OSTEOTROPIC BONE REPLACEMENT

Abstract

The invention relates to a method for producing an osteotropic bone replacement material from a starting material which substantially has portlandite, calcium oxide, aragonite; calcite and/or apatite. The starting material is introduced into an autoclave with a strontium, fluorine and/or gallium source, wherein when using a starting material which substantially has portlandite, calcium oxide, aragonite; calcite a phosphate source is introduced. In addition, H.sub.2O is added into the autoclave as part of a solvent and the pH value in the autoclave is set to a range above 7. Afterwards, the closed and filled autoclave is heated for at least 1 hour and then cooled. The osteotropic bone replacement material thus developed is subsequently cleaned from residues of the phosphorus, strontium, fluorine and/or gallium source. Furthermore, the invention relates to an osteotropic bone replacement material which substantially consists of apatite and in which strontium ions are incorporated into the crystal lattice.

Claims

1. Method for producing an osteotropic bone replacement material from a starting material which substantially has portlandite, calcium oxide, aragonite; calcite and/or apatite, in particular hydroxyl apatite, wherein portlandite, calcium oxide, aragonite and/or calcite are used in the form of burnt, unburnt and/or chemically treated biogenic skeletons and/or wherein as apatite vertebrate bones after pyrolytic or chemical maceration are used, wherein the starting material is introduced into an autoclave with a strontium, fluorine and/or gallium source, wherein when using a starting material which substantially has portlandite, calcium oxide, aragonite; calcite a phosphate source is introduced, wherein H.sub.2O is added into the autoclave as part of a solvent, wherein the pH value in the autoclave is set to a range above 7, wherein the closed and filled autoclave is heated for at least 1 hour and then cooled, and wherein subsequently the content of the autoclave is cleaned from residues of the phosphorus, strontium, fluorine and/or gallium source.

2. Method according to claim 1, characterized in that the closed and filled autoclave is heated to at least 30 degrees Celsius, preferably to over 190 degrees Celsius.

3. Method according to claim 1 or 2, characterized in that the content of the autoclave is cleaned mechanically using a filter apparatus.

4. Method according to any one of claims 1 to 3, characterized in that the content of the autoclave is washed with H.sub.2O until a pH value of preferably smaller than 8 is reached.

5. Method according to any one of claims 1 to 4, characterized in that to set the pH value in the autoclave to a range above 7 an alkaline solution, in particular an ammonia solution, is used.

6. Method according to any one of claims 1 to 5, characterized in that the strontium, fluorine and/or gallium source is introduced in excess in relation to the starting material.

7. Method according to any one of claims 1 to 6, characterized in that as phosphorus source ammonium dihydrogen phosphate or diammonium phosphate is added.

8. Method according to claim 7, characterized in that in the total mixture calcium and phosphorus atoms are present at a ratio not exceeding 10:5 (atomic ratio).

9. Method according to any one of claims 1 to 8, characterized in that the closed and filled autoclave is heated for at least 12 hours.

10. Method according to any one of claims 1 to 9, characterized in that as strontium, fluorine and/or gallium source a material easy to wash out or of bad water solubility is used.

11. Method according to any one of claims 1 to 10, characterized in that the strontium, fluorine and/or gallium source is added into the autoclave as solid matter in a container, and in that the container enables the exchange of ions of the strontium, fluorine and/or gallium source with the solvent wherein solids are retained.

12. Method according to any one of claims 1 to 11, characterized in that prior to or after introduction into the autoclave the starting material and/or the content of the autoclave is subjected to a pyrolytic treatment and/or a chemical cleaning method.

13. Osteotropic bone replacement material, in particular pursuant to a method according to any one of claims 1 to 12, which substantially has apatite, which is produced from portlandite, calcium oxide, aragonite and/or calcite in the form of burnt, unburnt and/or chemically treated biogenic skeletons and/or which is produced from vertebrate bones after pyrolytic or chemical maceration, wherein strontium, fluorine and/or gallium ions are incorporated into the crystal lattice of the apatite.

Description

[0033] The invention is explained in greater detail hereinafter by way of schematic exemplary embodiments with reference to the Figures, wherein show

[0034] FIGS. 1 to 6 results of the comparative tests

PRODUCTION METHOD

[0035] In the following an exemplary production of the osteotropic bone replacement material according to the invention pursuant to the method according to the invention is described.

