Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Scleroproteins – e.g. – fibroin – elastin – silk – etc.
Reexamination Certificate
1999-03-24
2002-05-07
Low, Christopher S. F. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Scleroproteins, e.g., fibroin, elastin, silk, etc.
C530S402000, C514S002600, C514S021800, C514S801000, C435S069100
Reexamination Certificate
active
06384196
ABSTRACT:
DESCRIPTION OF THE INVENTION
The invention relates to a process for the preparation of mineralized collagen fibrils, comprising forming the fibrils and mineralizing them in one process step. The invention farther relates to a process for the preparation of a collagen/calcium phosphate composite starting from mineralized collagen fibrils which are embedded in a calcium phosphate cement. The latter is then used as bone substitute material.
Collagenous tissue has many advantages as biomaterial compared with inorganic material. Collagen is an important protein in animals and accounts for about 30% of the proteins in vertebrates. It has the ability to assemble in vitro to fibrils, and these can be prepared in various modifications. In human bone, collagen represents, as structural material, the organic matrix in which minerals consisting of calcium, phosphate, hydroxyl, fluoride and carbonate ions, etc. are embedded. The similarity to human tissue affords an important advantage by comparison with alternative materials. This advantage is utilized for producing prostheses and biomaterials.
Numerous prior art documents dealing with the mineralization of collagen are known.
GB 1068 587 (Baxendale et al., 1963) discloses soluble collagen (from rat tails), which is reconstituted and mineralized by immersion in a supersaturated calcium phosphate solution. Assembly in the presence of ATP or chondroitin sulphate results in so-called SLS or FLS respectively, (cf. G. Reich “Kollagen”, Theodor Steinkopff, Dresden (1966) p. 134 et seq.) and not, as erroneously assumed, in collagen fibrils with a 64 nm periodicity.
U.S. Pat. No. 5,320,844 (Lui, 1992) describes crosslinked collagen which is dispersed in an acidic solution and is mineralized by adding a calcium and phosphate solution with neutral pH. Either both solutions are added simultaneously, or one of the two solutions is previously mixed with the collagen.
U.S. Pat. No. 5,455,231, U.S. Pat. No. 5,231,169 and WO 93/12736 (Constantz et al.) describe a crosslinked collagen which is solubilized in basic solution, and subsequently a calcium and a phosphate solution is added dropwise over several hours. No assembly step takes place here.
U.S. Pat. No. 5,532,217 (Silver et al., 1995) discloses collagen fibres which result from an acidic collagen solution which is extruded in a neutralizing buffer. The fibres, which have a diameter of from 20 to 500 &mgr;m are subsequently mineralized, for example, in a double diffusion chamber.
The present invention has an object of providing a process for the preparation of mineralized collagen fibrils resulting in a homogeneously mineralized collagen gel. It should be used only recombinant, not natural collagen.
Another objective is to provide a process for the preparation of a collagen/calcium phosphate composite using the mineralized fibrils, and ensuring a better union between collagen and calcium phosphate cement.
It has been possible to achieve the first object in a surprisingly simple manner by a process for the preparation of mineralized collagen fibrils in which fibril formation and mineralization take place in one process step. It has been possible to achieve the second object by a process for the preparation of a collagen/calcium phosphate composite by embedding the mineralized collagen fibrils in a calcium phosphate cement in a particular ratio by weight.
The mineralized collagen fibrils are prepared from soluble collagen, which allows a higher degree of purification by comparison with the use of insoluble collagen.
The possibility of reconstituting fibrils from soluble collagen has been known for a long time (A. Veis, K. Payne, “Collagen Fibrillogenesis” from “Collagen” ed. M.E. Nimni Vol. 1, Biochemistry, CRC Boca Raton 1988).
Uncrosslinked collagen is soluble at low pH (pH ≈2) and assembles to fibrils at neutral pH. Reconstitution takes place by mixing an acidic collagen solution with a neutral buffer solution. Suitable as acidic collagen solution are all collagen solutions which comprise dissolved calcium salt (e.g., calcium chloride) at an acidic pH, preferably pH=about 2. All neutral buffer solutions are suitable as buffer solutions. Any of the types of soluble (on a molecular level) collagens routinely used for producing prosthesis and biomaterials, e.g., as cited herein, can be used in the method of the invention.
The collagen fibrils are mineralized by precipitating calcium phosphate from a supersaturated calcium phosphate solution. The supersaturation is produced by mixing a calcium component and a phosphate component. By mixing the calcium component and the phosphate component at the same time with a collagen solution and the buffer solution there is simultaneous initiation of both processes. A suitable sequence of the processes must be achieved by a suitable choice of the parameters, that is to say the fibril formation must start before the mineralization because the collagen fibrils act as template (substrate) for the mineralization. At the same time, the formation of a dense collagen structure must be restricted to such an extent that adequate diffusion of calcium and phosphate ions into the collagen fibrils is possible within a time which is worthwhile in practice. The required sequence of processes is achieved within a narrow range of parameters. The applied parameters are dependent upon one another, e.g., if a higher pH is applied, the calcium and phosphate concentrations are lower, etc. Suitable combinations can be determined with at most a few routine experiments. In general, preferred ranges of the parameters are: pH about,5-10 (after mixing both components); calcium concentration about 1 mM-50 mM; phosphate concentration about 1 mM-50 mM; and buffer concentration about 10 mM-200 mM (at least the concentration of the acid in which the collagen is dissolved). In a most preferred embodiment, calcium and phosphate solutions are in concentrations such that the solution is supersaturated with respect to hydroxyapatite (in the neutralized state), yet the concentrations are low enough so that the precipitation occurs, not immediately, but after an induction period which allows collagen fibrils to form. The resulting collagen fibrils have, in contrast to collagen fibres, a diameter of only about 20-500 nm. During the mineralization it is possible to add to the collagen fibrils according to the invention, if desired, polyaspartate, polyglutamate, polyphosphoserine, other polycarboxylates or non-collagenous proteins or phosphoproteins, or any combinations thereof, in order to improve the mineralization and influence the kinetics of the mineralization process. Bone growth factors which stimulate new bone formation can also be added, e.g., bone morphogenetic proteins (BMP), transforming growth factors (TGF), vascular growth factors (VGF), or growth and differentiation factors (GDF).
It is furthermore possible according to the invention to embed the mineralized collagen fibrils in a calcium phosphate cement in a second process step. The calcium phosphate cement is prepared from tetracalcium phosphate, calcium hydrogen phosphate, calcium hydroxyapatite, calcium dihydrogen phosphate, calcium carbonate, sodium phosphates and/or mixtures thereof, with the collagen:calcium phosphate (CaP) ratio in the resulting composite being about 1:200 to about 1:1 by weight. The individual components of the calcium phosphate cement are reduced in the solid state to a suitable particle size (e.g., 0.1 &mgr;m to 10 &mgr;m) and converted by mixing with a liquid component into a plastic composition. Curing of the cement mixtures obtained in this way takes place in two steps:
Firstly in air at room temperature for a defined period
Subsequently in an aqueous phase, which may comprise phosphate or other mineral constituents, likewise for a defined period.
The embedding of the mineralized collagen fibrils takes place by mixing them into the cement precursor. The mineralized collagen fibrils can be added in the liquid component of the cement precursor or, in the freeze-dried state, mixed with powdered componen
Bradt Jens
Pompe Wolfgang
Weis Karl
Low Christopher S. F.
Merck Patent Gesellschaft
Millen White Zelano & Branigan P.C.
Mohamed Abdel A.
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