- BACKGROUND ART
The invention relates to a resorbable bone substituent material made of calcium phosphate particles of different phases which are embedded in a collagen matrix cross-linked being specific to the invention.
Bone defects which cannot be closed in natural healing through the organism require the application of bone substituent materials which can be of natural or artificial origins, and which will fill the defect and be restructured to endogenous bone.
The so-called “golden standard” represents the utilization of autologous bones then, i.e. the patient will be removed a piece of bone from the sound area, e.g. from the iliac crest (crista iliaca), which is used to fill the defect. Having greater bone defects or an inappropriate autologous bone, it is also fallen back upon foreign bone material which will be reconditioned accordingly. The advantage of providing foreign bones in larger quantities is confronted with their disadvantage that varying reconditioning processes may result in different mechanical properties (Palmer S. H., Gibbons C. L. M. H., Athanasou N. A.: JBJS (Br) 1999, 81-B, 333-5). Moreover, with foreign bones infections cannot be excluded (Boyce, T., Edwards J., Scarborough N.: Orthop. Clin. North Am. 1999, 30(4), 571-81). For the application of a debiologized animal bone, the same objections apply as for the application of foreign bones such that various synthetic bone substituents have been developed.
The advantages of the synthetic bone substituents are in that checking the chemical composition and structure exists in addition to the availability, and influencing the biologically effective properties can be carried out such that an optimum course of therapy is achieved.
The plurality of the synthetic bone substituents comprise calcium minerals such as calcium carbonate, calcium sulphate and different calcium phosphates. In the past, particularly calcium phosphates such as β-tricalcium phosphate and hydroxylapatite have been used, wherein hydroxylapatite is an essential constituent of the natural bone (Dorozhkin S. V., Epple M.: Angew. Chemie 2002, 114, 3260-77).
Calcium phosphate based bone substituent materials allow to be made in various embodiments for different ranges of application. Granulates are used for dental bone defect filling, e.g., whereas injectable cements are used with the stabilization of vertebral bodies. Load-carrying moulded ceramic articles are inserted with defects of the skull and the great long bones.
In the load-carrying case, particular difficulties arise for bone substituent materials made of calcium phosphates. The necessary structure is allowed to carry defined maximum forces only, and the naturally available brittleness of synthetic calcium phosphates has to be compensated by an adapted producing process. The increase of strength is achieved, e.g. by sintering. Disadvantageously, the sintered calcium phosphates are reabsorbed then substantially more slowly than it would correspond to the normal course of therapy (LeGeros R. Z.: Clinical Materials 1993, 14, 65-88). Moreover, with sintering loss of nanoporosity is notched up.
Another possibility to increase the strength of calcium phosphate based bone substituent materials is compression by cold isostatic pressing. However, the porosity of these mouldings then gets largely lost such that, for compensating this effect, macroscopic structuring has to be provided by mechanical remachining, e.g. bores (Tadic D., Epple M.: Biomaterials 2003, 24, 4565-71).
If the mechanical stressability does not play the important part, then calcium phosphate based bone substituents can be produced with further processes permitting a better adjustment of the porosity. If one confines to inorganic components as material constituents, then in particular sol-gel processes are just right to generate open-pored network structures (Brinker C J., Scherer G. W.: Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing, Academic Press, 1990).
In addition, if organic components are used as binding agents, then mouldings are allowed to be generated both in immediately shaping processes (Michnaa S., Wua W., Lewis J. A.: Biomaterials 2005, 26, 5632-9), e.g. as a three-dimensional grid pattern, and produced by press forming (Weihe S., Wehmöller M., Tschakaloff A., von Oepen R., Schiller C., Epple M., Eufinger H.: Mund-, Kiefer-, Gesichtschirurgie 2001, 5: 299-304). The porosity of these mouldings has to be set by the shaping process itself or by mechanical machining (D. Tadic D., M. Epple M.: Biomaterials 2003, 24, 4565-71).
Through the application of bone substituents which comprise adjustable components of calcium phosphates having different solubilities within the body fluid and thus various resorbence rates, the course of resorbence can be configured in a defined manner. As a result, the strain is additionally supported by the bone substituent material over a time period, in which new bone tissue is developing and solidifying. The calcium phosphates with less solubility will be completely reabsorbed later during remodelling of the bone (LeGeros R. Z.: Clinical Materials 1993, 14, 65-88).
