Method of producing a bone replacement material

p0001The present invention relates to a method for producing bone substitute material, of collagen-containing material and a mineral component, wherein the collagen-containing material is cross-linked. Such a method is summarized in Chemical Abstracts, vol 96, no. 20, May 17, 1982, pages 407, no. 168778 r described. The material described therein, ie gelatin, and calcium phosphate prepared from two different types of collagens, collagen hydrolyzate means much tanning. Collagen derivatives and specifically fibrillar collagens that have not been cleaned via an enzymatic resolution of the collagen fibers, are known to have an antigenicity that leads the organism to incompatibilities. Calcium phosphate in unsintered form has admixtures of strongly alkaline impurities, which give also after implantation to undesirable side effects and defensive reactions occasion. In addition, these structures will serve as prostheses and have different properties compared to the targeted bone substitute material. In addition, previously three different collagen derivatives must be made. Finally, in this citation, the material is tanned with 1.5% formaldehyde. This makes the material so strongly denatured that it is plastic-like and not absorbed by the body after implantation. but is recognized as a foreign body, which is known to cause undesirable side effects. Moreover, a stimulation of bone growth for the material is not described.

p0002Further, the method initially mentioned in EP-AO 030 583 of the same Applicant is described. Extensive clinical investigator obligations were published in the journal of Orthopaedics 121 (1983),. Page 115-123, entitled "Clinical experience with collagen-apatite implantation for local bone regeneration". H. means Meier, BD Katthagen, published, indicating that encouraging and satisfactory success has been achieved with it. These studies further revealed that the material is not yet clinically massive enough during insertion into the tissue, particularly impregnation with blood and tissue fluid still relatively deliquescent and bone formation in spite of heat treatment and HCl vapor deposition. Therefore, a stronger material would be desirable, which also still produces better stimulation of bone growth.

p0003In EP-A036 415, a method for producing a collagen product is described, which served as a bone substitute material for the above experiments, after admixing of fine apatite powder. This was a substantial increase in the absorptive capacity and mechanical strength than previously known collagen fabrics are achieved in that the collagen product to an additional heat treatment or treatment with gaseous hydrogen halide was subjected.

p0004From the same publication page 10 line 23 to page 11, line 3. It is also apparent. that it was known per se. to improve the physicochemical properties of collagen or gelatine products by chemical means, for example by cross-linking with aldehydes, especially formaldehyde or glutaraldehyde. However, these treatments have the disadvantage that the products obtained in the medical implant by the body very slowly or not at all absorbed and cause inflammation, immune reactions or the formation of foreign body giant cells, especially in the body. The cross-linking of collagen products with aldehydes has been doing exhaustive performed so that fully networked. plastic-like products resulted, which are recognized by the body as foreign and so cause the aforementioned harmful reactions. Residual amounts of bonded aldehyde in the implant are also damaging to tissue.

p0005From the publication Jap. Traumat. Surg (1982) 99, pp 265-269 by K. Hayashi et al. is a collagen-hydroxylapatite preparation is known which has been freeze-dried after crosslinking. On one hand, relatively fine-grained apatite was used and on the other hand been no indication that the addition of glutaraldehyde should be such that none of the reactions described above occur.

p0006It is accordingly an object of the present invention to provide a method for producing a bone substitute material with which it is possible to produce a stronger, slightly zerreißliches bone substitute material. the arrival and the ingrowth of the bone even more favorable and does not cause any unwanted side effects. The object is achieved by a method described in the claims.

p0007It has now been surprisingly found. that a very considerable improvement in the physicochemical properties of collagen could be caused by only partial cross-linking the collagen with formaldehyde or glutaraldehyde, without on the other hand, the finished product negative foreign body reactions triggers after implantation in the body, as previously described, or damage caused by Residues of aldehyde occur.

p0008In the production of the bone replacement material. which was the above-mentioned tests were based. a very fine powder apatite or tricalcium phosphate having particles in the size of 3-5 11m was used It has now been found that the osteogenic effect of the apatite is substantially increased. If this is in the form of powder aggregates or larger particles. This increase in particle size may preferably be achieved. that commercially available, in a finely crystalline form vorliegendes apatite or tricalcium phosphate into grains of about 20-1000 microns, preferably 50-300 microns diameter. are sintered.

p0009Meanwhile conducted tests have shown. that a bone substitute material. consisting of partially cross-linked with formaldehyde collagen and apatite or tricalcium phosphate sintered in the form of grains. already the collagen solution are added prior to lyophilization, is, significantly better results than the prior art provides Knochenersatzma material, whereby such a new bone substitute material is suitable for a larger range of indications. It is particularly advantageous to choose the distribution of apatite grains in collagen analogous to the distribution of the crossing points of the mineral structures in the natural bone, so that at the beginning of the formation of new bone, the bone replacement material is present as a kind of matrix of the forming bone is similar.

