WO1996012509A1

WO1996012509A1 - Process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts

Info

Publication number: WO1996012509A1
Application number: PCT/EP1995/003971
Authority: WO
Inventors: Giuseppe Oliva; Sergio Maggi
Original assignee: Giuseppe Oliva; Sergio Maggi
Priority date: 1994-10-24
Filing date: 1995-10-09
Publication date: 1996-05-02
Also published as: ITTO940852A0; ITTO940852A1; AU3840595A; IT1268641B1

Abstract

Process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts, comprising, according to the invention, at least one stage of elimination of cell bodies from the bone tissue being treated, by means of at least one step of chemical treatment, consisting of placing (immersion) of the tissue in a bath of hydrogen peroxide, for a period of time between a few minutes and some hours (for example, a period of time between 10 minutes and 36 hours), so as to obtain the complete deantigenization of the bone tissue being treated.

Description

"PROCESS FOR INACTIVATING AND ELIMINATING THE ORGANIC MATRIX FROM ANIMAL BONE FOR HETEROTOPIC XENOGRAFTS"

The present invention relates to a process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts.

When, through traumatic events or as a result of pathologic effects (for example, cystic tumours), a cavity forms inside bone tissue, either human or of some other animal, a repair process takes place naturally, involving two types of tissue: the bone tissue itself and the soft tissues that envelop it.

These two types of tissue exhibit different rates of growth: the soft tissues have very rapid growth, whereas the bone tissues have very slow growth.

Soft tissues and bone grow until they are in intimate contact, and this determines arrest of the repair process. (This effect does not occur, for example, in tumoral masses, which do not respond to inhibition of growth and give rise to masses of invasive tissue, which grow until death of the host individual supervenes.)

It is often observed in dentistry that following the removal of cysts, if they are not filled with materials having what is known as a "curtain effect" (i.e. filling materials based on hydroxyapatite, bioceramics, membranes made of collagen and the like), the granulation tissues invade the cyst cavity, hampering the formation of new bone. The result is a loss of bone tissue, with consequent diminished mechanical strength of the structure.

The aforementioned materials can be divided into natural biomaterials and synthetic biomaterials .

Synthetic biomaterials are mainly derived from ceramics or agglomerates of polymers containing substances such as calcium. Their sole merit is their biocompatibility. Their main shortcoming is that, because they cannot be absorbed (i.e. they are not metabolized and converted by the patient's body), they remain permanently in the newly formed bone tissue. In consequence, as bone is a continuous fabric of various mineral substances, deposited (alkaline phosphatase) on collagen fibres, the tissue that forms is of the osteoid type, with a mechanical strength that is lower than that of natural bone.

Natural biomaterials are derived, in the main, from.the mineral skeleton of some corals and from bones, primarily of bovine origin, from which the hydroxyapatite is extracted. This is a constituent of a crystalline nature that is present in all osseous tissues. The microporosity of the corals is similar to that of the spongy structure of human bone.

Grafts of natural biomaterials have been studied. After 4-5 years, the presence of these materials can still be detected in the graft, because they give rise to the formation of osteoid tissue and not bone.

It is important to emphasize that a graft of these substances is not a graft of bone tissue, but of one component of bone tissue, namely hydroxyapatite. There have been numerous studies of the phenomenon of recrystallization of hydroxyapatite during the processes of deantigenization of bovine bone and purification of coral derivatives.

In particular, conventional processes of deantigenization are based on exposing the starting material, for example bovine bone, to phenomena of calcination at high temperatures for prolonged periods, so as to obtain hydroxyapatite alone as the final product.

The phenomenon of recrystallization of hydroxyapatite and the resultant hardness are the main reason for the lack of absorption of these natural biomaterials by the human body.

The main object of the present invention is to provide a process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts, whereby it is possible to achieve, reliably and efficiently, the inactivation and elimination of those organic components present in animal bone, whether non-human or human bone, for transplanting to another subject, which components - if transplanted to humans - can give rise to phenomena of graft rejection, although the structure of the osseous tissues of the treated material remains intact.