In summary, it is to be noted that calcium phosphate based bone substituent materials are successfully used for the treatment of simple defects. However, at the moment application with heavy defects is not carried out due to of material-related problems. These problems above all rest in that the actual processes are not able to meet substantial requirements on bone substituent material and defect filling material bodies formed from it, respectively:
(1) on the one hand, to produce positive bone substitute mouldings, i.e. shaped anatomically and according to the defect, respectively, which completely fill the bone defect perfectly and non-positive as well, and
(2) on the other hand, to implement an easily resorbable structure.
- DISCLOSURE OF THE INVENTION
Moreover, with the application of the actual manufacturing processes it is very difficult to fit into the bone substituent material additional organic or inorganic matters having strongly varying concentrations as well as to provide an improvement of the load-carrying properties by means of embedded, permanent and resorbable supporting structures, respectively.
The problem formulation according to the invention is producing a positive non-brittle bone substitute moulding, i.e. being shaped anatomically and according to the defect, respectively, which completely fills the bone defect perfectly and which is resorbable as well.
The solution of the problem formulation is achieved by producing the bone substituent from a mixture of calcium phosphate particles embedded into a collagen matrix being cross-linked according to the invention.
In particular, collagen cross-linking is achieved by laccase-induced peptide cross-linking and suitable bridging molecules.
As bridging molecules the substituted dihydroxy aromatics and/or substrates of lygnolytical polyphenol oxidases such as laccases are generally suitable.
Therefore, monocyclic orthodihydroxy aromatics, monocyclic paradihydroxy aromatics, bicyclic monohydro aromatics, polycyclic monohydroxy aromatics, bicyclic dihydroxy aromatics, polycyclic dihydroxy aromatics, bicyclic trihydroxy aromatics, polycyclic trihydroxy aromatics or mixtures thereof are used. In contrast to the well-known mussel glues, the hydroxyl aromatics according to the invention are not part of a polymer chain.
These aromatics may be further substituted. Preferred functional groups are substituents selected from the group consisting of halogen, sulfo, sulfone, sulfamindo, sulfanyl, amino, amido, azo, imino and hydroxy. Then, it is to ascertain that substituted aromatics, in particular substituted dihydroxy aromatics, have very favourable polymerization properties such as fast polymerization, low natural linkage and good strength of cross-linking. Appropriate substitution of the aromatics results in that monohydroxy aromatics are also suitable as bridging molecule for cross-linking. Within the scope of this invention, substitution means that in addition to the hydroxyl groups 1, 2, 3 or 4 further groups are bonded to the aromatics. Further, monohydroxylated biaryl compounds are also suited as bridging molecules.
Particularly preferred as a bridging molecule are phenol derivates which comprise a hydroxyl group or a methoxy group corresponding to the formulas 1 and 2, on the ortho position or para position,
n=0-10, preferably 0 or 1, in particular 0;
R1═OH or NH2 or hal, preferably OH, Cl or Br, in particular OH;
R2═H, CH3, CHO, COCH3, CONH2, CON-alkyl, CON-alkyl-OH, COOH, COO-alkyl, alkyl, substituted aromatic, in particular CON-alkyl or COO-alkyl, and
R3═H, CH3, alkyl, substituted aromatic, in particular H or CH3.
Herein, alkyl means branched or non-branched aliphatic hydrocarbon chains having preferably 1 to 20 carbon atoms, more preferably 1 to 6 carbons, e.g. methyl, ethyl propyl, butyl, isobutyl, n-pentyl, n-hexyl.
Possible bridging molecules furthermore are compounds of formula 1 and of them the hydroquinone, which may be further substituted. Seen from a point of view of fast bonding reaction, if possible, substituted dihydroxy aromatics having a low natural linkage are particularly appropriate according to the invention. 2,5 dihydroxybenzamides are preferably used, wherein 2,5-dihydroxy-N-2-hydroxyethylbenzamide is particularly preferred.
In the case of aromatic trihydroxy compounds it is preferred that there are not more than two hydroxyl groups per unit of benzene. Particularly preferred are polyphenyls, i.e. biphenyl or triphenyl of the following formula 3:
- R1═H and R2═OH or R1═OH and R2═H.
n=0-10, preferably 0 or 1,
Then, the phenyls of formula 3 may be substituted, e.g., in the ortho position into a hydroxyl group having CH3, CHO, COCH3, CONH2, CON-alkyl, CON-alkyl-OH, COOH, COO-alkyl, alkyl, substituted aromatic in particular CON-alkyl or COO-alkyl and/or in the metha position into a hydroxyl group having CH3, alkyl, substituted aromatic in particular CH3.
