CA1142430A

CA1142430A - Bone substitute material and its use

Info

Publication number: CA1142430A
Application number: CA000341820A
Authority: CA
Inventors: Heinz-Helmut Werne
Original assignee: Intermedicat GmbH
Current assignee: Intermedicat GmbH
Priority date: 1978-12-16
Filing date: 1979-12-13
Publication date: 1983-03-08
Also published as: JPS619857B2; DE2962750D1; JPS5584161A; EP0012959A1; DE2854490B1; ES486893A1; DK530279A; ATE944T1; DK154892B; EP0012959B1; DE2854490C2; DK154892C

Abstract

Abstract of the Disclosure A collagen-based, bone substitute material is produced from bone, particularly calf bone, in a multistep process in which the bone is water washed, treated with H202 solution, water washed, degreased with an organic solvent, the mineral content eliminated, the salts are removed by a water rinse, and the product is sterilized.

Description

`` 3L1~2~30 This invention relates to a collagen-based material useful as a bone substitute material with improved biological acceptability and stability.

BONE IMPLANTATION BACKGROUND
-Very often during surgery it is necessary to fill bone defects with a filler material. This is necessary particularly as a result of injuries due to accidents which lead to bone fractures involving loss of bone substance or bone penetration. However, there are many other uses for bone filler material as, for example, filling cavities following removal of bone tumors or withdrawal of bone cysts.
Moreover, a bone filler material is required in those cases where, in the scope of corrective measures, a surgeon is required to fill up a cavity present initially in the bone, or created by the surgeon himself.
Based on the present state of the art of surgery, the most appropriate bone-filler material is autologous spongiosa or an autological cortico-spongeous chip (Vnfall-heilkunde [Traumatology], 81, 565 - 567 [1978]). Such bone filler material originates with the recipient. The donor of the bone and the recipient axe thus the same.
The homologous transplanation of human bones from one person to another one likewise is customary. For this purpose, bone banks are set up in certain places which keep appropriate bone material available. Moreover, the transplanation of animal bones, which are first subjected to an appropriate method of preparation like storage ïn

2 -ethylmercuri-mercapto-benzoxaZol-5-carbons~ure-natrium -cialite solution or the method disclosed in the German patent 961,654, is known and customary.

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The best surgical results have been achieved up to now at the site of the bone implantation by using bone removed from the same patient who is provided with the bone filler material (autologous transplantation). However, there are two important disadvantages with autologous trans-plantation and they are:
1. The bone substitute material must be obtained by an additional surgical operation. The additional surgical operation, which increases the operating time, may be very detrimental to the patient and, in individual cases, even may be life-threatening. This danger will affect primarily the seriously injured, older patients or patients pre-afflicted with other general ailments which are separate from that requiring the surgical operation. Moreover, the additional surgery leads to increased loss of blood and additional pain for the patient. Also, each surgical operation entails a certain amount of complications which may lead to additional damage which cannot always be anticipated in each individual case.
2. The quantity of autological bone material available is limited. The available reservoirs are the pelvic combs on both sides, ribs, the tibia head and the parts of the radius distal from the body. Even complete utilization of these sources, which requires a plurality of surgical operations, may not furnish an adequate quantity of bone substitute material in every case. If the bone need is greater than that obtainable from the patient, the surgeon must either utilize less bone replacement material than desired or he must resort to use of less suitable human or animal bones.

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In homologous bone transplantation from one human to another, the bone transplant frequently is not tolerated well by the recipient. Primarily responsible for this is the formation of antibodies against foreign protein. Moreover, appropriate donor bone material is hard to obtain. As a rule, it is necessary to resort to use of bones removed from bodies which have already deteriorated considerably.
In addition, the transmission of infections to the recipient by use of such bone cannot be ruled out.
The use of heterological transplantation material, like Kiel bone chip according to German patent 961,654, has demonstrated over the course of many years that this material is not well tolerated by some patients. Frequently, the bone fragments implanted by the surgeon are not converted into live bone tissue but remain in the tissue as dead material.

SUMMARY OF THE INVENTION
According to the invention, there is provided a collagen-based product, and process of making the product, which can be used as a bone substitute material and which can be made available in large quantities.
The material, upon implantation into the human or animal body, creates no antibodies, is of excellent tissue compatibility and after being implanted into the body is converted into bone tissue.