In particular, the present invention aims to provide a process for inactivating and eliminating the organic matrix from animal bone as specified above, which makes it possible to obtain a natural biomaterial for heterotopic xenografts, capable of controlling 1) anaphylactic reactions, 2) cytotoxic reactions, 3) immune-complex reactions and 4) cell-mediated reactions, in that the biomaterial itself, although still possessing the physicochemical characteristics of natural bone, is devoid of allergens, and so is perfectly biocompatible.

In view of these objects, the present invention envisages a process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts, whose essential characteristic forms the subject of the principal claim, which is understood to be wholly incorporated herein.

Further advantageous characteristics follow in the dependent claims, which are also understood to be wholly incorporated herein.

The object of the process is, essentially, the elimination of substances of an organic nature from bone tissues subjected to treatment, though without damaging the osseous structure of the actual tissues.

Specifically, the process aims to eliminate two groups of organic substances from the bone tissues being treated: lipids, which are particularly abundant in animal tissues and especially in those of bovine origin, and the cellular apparatus of bone, i.e. osteoblasts-osteoclasts , osteocytes and blood cells .

Whereas cells constituting the blood, if introduced into the human body, are eliminated from the recipient in about thirty minutes with little if any immune reaction, lipoproteins remain in the recipient's tissues for weeks. These therefore represent a quite potent and dangerous source of allergens, with a considerable capacity for activation of the immune system and consequent rejection of the graft and permanent immunization of the subject with respect to the substance.

The product obtained using the process according to the present invention is, to all intents and purposes, natural bone, but is devoid of lipids and cellular apparatus and contains: - Type I collagen approx. 30% by weight;

- Total minerals approx. 70% by weight, of which: . a) hydroxyapatite approx. 95% by weight and

. b) calcium, magnesium, sodium, fluorine, bicarbonates and potassium approx. 5% by weight.

The variability of the components is due, in the main, to the type of food eaten by the donor animal.

If blocks of osseous tissue obtained using the process according to the invention are implanted, the mechanism of their immobilization at the graft site is comparable to that of resolution of a fracture.

These characteristics mean that the product itself can be used by orthopaedic physicians and oncologists, who until now have avoided using biomaterials (apart from prostheses made of titanium) for transplant purposes, precisely on account of the difficulty in obtaining rapid bone integration with a mechanical supporting strength similar to that of natural tissues, preferring autografts and bone from a bone bank (from human donors from various sources). The process according to the invention makes it possible to produce slices of bone, even of large area, which can be adapted to the surgeon's precise requirements, or even entire sections of limb, standardized by means of a computerized milling device, connected to equipment for CAT (Computerized Axial Tomography), with mechanical support characteristics identical to those of natural human bone.

Some examples of implementation of the process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts are given below, in order to better illustrate the present invention.

Example I

In this example, the process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts, according to the invention, essentially comprises two stages, namely a) a stage of delipidization and b) a stage of elimination of the residual cell bodies for complete deantigenization of the bone tissue being treated.

In particular, in the delipidization stage a), most of the organic residues are eliminated from the bone tissue being treated by mechanical and not chemical extraction, which is the only way of preserving the mechanical strength of the actual bone tissue. In stage b), for elimination of residual cell bodies, the bone tissue being treated is placed in a bath of hydrogen peroxide. If desired, stage a) could be omitted. However,, it should be noted that hydrogen peroxide in large volumes, if applied for too long, damages the collagen contained in the bone tissue, reducing its mechanical strength. For this reason it is desirable for stage b) to be preceded by stage a) .

Accordingly, the process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts, according to the present example of embodiment of the invention, essentially comprises the following two stages:

