The invention relates to a bone graft substitute according to the preamble of claim 1 and a method for manufacturing a bone graft substitute according to the preamble of claim 24.
The following definitions shall be used throughout the description:
Resorption=degradation=process by which a material is removed from the human body.
Macropores=here, we define macropores as pores that have a diameter superior to 30-50 microns
Micropores=pores with a diameter in the range of 0.1 to 20-30 microns
Nanopores=pores with a diameter smaller than 100 nm
Tortuous=tortuous pores are pores that do not have a straight shape (e.g. cylindrical, sphere), but a complex shape, such as a helix, with a large aspect ratio (ratio between the longest and the shortest pore dimension).
Tortuosity=Tortuosity is defined as the ratio between the distance required to join two points in a porous structure through the porous network and the direct distance (with a straight line). Tortuosity values are by definition larger than 1 and often larger than 3.
Calcium phosphate bone graft substitutes have proved to be very good bone graft substitutes: the materials have an excellent biocompatibility and depending on their exact composition, might also be degraded over time and replaced by new bone. One particularly successful material is β-tricalcium phosphate [β-Ca3(PO4)2] or shortly [β-TCP].
In past years, many studies have showed the importance of calcium and phosphate ions on the cellular response of bone cells, such as osteoblasts (“bone-forming cells”) and osteoclasts (“bone-resorbing cells”). For example, it is known that a small increase of calcium concentration down-regulates osteoclast activity and up-regulates osteoblast activity. Also, it has been shown that increased calcium ion concentrations could trigger osteoblasts to produce bone morphogenetic proteins such as BMP-2 and BMP-4. We have therefore surprisingly found that calcium phosphate bone graft substitutes can be used as drug delivery systems (Ca and phosphate ions being the drugs). The control of calcium and/or phosphate ions release enables also a control of the in vivo properties of calcium phosphate materials.
Generally, it is desirable to have a cell-mediated degradation (e.g. osteoclasts) rather than having a purely physico-chemical degradation, i.e. dissolution, because a cell-mediated degradation ensures that material degradation is not too fast compared to bone formation. However, by just relying on cells to reach material degradation and hence calcium and phosphate release, it is not possible to control the up-regulation or down-regulation of cells in the close surroundings of the material.
It is an object of the invention to provide a bone graft substitute in the form of an implantable three-dimensional scaffold comprising calcium phosphate and having pores and which is impregnated with a calcium and/or phosphate containing substance whereby the dissolution rate DRS of said scaffold is slower than the dissolution rate DRD of said calcium and/or phosphate containing substance.
The advantage of the bone graft substitute according to the invention lies in the improved in vivo response of calcium phosphate bone substitutes through selective calcium or phosphate release.
It is a further object of the invention to provide a method for manufacturing a bone graft substitute characterized by impregnating a three-dimensional scaffold comprising calcium phosphate having interconnected pores with a calcium and/or phosphate containing substance; whereby the chemical composition and integrity of said scaffold remains essentially unaffected by said impregnation with said calcium and/or phosphate containing substance. The impregnation can be effected e.g. by spraying, soaking, tipping.
It is a further object of the invention therefore to load a matrix or scaffold that is degraded by cells like β-TCP with a compound that can spontaneously dissolve in vivo, like calcium chloride (CaCl2). The main condition for that purpose is to use a compound that is soluble in vivo. Further in the text, the term of “scaffold” will be used to designate a material resorbed by cell-mediation and the term of “drug” when reference is made to the compound that is soluble in vivo and contains calcium and/or phosphate ions.
Typical calcium phosphate bone graft materials of interest for the scaffold (beside β-TCP) are hydroxyapatite (Ca5(PO4)3OH; HA; sintered or non-sintered), dicalcium phosphate (CaHPO4; DCP), octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP), α-tricalcium phosphate (α-Ca3(PO4)2; α-TCP), α-calcium pyrophosphate (α-Ca2P2O7; α-CPP), and β-calcium pyrophosphate (β-Ca2P2O7; β-CPP). Of interest are also all calcium phosphates having the general apatite structure according to x-ray diffraction, but not having the exact stoichiometry of hydroxyapatite. This includes for example calcium-deficient hydroxyapatite (Ca9(PO4)5(HPO4)(OH); CDHA—sometime called “tricalcium phosphate”), carbonated apatites, and more generally all ion-substituted apatites.
All potential scaffolds can also contain some foreign ions in their structure (not only hydroxyapatite). Surprisingly it has been found that many ionic substitutions exist in calcium phosphates. Of particular interest are Mg, Sr, Zn, Si, Na, K, Li and Cl as potential ions for b-TCP, b-CPP, a-CPP and a-TCP. For HA, OCP, DCP and DCPD, the latter ions as well as CO3−2 ions or SO4−2 ions can be used.