[0036] For this purpose, a Teflon container with a capacity of 150 ml is used. This is filled with the following substances:

TABLE-US-00001 Burnt algae material 19.822 g Ammonium dihydrogen phosphate 26.38 g Strontium fluoride 2.212 g Potassium fluoride 2.212 g Ammonia solution (25%) 50 ml Deionized H.sub.2O 50 ml

[0037] As starting material a skeleton of lime-encrusting algae is used as an example for aragonite. When using starting materials containing CO.sub.2 it is usually advantageous if these are burnt, in which case preservation of the external structure of the material is desirable. As described, however, the method according to the invention can also be applied to an apatite material e.g. from vertebrate bones as starting material, in which case the presence of a phosphate source has proved to be advantageous on the one hand for the introduction of the osteotropic ions and on the other hand, however, also for preserving the structure of the starting material.

[0038] Before being added into the Teflon container the algae skeleton is cauterized so that any foreign proteins, proteins or the like are removed. Furthermore, ammonium dihydrogen phosphate, strontium fluoride, potassium fluoride and a 25 percent ammonia solution are added. In addition, deionized water is also added.

[0039] The respective weights or respectively the volumes of the added substances can be gathered from the table.

[0040] In the present case, ammonium dihydrogen phosphate serves as phosphate source, wherein, as set out, other phosphate sources are possible too. Strontium fluoride is used as strontium source and as fluorine source, too. Potassium fluoride is also employed as fluoride source.

[0041] After the substances have been introduced into the Teflon container and a possible gas formation has been awaited the said container is closed. Afterwards, the Teflon container is placed into an autoclave, e.g. into a pressure digestion container. This container is preferably made of stainless steel. Subsequently, the cover is screwed tightly so that an autoclave is created.

[0042] The correspondingly firmly closed pressure digestion container is then placed into a preheated heating cabinet or a heating block that has a temperature of 190° C.

[0043] The pressure digestion container remains in the heating cabinet for 5 days wherein the temperature of 190° C. is maintained. After expiration of this time the heating cabinet is switched off. The pressure digestion container then cools down slowly in the heating cabinet or respectively in the heating block. This takes approximately one day.

[0044] Following complete cooling-down the pressure digestion container and the Teflon container are opened and the resultant osteotropic bone replacement material is cleaned. For this purpose, the osteotropic bone replacement material is applied together with water onto a filter paper and washed. Several cleaning processes, e.g. rinsing processes, are carried out until a pH value below 8 is reached.

[0045] Subsequently, the osteotropic bone replacement material is again introduced into the heating cabinet but only dried at 40° C.

[0046] Afterwards, the osteotropic bone replacement material is ready for further use. For instance it can then be brought into desired shapes and sterilized.

[0047] Tests and Results

[0048] In the following the effect of the new osteotropic bone replacement material produced according to the method pursuant to the invention is explained in greater detail and respectively the osteoinductive effectiveness is proved on the basis of test results. The results show that by the bone replacement material according to the invention the local formation of new bone in the bone defect should be additionally stimulated due to the fact that the most important bone formation marker in human bone cells, the alkaline phosphatase, is stimulated.

[0049] In vitro tests with primary human osteoblastic bone cells were carried out to examine the direct influence of the osteotropic bone replacement material according to the invention when in contact with primary human cells. This was also carried out to avoid overlooking detrimental influences of the osteotropic bone replacement material according to the invention on human bone cells that would involve cell death.

[0050] Hence, before clinical use of new bone replacement materials in vivo primary human osteoblastic bone cells are suitable as sensitive test system to examine the effect of a novel bone replacement material on human bone cell metabolism.

[0051] A cell in vitro—thus also a primary human bone cell—has four possible ways of reaction: [0052] no reaction at all [0053] apoptosis (cell death) [0054] increased/reduced cell division [0055] increased/reduced production of differentiated cell products (for example alkaline phosphatase in the case of bone cells) that are necessary for the build-up of new bone tissue and for the mineralization and formation of hydroxyl apatite in vivo.

[0056] To carry out the tests co-cultures of primary human bone cells were used. In order to compare the effect commercially available bone replacement materials and osteotropic bone replacement material produced according to the method pursuant to the invention were used. In the in vitro experiments the following bone replacement materials were comparatively tested with regard to their effect on primary human bone cells: [0057] 1. BioOss® (bovine bone granulate, commercially distributed by Geistlich Biomaterials) [0058] 2. Algipore® (based on EP 230 570 B1, commercially distributed by Dentsply) [0059] 3. New Algipore 1 (=NA1) osteotropic bone replacement material according to the invention [0060] 4. New Algipore 2 (=NA2) osteotropic bone replacement material according to the invention [0061] 5. New BioOss1 (BioOss® which was subjected to the method according to the invention)