PRODUCTION OF THE PRODUCT OF THE INVENTION

Bones, and specifically bovine bone material, for example from the calf, preferably are used as the starting 11fl~2~

material in making the bone substitute according to the invention. However, other starting materials such as tendons, skins, human bones and similar material containing collagen, also may be used. The subsequently described method for the production oE the material according to the invention is based on the use of calf bones as starting material by way of an examp]e. The method of the invention represents a combination of steps which are carried out in the disclosed sequence, to obtain the bone substitute material with improved biological stability, according to the invention.
In a first step of the method, the bones are rinsed with water sufficiently long to remove blood pigment and other water-soluble proteins. This rinsing period usually takes 2 to 3 days During this rinsing, the washing water is renewed frequently. The rinsing also can be carried out by rinsing the bones in running water. No particular temperature is required, but the rinsing may be carried out appropriately at ambient temperatureO
In a second step of the method, removal af an additional protein content, not soluble in water, is effected.
It lS important that foreign proteins outside the collagen, like bone cells for example, be removed to prevent anti-reactions by the formation of antibodies, even those of an enzymatic kind. The removaI of the protein content in this step is accomplished by putting the bone material in a protein remover as, for example, in an H2O2 soluticn.
This H2O2 solution should have a concentration of more than 10%. The duration of the treatment normally amounts to 2 to 3 days. Here again, the H2O2 solution should be renewed repeatedly to assure complete removal of the protein content.

~ 42430 Step thxee again comprises a rinsing step. The material from step two is rinsed with cold water to rinse out and remove the dissolved soft parts. Rinsing under running water is advantageously used in this step.
In step four of the method, the material thusly obtained from step three is degreased. The degreasing is done with an organic solvent or mixture thereof. Appropriate solvents are, for example, acetone or ether or specifically a combinatin of both solvents. The degreasing may be accom-plished by contacting the bone material with the solvent in any equipment appropriate therefor. A Soxhlet apparatus is particularly appropriate for this degreasing.
In step five of the method, the bone material from ste~ four is treated with a complexing agent or an ion exchanger. The purpose of this step is to eliminate the mineral content of the bone. The salts of ethylenediamine tetraacetic acid are appropriate complex formers which can be used.
A suitable solution of a complexin~ agent can be produced according to a method disclosed in Romeis "Lehr-buch der Mikroskopischen Technik" (Textbook of Microscopic Technique). Thus, 250 grams of disodium ethylenediamine tetraacetic acid are treated with 200 cc of distilled water and heated. With continuous agitation, 50 cc of a 40% sodium hydroxide solution are added and then the mixture volume is increased to 800 cc with distilled water. By dropwise addition of 40% sodium hydroxide, the pH is adjusted to 7.4 and then the volume is adjusted with distilled water to a total volume of 1000 cc.

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The bone material is immersed into a complexing solution, such as the one just described, and left therein for quite some time, for example up to several weeks, until a rubber-like elastic product is obtained.
The rubber-like elastic product thusly obtained is washed in a sixth step of the method to remove the salt residues present. The washing is done by rinsing ~nder running water for an adequate time, like 2 to 3 days for example.
In step seven of -the method, the material thusly obtained is then subjected, if applicable, to freeze drying using conventional procedures.
In step eight of the method, the material obtained is subjected to sterilization, particularly by use of radia-tion sterilization. A radiation sterilization can be effected with the usual dose of 2.5 megarads.
In the above disclosed combination method, steps 1 to 3 and step 4 as such are known. According to German patent 961,954 a drying step is inserted after the operating step 3 and it is carried out at not more than 40C (104F) over a period of 12 to 24 hours. This drying step must not be applied in the present method because, if it is, the product according to the invention will not be obtained.
Thus, it is essential, after the step 3 rinsing to go to step 4, that is degreasing with an organic solvent, without first drying the product.
Step 5 of the method is known as such, for example, from the textbook by Romeis "Textbook of Microscopic Technique".
However, the combinatin of method steps 1 to 4 of the present invention, with method step 5 of the invention, was not previously known.

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Method steps 6 to 8 relate to method steps previous-ly known per se. However, these steps too were not previous-ly used in combination with the method steps 1 to 5 of the present invention.

USE O~ THE PRODUCT AS A BONE SU~STITUTE MATERIAL
The product produced as described above is a useful bone substitute material of purified collagen, and especially purified calf collagen. The process preserves its molecular structure, to a large extent by carefully avoiding all detri-mental factors, like acid, during the decalcification process.
The product can be used as a bone implant by placing it-in a bone defect in the dry state as obtalned by the described process, or as a paste formed by mixing it with saline solution.
The bone suhstitute material thus obtained, upon implantation into the body, has surprisingly good qualities.
For example, the resorption time is substantially higher than in other previously known and used materials. Moreover, fine-tissue examinations at various points in time following implantation into guinea pigs demonstrated an excellent compatibility without rejection reactions by the body.
As soon as two weeks after implantation, the start of conver-sion of the material into bone tissues was observed. Fine tissue examinations showed deposits of mineral substance, connective tissue cells, sprouting blood vessels and bone-forming cells.