Cross-linking of collagen occurs according to the invention under the influence of polyphenol oxidases such as lignolytic polyphenol oxidases, in particular laccases (EC 184.108.40.206). Laccases are known as cross-linkers. They are allowed to arise from plants, mushrooms, bacteria or insects or be derived from natural enzymes. The laccases to be used within the scope of this invention may be produced recombinantly or can be cleaned up.
Examples thereof are laccases being extracted from the species of aspergillus, neurospora, podospora, botrytis, collybia, fomes, lentinus, pleurotus, pycnoporus, pyricularia, trametes, rhizoctonia, coprinus, psatyrella, myceliophthora, schtalidium, polyporus, phlebia or coriolus. The preparation of laccases is disclosed in EP 0947142.
By the application of polyphenol oxidases such as lignolytic polyphenol oxidases, preferably laccase (EC 220.127.116.11), the substrate spectrum thereof can be employed for the cross-linking reaction. Therefore, the invention particularly distinguishes in that a wide range of bridging molecules can be used for cross-linking.
Variations of the concentrations approximately of 1 to 50 mM are possible both with the individual component collagen and the bridging molecule matter.
Then, it must be considered, that according to the selected bridging molecule, an interfering natural reaction of the bridging molecule takes place decreasing the formation of cross-linking bonds. An excessively low concentration of the bridging molecules results in a too slow reaction, an excessively high concentration results in stronger side reactions through natural linkage. The concentration of the polyphenol oxidase influences the reaction rate wherein, according to application, faster cross-linking or a longer processability of the combination can be achieved by varying the concentration. A preferred volume ratio for cross-linking of soft tissues, for example, is implemented in the formulation of 8.5 nM of collagen, 12.5 mM of 2,5-dihydroxy-N-2-hydroxyethylbenzamide, 0.32 U (156 nmol ml−1 min−1) of polyphenol oxidase.
Cross-linking of a collagen matrix by means of bridging molecules with embedding calcium phosphate particles of different phases and concentrations into the matrix results in a solid material with the suitability as bone substituent.
The polyphenol oxidase and the polyphenols are dissolved preferably in phosphate buffer such as calcium phosphate or sodium phosphate buffer or PBS. The consistency of the used components of laccase and polyphenol is from liquid to pasty. The viscosity of the used individual components can be varied by solvents. The concentration of the solvents influences cross-linking.
Cross-linking reaction preferably occurs at pH value of 5 to 7. The reaction is allowed to proceed within the temperature range of 2 to 80 degrees centigrade, however, a temperature is preferred within the range of 20 to 37 degrees centigrade, in particular within the range of 25 to 30 degrees centigrade.
In support of cross-linking another shorter-chain peptides can be used. In a particularly preferred embodiment of the invention about 50 percent of the amino acids of the peptide consist of lysine. Lysine and another amino acid may be arranged as a repeating dipeptide unit. Another succession or absorbing of further amino acids of, in particular arginine, asparagine, glutamine or histidine (instead of lysine or in addition thereto), serine or threonine (instead of tyrosine or in addition thereto), of cysteine or other amino acids is also possible.
In a likewise preferred embodiment it exclusively concerns with polymers consisting of two amino acids such as (lysine tyrosine)n, wherein n can assume values of between 5 and 40 such as 5, 10 or 20.
Furthermore, (lysine tyrosine), is advantageously used as a peptide. With the use of (lysine tyrosine)n within the matrix, lysine exists in a high concentration in the bone substituent material and has a positive effect on the proliferation and differentiation of bone cells.
Prior to cross-linking, the substance mixture has viscous properties and can be arbitrarily shaped out. Then, simple shapes such as globules as well as complex mouldings can be represented.
Furthermore, filling the porous mouldings with the viscous material and curing thereof is possible.
In one preferred embodiment the material is configured as an interconnectingly porous body.
Preferably, the part by weight to the total weight of calcium phosphate particles is 70 to 95 percent, of collagen and oligopeptides is 5 to 15 percent if necessary, and of the bridging molecule substance is about 0.5 to 5 percent.
The bone substituent materials according to the invention are allowed to include further substances. In a preferred mode of implementation the material additionally includes SiO2.
The bone substituent material can be doped with antibiotics. Then, antibiotics having free amino groups which have been mixed to the bone substituent prior to crosslinking will be bonded to collagen during the laccase-induced cross-linking. On that occasion, it could be demonstrated that the collagens doped in this manner with antibiotics have a strong antimicrobial effectiveness.