TABLE-US-00002 NA1 NA2 BioOss1 Starting material [g] 7.21 7.51 0.5 Duration [h:min] 116.9666667 221.75 116 Ammonium hydrogen 10 10.001 10.0521 phosphate [g] Potassium fluoride [g] 0.4 0.821 1.996 SrF.sub.2 [g] 0.35 0.688 0.821 Ammonia solution 25% [g] 6 g 50.278 18.183 H.sub.2O filled up filled up filled up to 75% to 75% to 75% Autoclave volume [ml] 57.7 57.7 57.7

[0062] The following determination methods were chosen to examine the influence of the above materials on cell functions of primary human bone cells: [0063] a. Alkaline phosphatase [0064] b. Cell protein

[0065] 1. Test

[0066] Alkaline Phosphatase

[0067] The comparative results are expressed in % of the control +/− standard deviation. Initially, the activity of alkaline phosphatase was examined in the medium supernatant after culture of the human bone cells in the presence of the different materials. As control served an aliquot of the employed cell culture growth medium by itself without cells because in the cavities of the culture plates always remain residues of the serum contained in the culture medium despite serum-free rinsing prior to the exposition of the cells with the materials. The serum also always contains small amounts of alkaline phosphatase. In this first test the new BioOss1 had not yet been available.

TABLE-US-00003 Cells alone BioOss ® Algipore ® NA1 NA2 243 +/− 216 +/− 240 +/− 291 +/− 443 +/− 9% 3% 8% 9% 15%

[0068] These results are illustrated graphically in FIG. 1.

[0069] Interpretation

[0070] BioOss® is prone to reduce the activity of the enzyme alkaline phosphatase which is indispensable for the mineral and bone formation of human bone cells, whereas the bone replacement material according to the invention brings about a highly significant increase in the activity of alkaline phosphatase secreted by human bone cells.

[0071] Thus, the bone-specific alkaline phosphatase as the most important osteoblast marker protein is stimulated by the bone replacement material according to the invention to a extreme significantly stronger degree in the human bone cell model than in the presence of the conventional materials. The different effectiveness of NA1 and NA2 on alkaline phosphatase activity can be ascribed to an increased fluoride and strontium content in NA2 as compared to NA1.

[0072] When measuring the activity of alkaline phosphatase in the cell culture supernatant account must be taken of the fact that also in vivo the mineral or bone formation takes place extracellularly through secretion of alkaline phosphatase into the osteoblastic microenvironment.

[0073] Cell Division/Cell Protein

[0074] As an indication of an effect of the bone replacement materials on cell division the protein content in the individual “cavities”, i.e. reaction chambers of the employed multi-perforated plates was analyzed. For this, a triton extract of the respective cavities was used to determine the protein content according to the BCA method. The higher the protein content in the individual cavities, the more cell material that corresponds to protein material must be present in the cavities. This means that the cell count has increased since a bone cell always has a similar size and proteins are not stored to a larger extent intracellularly in bone cells. A reduction of the protein content in a cell culture cavity, i.e. a reaction chamber, would therefore be tantamount to a reduction of the cell count located adherently on the cell culture base or on the bone replacement materials (apoptotic cells, i.e. dead cells, do not stay adherent and are flushed away before the addition of triton). The results are again stated in % of the control +/− standard deviation.

TABLE-US-00004 Cells alone BioOss ® Algipore ® NA1 NA2 96 +/− 108 +/− 103 +/− 103 +/− 103 +/− 3% 4% 3% 3% 3%

[0075] These results are illustrated graphically in FIG. 2.

[0076] Interpretation

[0077] There is no difference between the examined materials with regard to the cell protein content in the cell culture cavities. Hence, the materials do not have an impact on cell division, nor on cell death, because apoptotically degenerating cells do not stay adherent but become detached and would be flushed away prior to the assay method.

[0078] Overall View

[0079] These observations indicate that the materials obtained according to the method pursuant to the invention have a very positive effect on the alkaline phosphatase activity of human bone cells.

[0080] This observation is consistent with a very favorable and sustained effect of the bone replacement material according to the invention stimulating bone mineral formation in vivo without cell division being impacted, i.e. stimulated or inhibited.

[0081] 2. Test, Activation of the Conventional BioOss® Material by the Method According to the Invention

[0082] The material BioOss® hitherto employed as bone replacement material in dental medicine or oral surgery consists of natural hydroxyl apatite material of inorganic bone tissue of cattle. By way of the method according to the invention, even in the case of a natural calcium phosphate crystal lattice, a partial exchange of calcium ions in the hydroxyl apatite crystal with strontium and fluoride ions can be carried out in a controlled manner. In this case, calcium and phosphorus atoms should be present at a ratio not exceeding 10:5 in the total mixture of the autoclave net weight.

[0083] In the following experiment with primary human osteoblastic cells the effect of the conventional BioOss® material on alkaline phosphatase activity is examined in parallel to the effects of the BioOss1 material activated by the method according to the invention.