p0010The starting material required for performing the method according to the invention can be prepared in the manner described below:<ul><li>1 kg bovine tendon was frozen at -10 to -20 ° C and while very finely comminuted 10-20 min. With a Messerhomogenisator high revs. The temperature of the ground material was maintained by the addition of ice chips below + 40 ° C. The tough fibrous Gewebebrei was dissolved in 5 liters of 10% NaCl solution containing 2.5 g of sodium azide and 50 ml of a 10% aqueous solution of the nonionic surfactant NP 55/52 (Polyoxyäthylennonoylphenyläther) was suspended under vigorous stirring. The suspension was further stirred at room temperature for 2 hours and then centrifuged. The gray to brownish cloudy supernatant containing fat and unwanted water-soluble dietary fiber, was discarded. The residual white Hautfaserbrei was extracted twice more in the same manner, wherein the extraction liquid per liter of 0.1 mol of disodium hydrogen phosphate was added.</li></ul>

p0011The starting material can also be used pigskin instead of bovine tendon.

p0012A in the manner described above from 1 kg of beef tendon preserved defatted and extracted fibrous Gewebebrei was suspended 0.5M acetic acid in 5 volumes. To the suspension was added a solution of 1 g of technical pepsin in 100 ml of 0.01N HCI. The pH of the suspension was adjusted with HCI to 2.9. The suspension was digested with frequent stirring for 48 h. The viscous collagen solution was filtered through a suction filter G. 1 to remove undigerierte residues. The collagen was precipitated by addition of 30% aqueous sodium hydroxide from the suspension and separated by centrifugation. The collagen was purified by dissolving it in 0.5 M acetic acid and precipitation by slow addition of 3% sodium chloride. The purified collagen was dissolved in 0.5 M acetic acid and diluted with water. Still present in the collagen residual sodium chloride was removed by washing on an ultrafilter. The ultrafiltration was continued until the eluate with the addition of silver nitrate no more chloride ions were detected, and the collagen concentration was about 1%. The collagen solution was filtered. with 0.2 wt.% formaldehyde and 5 parts by weight of sintered apatite grains the size of 20-1000 microns. both based on the collagen content of the collagen solution. added. stirred for about 20 minutes. poured into suitable molds. allowed to stand for several hours, lyophilized and then sterilized by y irradiation.

example 1

p0013600 g of a prepared according to the method described above, ultra-filtered collagen solution with a collagen content of 0.88 wt .-% was klartiltriert and treated with 10.6 ml of a 0.1 wt .-% aqueous formaldehyde solution and stirred. While stirring the mixture 26.4 g of sintered apatite granules were added to about 50-150 microns in diameter and stirred for 22 minutes. The pH of the solution increased during the stirring of original pH 3.5 to 6.35. The mixture was poured in portions of 70 g in polystyrene trays of size 12 x 7.5 cm, left to stand overnight. the next day lyophilized, then packed and finally sterilized by y-irradiation at a dose of 2.5 Mrad.

example 2

p0014800 g of a prepared according to the method described above, ultra-filtered collagen solution with a collagen content 1.07 wt .-% collagen was charged with 6.83 ml of a 0.25 wt .-% aqueous solution of glutaraldehyde and with 17.12 g = twice the amount , based on the collagen content, added to the sintered apatite grains or tricalcium phosphate with about 300-700 microns in diameter, stirred for half an hour, allowed to stand for 10 hours, 6.4 x 3.8 cm filled in portions of 18 g in cups the size, lyophilized, packaged and sterilized by irradiation with 2.5 Mrad gamma rays.

example 3

p0015200 g of a prepared according to the method described above, ultra-filtered collagen solution with a collagen content of 0.97 wt .-% were with 5.82 g = three times the amount. based on the collagen content. mixed on sintered apatite grains of about 100 microns in diameter, stirred for half an hour, filled in portions of 40 g in round dishes with a diameter of 8 cm and lyophilised. The resulting material was not resistant to aqueous solutions and was therefore partially crosslinked by a charged in, with 35 wt .-% hydrochloric Formaldehdlösung. closed chamber of formaldehyde vapor was exposed for 2 hours. The formaldehyde solution was removed from the chamber and the chamber evacuated and 6 times. to remove the unbound formaldehyde gas from the collagen-apatite material. In the third aeration air was passed through an aqueous ammonium hydroxide solution, to bind final residual amounts of formaldehyde in the inflowing ammonia. The bone substitute material obtained was suitable after this treatment in aqueous solutions and stable after sterilization for implantation into bone defects.

p0016From the above example it is clear that one can achieve good results with an amount of formaldehyde. the below 1 wt .-%. is based on the dry weight of the collagen. preferably in an amount of 0.05-0.5 wt .-%. When using other aldehydes, the corresponding amount to be used.

p0017In addition to the mentioned form and glutaraldehyde may also be used a saturated or unsaturated, mono- or polyfunctional aliphatic aldehyde, glyoxal, unsubstituted or substituted di- or Trichlortriazine, wherein as gaseous crosslinking agent ethylene oxide also comes into consideration.

p0018It is desirable to reinforce the mechanical strength of the bone substitute material even further by treating it with honeycomb, reticular layer-like or woven-fabric-like (woven, twisted, knitted) supporting elements of plastic, textile, metal, aluminum, ceramics, carbon fiber, carbon, bone cement, fiberglass or Bioglasfaser combined. The support elements may have the shape of the bone part to be replaced. It can also be expedient, the new bone substitute material shall be included antibiotics.

p0019Purpose compacted and solidified the bone replacement material can be optionally compressed in the dry or wet state by heating.