The bone substituent material can be inserted with and without a supporting structure. If a resorbable organic material is inserted as supporting structure, it is of particular advantage according to the invention to select an organic material carrying free amino groups. In this case, cross-linking occurs by means of bridging molecules not only toward the collagen but toward the supporting structure as well such that a solid connection is obtained, however, which will be substituted by endogenic tissue during the healing process. The mechanism of resorbence can be in a hydrolytical or enzymatical manner. In particular, the peptides can be decomposed, and individual fragments can be taken away and be separated. Alternatively, the fragments or amino acids are also allowed to be incorporated into the regenerating tissue.
In a further embodiment, the porosity of the calcium phosphate moulding is directed. Consequently, the mechanical properties of the resulting composite moulding are depending on the direction as well.
By cross-linking the contacting surfaces of two mouldings having directionally depending mechanical properties which result in a solid connection of the mouldings, the directional dependence of the mechanical properties of the resulting moulding will be reduced.
WAY (S) FOR IMPLEMENTING THE INVENTION
By means of the solid combination of a plurality of two-dimensional mouldings through cross-linking of their paired contacting surfaces, a multi-layer composite moulding is made which has a high mechanical stressability.
- Second Embodiment
A mixture from particles of different modifications of calcium phosphate and collagen is homogeneously mixed with 2,5 dihydroxy-N-2-hydroxyethylbenzamide such that a concentration of 12.5 mM is achieved. Cross-linking is initiated by adding of laccase having an activity of 0.32 U (156 mmol ml−1 min−1). After curing of the adhesive the mineral constituents together with the adhesive material form, depending on the solid matter components, a high viscous or solid material.
- Third Embodiment
An experimental arrangement such as in the first embodiment is selected, however, in addition to the collagen an oligopeptide (length of 2 to approx. 100 amino acids, preferably approx. 4 to appr. 20 amino acids) is added. Compared with usual proteinaceous amino acids, modified and atypical amino acids, respectively, such as hydroxylisine can also be comprised in the oligopeptide. Lysine containing oligopeptides are preferably used, and it is of particular advantage when lysine is approximately 50 percent of the amino acids of the peptide. Additionally, approximately 50 percent of the amino acids of the peptide may be tyrosine. Lysine and tyrosine may be arranged, e.g. as a repeating dipeptide unit. In particular, by adding peptides which consisted of repeating dipeptide units of lysine and tyrosine ([lys-tyr]n or [tyr˜lys]n, n=5 or n=10, good cross-linking has been achieved with the incorporation of the collagen and inclusion of the calcium phosphates.
- Fourth Embodiment
The oligopeptides described in the second embodiment are received in PBS (phosphate buffered saline, 2.7 M NaCl2, 54 mM KCl, 87 mM Na2HPO4, 30 mM KH2PO4, pH 7.4) and laccase is added (Component 1). 2,5-dihydroxy-N-2-hydroxyethyl-benzamide is dissolved in PBS (Component 2). After combining both components, mixing with particles of different modifications of calcium phosphate and collagen is carried out. The quantity of solvent is selected minimally in order to achieve a solution of the components as concentrated as possible.
The viscous mixture of particles of different modifications of calcium phosphate and collagen, if necessary, and of further oligopeptides according to the second embodiment 2,5-dihydroxy-N-2-hydroxyethyl benzamide and laccase, is inserted by means of capillary action, pressurizing or vacuum into the pores of an interconnectingly porous brittle moulding prior to sintering based on calcium phosphate, which after curing forms a solid composite moulding in compound with the CaP moulding. In a specific embodiment, the interconnectingly porous moulding is formed such that the inner surface thereof is largely covered with collagen being locally determined, cross-linked.
- Fifth Embodiment
After cross-linking initiated by laccase, a material being interconnectingly porous with regard to both the mineral and the collagen is obtained. When implanted, due to its osteoconductive action this material serves as a biological guide bar for the regeneration of missing bone and thus for healing the defect.
- Sixth Embodiment
A composite moulding according to the third embodiment is prepared with the specific feature that a simply open or multiply open hollow body or a closed hollow body is created by means of a multilayer cross-linking of contacting surfaces. After the implantation the recovery of the bone defect occurs while maintaining dimensional stability.
- Seventh Embodiment
Preparation of a bone substituent according to the first embodiment with the specific feature that an amino group carrying antibiotic has been homogeneously distributed in the mixture before adding of laccase. After cross-linking with the laccase the collagen is doped with the antibiotic. Thus, a bone substituent is formed which is active ingredient loaded both inside and on the surface.
Preparation of a multilayer composite moulding according to the sixth embodiment with the specific feature that identical or different active ingredients will be embedded into one or several layer boundaries.