- stage a) of delipidization by mechanical extraction, in which: al) the bone material to be treated (for example, bovine bone) is placed in an autoclave, for example in a 3 or 5% solution of water and ammonia, for a time varying from 30 minutes to about 6 hours, under a pressure of approx. 2 bar; a2) the bone material removed from the autoclave is washed by means of a jet of saturated steam, at a temperature of approx. 120-150°C and under a pressure of approx. 2-3 bar; a3) on completion of washing, the material is cut to the desired shapes and dimensions; a4) the cut pieces of bone material are then boiled in water, preferably with the addition of a surfactant, for about 5-20 minutes at a temperature of about 90°C; a5) after boiling, the cut pieces of bone material are washed using purified water and ultrasound, at a temperature of about 30-50°C for about 10-30 minutes; aβ) the cut pieces of bone material are again placed in the autoclave in a water bath, for a time of about 25-35 minutes, at a temperature of about 110-130°C and under a starting pressure of approx. 5 bar and a final pressure of approx. 9 bar; a7) after removal from the autoclave, the cut pieces of bone material are washed using purified water and ultrasound, according to the method stated in point a5) above; we then move on to:-

- stage b) for the elimination of residual cell bodies, adopting the following procedure: bl) the cut pieces of bone material, pretreated as described above, are immersed in a bath of hydrogen peroxide at 100-140 volumes, at a temperature between 10 and 45°C, for a period of time varying from 10 minutes to 36 hours; b2) the cut pieces are removed from the bath and are washed, for example as indicated in point a5) above; b3) a further washing is then carried out in physiological saline, for example at a temperature between 10 and 45°C; b4) next, the cut pieces of bone material are dried at a temperature between 50 and 90°C; b5) the cut and dried pieces are then fragmented or cut to the desired size; b6) finally, the product obtained is sterilized and packaged. The product obtained is, for example, in the form of granules, or of cortical or cancellous bone tissue for implants in cyst cavities, or in the form of bone lamellae or stackable blocks for dental and orthopaedic use.

This product, when grafted, after natural vascularization and physiological remanagement of the bone life cycle, is replaced by newly formed natural human bone tissue.

Accordingly, preformed pieces can replace the usual metal prostheses used in orthopaedics.

Remarks on the process steps presented above: Step al): Boiling in the autoclave varies from a minimum of 30 minutes to a maximum of 6 hours, depending on the pressure and temperature of treatment: the higher the temperature and, therefore, the pressure, the shorter the required treatment time. The purpose of this process step is to eliminate residues of tendons, muscles and fats, which normally remain on the bones after the animal has been boned by the butcher.

The same result could be obtained by scraping the bone with sharp cutting tools. If the bone does not have such residues, it is possible to start directly with step a2).

Step a2): The purpose of this process step is to eliminate, by means of a jet of steam at considerable pressure, the periosteum which covers the bone and is extremely tenacious, as well as any tissue residues from natural cavities and crevices of the bone. The heating applied during the preceding step al) softens these residues and makes it possible to remove them more quickly and efficiently. It is therefore recommended to carry out this process step a2) by removing blocks of bone directly from the autoclave, keeping them hot and bringing them under the steam jet immediately. The greater the pressure of the jet, the better the resulting cleaning action, though temperatures above 120°C damage the structure of the collagen fibres, causing softening of the tissue obtained.

This process step can be repeated several times.

Step a3): The blocks of bone (femurs, tibias, pelvis, ribs and the like) are cut up by means of cutting-off saws, band saws or saws with a diamond-impregnated disc. In this way the required blocks are obtained, in the right shape and thickness, according to the site of transplantation or on the basis of standard pieces, determined by market demands.

For the preparation of material for use in dentistry, blocks of bone are cut to a thickness varying from 10 to 20 mm. For the preparation of materials for orthopaedic use, long bones of suitable diameter are, for example, cut longitudinally into two matching halves.

Step a4): Defatting agents, such as those derived from benzene, toluene, methyl amines and the like, could also be mixed with the water, but these have the drawback that they adhere tenaciously to the tissues and so require prolonged rinsing in running water, sometimes for several days, to ensure that they are eliminated completely. Benzene derivatives are potent carcinogens. It is therefore preferred to use surfactants based on neutral soaps and ammonia, which can be removed much more easily at the end of the cycle.

This treatment can be repeated several times as a function of the thickness of bone fragment being treated and of the quantity of fats it contains, which vary depending on the age and weight of the donor animal.

