupted, the osseous ce¬ ramic settled and the supernatant was analysed. Since no collagen was detected therein, more tropocollagen solution was added until an excess of collagen was found in the supernatant, which showed that the complexing reaction was finished and the osseous ceramic-collagen complex was ready for use.

Example 5

Preparation of the composition of osseous ceramic and collagen in the form of a gel

A fibrous collagen gel was prepared as described in Example 3 and, immediately after the dialysis step, the osseous ceramic was placed onto a layer of this collagen gel, in equal parts based on the weight of collagen, and the re¬ sulting material was mechanically mixed until a milky and white mass was formed, which showed that the complexing re¬ action of the osseous ceramic and collagen was completed.

In a later step, the complex was subjected to a gamma-radiation treatment and, as a result, a rubbery mass was obtained, which could be cut and used for filling bone fail¬ ures.

Although the present invention has been described in relation to the parameters and conditions regarded as giving the optimum results, both relating to the osseous ceramic as well as to the bone regenerating and growth promoting composi¬ tion, it must be observed that the invention encompasses every embodiment which make it possible to prepare an osseous mate¬ rial from natural bones, which maintains the microanatomy and physical-chemical property of the minerals of the original bones used as the starting material, thereby overcoming the inconveniences of the analogous materials already known from the prior art.

Claims

1 - Process for preparing an osseous ceramic mate¬ rial useful in a bone regenerating and growth promoting compo¬ sition, said process comprising grinding bones to produce bone particles, cleaning said bone particles to remove fat and any other organic material and heating the thus cleaned bone par¬ ticles, characterized in that the heating step comprises an initial step of gradual heating up to a temperature of about 200°C during approximately 1 hour, followed by a plurality of additional heating steps, these additional steps being sepa¬ rated from each other by intervals of constant temperature, until a final temperature of about 1200°C, whereby the osseous ceramic thus obtained presents a hystological microstructural distribution and mineral physical-chemical properties substan- tially similar to those of the bones from which it was ob¬ tained.

2 - Process according to claim 1, characterized in that a first of said additional heating step comprises the continuation of the said initial step of gradual heating in order to achieve about 400°C in the end of the second hour.

3 - Process according to claim 1 or 2, characterized in that each of said additional heating steps increases the temperature of about 200°C and each of said intervals of con¬ stant temperature is of about 2 hours. 4 - Process according to any of claims 1 to 3, char¬ acterized in that the material is maintained at 1200°C for about 3 hours.

5 - Process according to any of claims 1 to 4, char¬ acterized in that the final ceramic material is allowed to cool slowly down to room temperature.

6 - Process according to claim 5, characterized in that it includes a further step of grinding the final ceramic material.

7 - Process according to claim 6, characterized in that the material is ground into particles, the size of which ranges from 250 to 1000 um.

8 - Bone regenerating and growth promoting composi¬ tion comprising collagen and an osseous ceramic material, characterized in that the osseous ceramic material presents hystological microstructural distribution and mineral physical-chemical properties similar to those of the bone from which it was obtained.

9 - Composition according to claim 8, characterized in that the osseous ceramic material is obtained by a process as defined in any one of claims 1 to 7.

10 - Composition according to claim 8 or 9, charac¬ terized in that the collagen is used in the form of a sol¬ ution. 11 - Composition according to claim 8 or 9, charac¬ terized in that the collagen is used in the form of a gel.

12 - Composition according to claim 11, character¬ ized in that the weight ratio of the osseous ceramic material to the collagen gel is of 1:1 to 1:2 13 - Composition according to any one of claims 8 to

12, characterized in that the collagen is prepared from bovine tendons