Typical calcium-containing ionic materials that can be used as calcium “drug” are calcium chloride (anhydrous: CaCl2, monohydrate: CaCl2.H2O, dihydrate: CaCl2.2H2O, or hexahydrate: CaCl2.6H2O), dicalcium phosphate dihydrate (CaHPO4.2H2O; DCPD), calcium sulphate dihydrate (CaSO4.2H2O; CSD), calcium sulphate hemihydrate (CaSO4.½H2O; CSH), calcium sulphate (CaSO4), calcium acetate (anhydrous: Ca(C2H3O2)2, monohydrate: Ca(C2H3O2)2.H2O, or dihydrate Ca(C2H3O2)2.2H2O), calcium citrate (Ca3(C6H5O7).4H2O), calcium fumarate (CaC4H2O4.3H2O), calcium glycerophosphate (CaC3H5(OH2)PO4), calcium lactate (Ca(C3H5O3)2.5H2O), calcium malate (dl-malate: CaC4H4O5.3H2O, 1-malate: CaC4H4O5.2H2O, or malate dihydrogen: Ca(HC4H4O5)2.6H2O), calcium maleate (CaC4H2O4.H2O), calcium malonate (CaC3H2O4.4H2O), calcium oxalate (CaC2O4), calcium oxalate hydrate (CaC2O4·H2O), calcium salicylate.(Ca(C7H5O3)2-2H2O), calcium succinate (CaC4H6O4.3H2O), calcium tartrate (d-tartrate: CaC4H4O6.4H2O; dl-tartrate: CaC4H4O6.4H2O; mesotartrate: CaC4H4O6.3H2O), and calcium valerate (Ca(C5H9O2)2).
Typical phosphate-containing ionic materials that can be used as phosphate “drug” are DCPD, sodium phosphate (Na2HPO4, NaH2PO4 or a mixture thereof; non-hydrated or hydrated species like Na2HPO4.2H2O, Na2HPO4.7H2O, Na2HPO4.12H2O, NaH2PO4.H2O, NaH2PO4.2H2O), calcium glycerophosphate (CaC3H5(OH2)PO4), potassium orthophosphate (K3PO4), dihydrogen potassium orthophosphate (KH2PO4), monohydrogen potassium orthophosphate (K2HPO4), and sodium orthophosphate (Na3PO4.10H2O and Na3PO4.12H2O).
The solubility of the drug in an aqueous solution having a physiological ionic strength (0.15M) and a pH of 7.4 at 37° C. should be in an adequate range, typically superior to 2 mM, preferably superior to 10 mM. An adequate range appears to be between 10 mM and 1M.
It is particularly useful to have a rather low solubility in physiological conditions because the release rate is accordingly low. However, a low solubility is not adequate for loading because the loaded amount is limited. So, compounds that present a rather low solubility at physiological conditions and a high solubility in other conditions (e.g. Na2HPO4.12H2O is much more soluble at 90° C. than at 37° C.) are interesting because loading can be made in these advantageous conditions and release in physiological conditions is still slow.
Porosity and Pore Size
To load the scaffold with calcium and/or phosphate ions, it is necessary to have a porous scaffold, preferably a scaffold with interconnected pores to allow drug invasion into the pores. A porosity in the range of 40 to 95%, preferably of 55 to 80% is advantageous. It is important to have a slow release, hence implying that the pores should be relatively small (the smaller they are, the slower the release of calcium and phosphate ions will be). Therefore the scaffold should preferably contain micropores or even nanopores. Ideally, at least 10% of the total volume (preferably 20%) should be constituted of micropores or nanopores. It is further advantageous to have tortuous pores. Tortuosity values larger than 5 are preferred.
The ideal pore size depends on the purpose of the bone graft substitute. Small pores (or a large specific surface area) will favor a rapid resorption. So, the resorption rate will increase in the order macropore<micropore<nanopore. However, since the scaffold is meant to be resorbed by cells, a fast resorption also requires the presence of cells. In other words, scaffolds that have a size superior to a few millimeters should preferably have an interconnected porous network with interconnections larger than 30 to 50 microns to allow bone vessel ingrowth hence leading to rapid bone ingrowth and scaffold resorption. In this case, it is important to have about 30 to 70% of the scaffold volume constituted of macropores, preferably 40 to 60%.
Two main loading methods can be used for manufacturing the bone graft substitute according to the invention
(i) Soaking in a Concentrated Drug Solution—
The first possibility is to create a solution containing calcium and/or phosphate ions, soak the porous scaffold into this solution, and let it dry. The pores are then filled with the salt used for the preparation of the calcium and/or phosphate containing solution.
In that respect, it is advantageous to soak the scaffold in a small amount of solution (for example by placing the scaffold vertically into a solution—the solution reaching only the bottom third of the scaffold) and let this dry. During drying, there is constantly a capillary rise from the solution to the top of the scaffold, leading finally to a very large loading of the scaffold with the soluble calcium and/or phosphate entities.
The temperature of the soaking solution is important. Some compounds are much more soluble at low or at high temperature in water. So, it can be advantageous to prepare a solution at e.g. 80° C. and perform the impregnation and drying at the latter temperature. For other compounds, it can be advantageous to soak the sample with a cold solution (e.g. of 5° C.) and then perform drying at e.g. 60° C. So the solution, soaking and drying temperatures can be varied and the temperature at which the impregnation solution is prepared is of some importance due to the temperature dependence of some solubilities.
It is further advantageous to dry the soaked sample in such a way that the solution can only evaporate through the sample (or scaffold). The beaker or flask containing the soaking solution should be preferably fully covered with a water proof membrane (or material) except where the scaffold is. This approach improves the soaking efficiency.
(ii) Soaking the Scaffold with a Slurry—
The second possibility is to create a slurry containing drug particles and soak the scaffold with the slurry. A requirement is to have drug particles small enough to penetrate the scaffold porosity. Impregnation may be performed under vacuum or under varying pressure cycles, e.g. vacuum—room pressure cycles.
The procedures of impregnation and drying can require different conditions. Impregnation is preferably performed at slower rate than drying (drying may start when there is no more liquid surrounding the scaffold, but still some liquid within the scaffold).