Step a5) : The temperature of the purified water and the treatment time vary according to the thickness and amount of bone being treated. Use of ultrasound during rinsing greatly facilitates elimination both of the surfactant and of any cell residues present, dramatically reducing (by about 70%) the rinsing time. Step a6): This treatment makes it possible to dissolve the remaining fats at high temperatures. The use of an autoclave, and therefore of temperatures up to about 130°C in a water bath, avoids calcination of the tissue, leaving all the mineral constituents of the bone intact.

This treatment can be repeated several times with fresh liquid, depending on the quantity and the thickness of the tissue being treated. Step a7): By using ultrasound, this treatment provides better removal of cells and fats that are still present in the bone tissues being treated.

Step bl): In this process step, the extreme variability of the treatment times and temperatures is explained as follows: Hydrogen peroxide is a compound that has a pH close to 4 at a volume of 120 and which becomes active only and exclusively in the presence of organic substances. Moreover, in contrast to other acids, it does not cause demineralization of osseous tissues. These characteristics, and its liquid state, enable it to reach the organic components which are still present in the osseous tissues after the treatments carried out in process steps from al) to a7), rapidly and with great precision.

Its behaviour is influenced by the concentration and the temperature, as well as by the duration of action. According to tests that have been carried out, contact of hydrogen peroxide with bovine collagen causes reversible softening of the latter, not its destruction. This turned out to be, according to the tests undertaken, the only substance that is capable of avoiding recourse to calcination treatments, which are destructive, but unavoidable - without resorting to hydrogen peroxide - for eliminating cell bodies of infinitesimal dimensions. If process steps a2), a4) and a5) are repeated several times, at this process step it is possible to reach a fairly high degree of cleanness of the treated bone tissue (equal to about 95%). This makes it possible to limit the treatment in the hydrogen peroxide bath even down to a time of just 15 minutes. In this way a finished product is obtained with mechanical characteristics almost identical to those of untreated bone tissue. Step b2) : This treatment facilitates removal of the residues of hydrogen peroxide.

Step b3) : The purpose of this process step is to normalize the pH of the tissue obtained, bringing it as close as possible to the physiological pH. The treatment time depends on the amount of bone treated, whereas the temperature of the solution is irrelevant.

Step b4) : The operation involving drying of the bone tissue is performed at a temperature that varies from 50 to 90°C, depending on the amount of material being treated, and for drying times that vary according to whether use is made, for example, of drying stoves or infrared drying systems, or a hot-air cabinet.

Note: this step b4) , as well as the next process steps b5) and b6), are not crucial for the purposes of the present invention, but are among the conventional production techniques of a pharmaceutical laboratory. Steps b5 and bβ): see the note relating to step b4) .

Example II

Stage a): this comprises the process steps from al) to a7) as described in Example I above, with the following variations only for the steps enumerated below: Step a4) : Boiling is carried out in a solution of water and ammonia, at a concentration varying between 3 and 10% by weight (a concentration of 7% by weight of ammonia proved to be advantageous), at a temperature of approx. 90^eC, for 10-20 minutes.

Step a5) : The cut piece of bone is washed using a jet of steam at a pressure of 3-5 bar, at a temperature between 120 and 150°C and for a time that varies as a function of the thickness of the piece being treated.

Step a6): The piece of bone being treated is washed in purified water and by means of ultrasound. Step a7) : Steps a4) , a5) and aβ) are repeated several times in succession. Every 3 treatment cycles, a fragment of treated bone is examined under the microscope to assess it - using known systems of coloration - for the presence or absence of residual fats in the tissues. Stage b) : This comprises the process steps from bl) to b6) as described in Example I above.

In this example II, owing to the repetition of process steps a4), a5) and aβ) , the bone tissue being treated arrives at stage b) completely defatted. Accordingly, the duration of immersion of the bone tissue in the bath of hydrogen peroxide varies only according to the mechanical properties of the product that it are [sic] desired to obtain. For example, lower hardness of the tissue (resulting from a longer period of immersion in the bath) corresponds to much faster absorption of the transplanted tissue. This makes it possible to programme the healing times either by replacement of the bone supplied or by integration (not absorption) of the residual transplant bone tissues.

Example III

Preparation of cortical tissue is carried out in this example.