[0084] At the same time the materials already tested above were also used in the same experiment in order to allow a ranking of the effectiveness of all materials produced by way of the method according to the invention in a parallel test approach. In this case, too, the alkaline phosphatase activity in the cell culture supernatant was measured after an incubation time of the cells with the new material 24 hours.

[0085] This experiment was again carried out with primary human bone cells of a different individual than in the first experiments (1. test).

[0086] Alkaline Phosphatase

TABLE-US-00005 Cells alone BioOss ® BioOss1 Algipore ® NA1 NA2 393 +/− 368 +/− 645 +/− 431 +/− 418 +/− 747 +/− 19% 16% 20% 24% 9% 51%

[0087] These results are illustrated graphically in FIG. 3.

[0088] Cell Division/Cell Protein

TABLE-US-00006 Cells alone BioOss ® BioOss1 Algipore ® NA1 NA2 96 +/− 106 +/− 97 +/− 93 +/− 98 +/− 107 +/− 5% 5% 3% 6% 3% 5%

[0089] These results are illustrated graphically in FIG. 4.

[0090] Interpretation

[0091] While the conventional BioOss® material has no significantly stimulating effect on the alkaline phosphatase activity in the cell culture supernatants as compared to human bone cells without contact to a bone replacement material, the BioOss® material (BioOss1) pretreated by way of the method according to the invention brings about almost a doubling of alkaline phosphatase activity. Therefore, the method according to the invention is also suitable for activating commercially available bovine hydroxyl apatite material and lends osteoinductive properties to the BioOss® material that has so far only been of osteoconductive nature.

[0092] Furthermore, this experiment reproduces the results of the first test already analyzed above: Commercial BioOss® shows no substantial stimulation of alkaline phosphatase while BioOss1 produced by way of the method according to the invention shows an activation of alkaline phosphatase activity that is almost twice as strong. Likewise, the “algae hydroxyl apatite materials” NA1 and in particular NA2 produced by way of the method according to the invention show an even more potent stimulation of alkaline phosphatase activity.

[0093] In this comparative experiment there is again no reliable indication as to a significantly different effect of the tested bone replacement materials on cellular total protein production in the cell supernatants in the reaction chambers (see FIG. 4).

[0094] 3. Test, Reproduction of the Results

[0095] In another approach all experiments are once more repeated with different primary human osteoblastic cells of a third healthy donor and the results are reproduced in a consistent manner. Hence, in this case the effects of all materials hitherto produced according to the method pursuant to the invention are again reproduced in parallel in a further test approach and compared with the effects of commercially available bone replacement materials.

[0096] The alkaline phosphatase activities were again measured in the cell culture supernatants in the reaction chambers.

[0097] Alkaline Phosphatase

TABLE-US-00007 Cells alone BioOss ® BioOss1 Algipore ® NA1 NA2 566 +/− 488 +/− 708 +/− 683 +/− 683 +/− 867 +/− 32% 14% 18% 31% 61% 63%

[0098] These results are illustrated graphically in FIG. 5.

[0099] Cell Division/Cell Protein

TABLE-US-00008 Cells alone BioOss ® BioOss1 Algipore ® NA1 NA2 96 +/− 113 +/− 98 +/− 103 +/− 100 +/− 102 +/− 5% 4% 5% 3% 7% 3%

[0100] These results are illustrated graphically in FIG. 6.

[0101] Interpretation

[0102] In this experiment, too, the bone replacement materials NA2 and BioOss1 “activated” by the method according to the invention prove to be of significantly stronger effect as to the stimulation of alkaline phosphatase activity than the commercially available bone replacement materials BioOss® and Algipore®. The NA1 material treated in an initial process shows an effect comparable to the commercially available Algipore® bone replacement material.

[0103] Consequently, every bone replacement material “activated” by the production method according to the invention proves to be superior to the previous products with regard to the stimulation of the osteoblastic standard bone formation marker alkaline phosphatase.

[0104] The production method according to the invention is therefore suitable to produce an activated bone replacement material both during a conversion process from a calcium carbonate or respectively a mixture of portlandite, calcium oxide and calcite into an apatite material and directly from an existing hydroxyl apatite material by incorporating activated ions, such as strontium ions, into the crystal lattice. Within the framework of the invention a material can in particular be considered as activated that has osteoinductive or respectively osteotropic or also antiresorptive properties.

[0105] Consequently, by the method according to the invention and respectively accordingly by the osteotropic bone replacement material produced using the method according to the invention a material is provided that has long-acting and localized osteoinductive or respectively osteotropic properties and is excellently suitable as implant material in bone tissue.