ENHANCED RESULTS BY CALCITONIN ADDITION

The outstanding properties of the material obtained according to the invention can be improved substantially ~ L4Z~3t~
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as a bone substitute material by blending this material with a small quantity of the hormone calcitonin, also called thyrocalcitonin. For example, calcitonin solutions commer-cially available are appropriately used. The subs-tantially improved results obtained by such a combination apparently are due to a s~nergistic effect between the basic bone substi-tute material and the hormone calcitonin. The time required for new bone formation is reduced considerably when the bone substitute material is treated with calcitonin.
The followinq example is presented to further illustrate the invention.

Example One kilogram of calf bones (spongiosa pieces) are washed carefully and thus freed of coarse blood residues.
This quantity of bones is then placed in an appropriate vessel, having inlet and outlet connections, and rinsed for 72 hours with running tap water. The bone is then im-mersed for 72 hours in an H2O2 solution (15%). The solution is changed 3 to 4 times. The bones are then rinsed with running tap water for 24 hours. Subsequently, the bones are degreased in a Soxhlet apparatus using 60% acetone and 40~ diethyl ether over a period of about 12 hours.
1250 grams of disodium ethylenediamine tetraacetic acid is added to 1000 cc of distilled water and the mixture is then heated. With continuous mixing, 200 cc of a 40 sodium hydroxide solution is added and then the mixture is brought to a 4000 cc volume by addition of distilIed water. The mixture is adjusted to a pH of 7.4 by the dropwise addition of a 40% sodium hydroxide solution. Then distilled water is added to attain a 5000 cc volume.

~ L42~30 The bones treated as described above are immersed in the solution so produced until several samples taken out prove to be free from minerals. About 3 to 4 weeks at room temperature will be required, depending on the size of the individual pieces.
Another degreasing of the bone material is carried out with 60% acetone and 40~ diethyl ether in a Soxhlet apparatus over about 12 hours. Subsequently, the bone mate-rial is freeze-dried. This is followed, after appropriate foil wrapping, by radiation with 2.5 Mrad. for sterilization.
The following experiments used the bone substitute material produced according to this example, with the de-scribed results.
The bone substitute material according to the invention was tested in rats and rabbits for its properties.
The collagen matrix was implanted into the animals subcutane-ously and into the dorsal muscular system. The implants were removed from various groups after 1, 2, 4, 8, and 12 weeks and examined histologically.
The growth of fine blood vessels and cells as soon as one week after implantation was observed. Chondro-blasts, fibroblasts and osteoblasts (cartillage, connective tissue and bone-forming cells) was observed after 2 weeks.
As time passed there was progressive structuring and restruc-turing until regular bone structures formed.
The foregoing detailed description has been given for clearness of understanding only, ànd no unnecessary limitations should be understood therefrom, as modifications will be obvious to those skilled in the art.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINES AS FOLLOWS:

1. A product useful as a bone substitute material with improved acceptability and stability consisting of natural collagen, which is preserved to a large extent as to its molecular structure, produced by the following method:
a) removing the blood pigment and other water-soluble proteins from natural collagen-containing material by treatment with water;
b) removing proteins from the material by treatment with H202 solution;
c) rinsing the material in cold water;
d) degreasing the material with an organic solvent;
e) eliminating the mineral content of the collagen material;
f) removing the residual salts from the material by rinsing with water; and g) sterilizing the product.

2. A product according to claim l, characterized by using a bone material as starting material.

3. A product according to claim 2 in which the bone material is calf bones.

4. A product according to claims 1 and 2, contain-ing the hormone calcitonine.

5. A process of producing a product useful as a bone substitute material, which comprises:

a) removing the blood pigment and other water-soluble proteins from animal bones by rinsing them with water;
b) removing proteins from the bones by treating them with a H2O2 solution;
c) rinsing the bones in cold water;
d) degreasing the bones with an organic solvent;
e) eliminating the mineral content from the bone material;
f) removing the residual salts from the bone material by rinsing with water; and g) sterilizing the bone material.

6. A process according to claim 5 in which the bone material is freeze-dried between steps f and g.

7. A process according to claim 5 in which the hormone calcitonin is added to the product.

8. A process according to claim 5 in which the mineral content is removed in step e by use of a complexing agent or an ion exchanger.

9. A process according to claim 5 in which the bones are calf bones.