Stage a) : the procedure is the same as in Example I above.

Stage b) : the procedure is the same as in Example I above, but with the insertion, between process steps bl) and b2), of an operation involving crushing of the cut pieces of bone into particles with an average size varying between 0.25 and 2 mm.

As follows from the above examples, in the process according to the invention the treatment of calcination of the bone tissue to be treated is eliminated entirely, and no substances are used that are harmful to the health of humans or animals. Furthermore, no acids or substances are used which cause demineralization of bone.

In stage a), treatment with the jet of steam at high pressure (Example I, step a2) is especially important.

In stage b) , attention should be drawn in particular to the use of a bath of hydrogen peroxide as a caustic agent for the complete removal of organic substances from the bone tissue being treated (Example I, step bl).

Claims

1. Process for inactivating and eliminating the organic matrix from animal bone for heterotopic xenografts, characterized in that it comprises at least one stage of elimination of cell bodies from the bone tissue being treated, by means of at least one step of chemical treatment, consisting of placing (immersion) of the tissue in a bath of hydrogen peroxide, for a period of time between a few minutes and some hours (for example, a period of time between 10 minutes and 36 hours), so as to obtain the complete deantigenization of the bone tissue being treated.

2. Process according to Claim 1, characterized in that it further comprises a stage of delipidization of the bone tissue being treated, which precedes the said stage of elimination of cell bodies, and which basically consists in the mechanical extraction of most of the organic residues from the tissue.

3. Process according to Claim 2, characterized in that the said stage of delipidization comprises at least one step of washing of the bone tissue being treated, by means of a jet of steam, for example at a temperature between 100 and 150°C and under a pressure between 1 and 5 bar.

4. Process according to Claim 1, characterized in that the said stage of elimination of cell bodies further comprises one or more of the following steps of treatment of the bone tissue: washing, desiccation or drying, fragmentation or cutting, sterilization and packaging.

5. Process according to Claim 2 or 3, characterized in that the said stage of delipidization comprises at least one stage of treatment of the bone tissue in an autoclave, for example in a solution of water and ammonia (for example, at 7%), at a temperature above 100°C, for a period of time between 15 minutes and 12 hours, under a pressure that can vary from 1 to 4 bar.

6. Process according to Claim 2 or 3, characterized in that it further comprises one or more of the following steps of treatment of the bone tissue: cutting the bone tissue to the desired shape and dimensions, boiling the bone tissue in a water bath, for example with addition of a surfactant, washing the bone tissue, for example using purified water and ultrasound.

7. Process according to Claims 1 and 2, characterized in that it comprises the following two stages: - stage a) of delipidization by mechanical extraction, in which: al) the bone material to be treated (for example, bovine bone) is placed in an autoclave, for example in a solution of water and ammonia (for example, at 7%), for a time varying from 30 minutes to about 6 hours, under a pressure of approx. 2 bar; a2) the bone material removed from the autoclave is washed by means of a jet of saturated steam, at a temperature of approx. 120-150°C and under a pressure of approx. 2-3 bar; a3) on completion of washing, the material is cut to the desired shapes and dimensions; a4) the cut pieces of bone material are then boiled in water, preferably with the addition of a surfactant, neutral soap, ammonia and the like, for about 5-20 minutes at a temperature of about 90°C; a5) after boiling, the cut pieces of bone material are washed using purified water and ultrasound, at a temperature of about 30-50°C for about 10-30 minutes; a6) the cut pieces of bone material are again placed in the autoclave in a water bath, for a time of about 25-35 minutes, at a temperature of about 110-130°C and under a starting pressure of approx. 5 bar and a final pressure of approx. 9 bar; a7) after removal from the autoclave, the cut pieces of bone material are washed using purified water and ultrasound, according to the method stated in point a5) above; we then move on to: - stage b) for the elimination of residual cell bodies, adopting the following procedure: bl) the cut pieces of bone material, pretreated as described above, are immersed in a bath of hydrogen peroxide at 100-140 volumes, at a temperature between 10 and 45°C, for a period of time varying from 10 minutes to 36 hours; b2) the cut pieces are removed from the bath and are washed, for example as indicated in point a5) above; b3) a further washing is then carried out in physiological saline, for example at a temperature between 10 and 45°C; b4) next, the cut pieces of bone material are dried at a temperature between 50 and 90*C; b5) the cut and dried pieces are then fragmented or cut to the desired size; bβ) finally, the product obtained is sterilized and packaged.

8. Process according to Claims 1 and 2, characterized in that it comprises the following two stages:

- stage a) of delipidization by mechanical extraction, in which: al) the bone material to be treated (for example, bovine bone) is placed in an autoclave for a time varying from 30 minutes to about 6 hours, under a pressure of approx. 2 bar; a2) the bone material removed from the autoclave is washed by means of a jet of saturated steam, at a temperature of approx. 120-150°C and under a pressure of approx. 2-3 bar; a3) on completion of washing, the material is cut to the desired shapes and dimensions; a4) the cut pieces of bone material are then boiled in water, preferably with the addition of ammonia at a concentration varying between 3 and 10% by weight, for about 10-20 minutes at a temperature of about 90^βC; a5) after boiling, the cut pieces of bone material are washed by means of a jet of steam at a pressure of 3-5 bar, at a temperature between 120 and 150°C and for a time that varies in relation to the thickness of the piece being treated; a6) the cut pieces of bone material are then washed in purified water and by means of ultrasound; a7) steps a4), a5) and aβ) are repeated, in succession; we then move on to:

- stage b) for the elimination of residual cell bodies, adopting the following procedure: bl) the cut pieces of bone material, pretreated as described above, are immersed in a bath of hydrogen peroxide at 100-140 volumes, at a temperature between 10 and 45°C, for a period of time varying from 10 minutes to 36 hours; b2) the cut pieces are removed from the bath and are washed, for example as indicated in point a5) above; b3) a further washing is then carried out in physiological saline, for example at a temperature between 10 and 45°C; b4) next, the cut pieces of bone material are dried at a temperature between 50 and 90°C; b5) the cut and dried pieces are then fragmented or cut to the desired size; b6) finally, the product obtained is sterilized and packaged.

9. Process according to Claims 1 and 2, for the preparation of cortical tissue, characterized in that it comprises the following two stages: - stage a) of delipidization by mechanical extraction, in which: al) the bone material to be treated (for example, bovine bone) is placed in an autoclave, for example in a solution of water and ammonia (for example, at 7%), for a time varying from 30 minutes to about 6 hours, under a pressure of approx. 2 bar; a2) from the autoclave the bone material removed is washed by means of a jet of saturated steam, at a temperature of approx. 120-150°C and under a pressure of approx. 2-3 bar; a3) on completion of washing, the material is cut to the desired shapes and dimensions; a4) the cut pieces of bone material are then boiled in water, preferably with the addition of a surfactant, neutral soap, ammonia and the like, for about 5-20 minutes at a temperature of about

90°C; a5) after boiling, the cut pieces of bone material are washed using purified water and ultrasound, at a temperature of about 30-5 °C for about 10-30 minutes; a6) the cut pieces of bone material are again placed in the autoclave in a water bath, for a time of about 25-35 minutes, at a temperature of about 110-130°C and under a starting pressure of approx. 5 bar and a final pressure of approx. 9 bar; a7) after removal from the autoclave, the cut pieces of bone material are washed using purified water and ultrasound, according to the method indicated in point a5) above; we then move on to:

- stage b) for the elimination of residual cell bodies, adopting the following procedure: bl) the cut pieces of bone material, pretreated as described above, are immersed in a bath of hydrogen peroxide at 100-140 volumes, at a temperature between 10 and 45°C, for a period of time varying from 10 minutes to 36 hours; b2) the cut pieces of bone are fragmented into particles, with an average size varying between about 0.25 and 2 mm; b3) the cut pieces are removed from the bath and are washed, for example as indicated in point a5) above; b4) a further washing is then carried out in physiological saline, for example at a temperature between 10 and 45°C; b5) next, the cut pieces of bone material are dried at a temperature between 50 and 90^βC; bβ) finally, the product obtained is sterilized and packaged.