Biomimetic collagen-hydroxyapatite composite material

Abstract

The invention relates to: a biomimetic collagen-hydroxyapatite composite material comprising an at least partially fibrous collagen scaffold including mature native collagen fibers possessing triple helicity as shown by Circular Dichroism Spectroscopy, wherein those mature native collagen fibers are at least partially covered with epitactically grown crystals of nanocrystalline hydroxyapatite, whereby the epitactically grown nanocrystals have the same morphology as human bone mineral and the same size as human bone mineral, i.e. a length of 30 to 50 nm and a width of 14 to 25 nm, a process of preparing the above biomimetic collagen-hydroxyapatite composite material comprising the steps of a) immersing an at least partially fibrous collagen scaffold including the above mature native collagen fibers in a saturated aqueous solution of saturated Ca.sup.2+/Hx-PO4.sup.(3-x) to start the formation process of the composite implant material whereby epitactically grown nanocrystals will be formed on the mature native collagen fibers, the epitactically grown nanocrystals having the same morphology and same size as human bone mineral, b) stopping the formation process of the composite implant material by separating solid material from the aqueous solution, rinsing with water and drying, and c) optionally sterilizing the separated material coming from step b), as well as the use of the above biomimetic collagen-hydroxyapatite composite material as an implant or prosthesis for bone formation, bone regeneration, bone repair and/or bone replacement at a defect site in a human subject or in an animal, or as an implant for combined bone and cartilage regeneration.

Claims

1. A biomimetic collagen-hydroxyapatite composite material comprising an at least partially fibrous collagen scaffold including mature native collagen fibers possessing triple helicity as shown by Circular Dichroism Spectroscopy, wherein those mature native collagen fibers are at least partially covered with epitactically grown crystals of nanocrystalline hydroxyapatite, whereby the epitactically grown nanocrystals have the same morphology as human bone mineral and the same size as human bone mineral, namely a length of 30 to 50 nm and a width of 14 to 25 nm and wherein the epitactically grown crystals of nanocrystalline hydroxyapatite form a layer having a thickness of at least 30?15 nm, as determined by X-ray diffraction analysis.

2. The biomimetic collagen-hydroxyapatite composite of claim 1, wherein the at least partially fibrous collagen scaffold comprises on its external surface at least 2% of mature native collagen fibers as determined by picture analysis on SEM micrographs and Circular Dichroism Spectroscopy.

3. The biomimetic collagen-hydroxyapatite composite of claim 1, wherein the at least partially fibrous collagen scaffold comprises on its external surface at least 10% of mature native collagen fibers as determined by picture analysis on SEM micrographs and Circular Dichroism Spectroscopy.

4. The biomimetic collagen-hydroxyapatite composite material of claim 1 wherein the fibrous collagen scaffold has a w/w ratio to the epitactically grown crystals of nanocrystalline hydroxyapatite between 5:95 and 95:5.

5. The biomimetic collagen-hydroxyapatite composite material of claim 1 which is a shaped body.

6. The biomimetic collagen-hydroxyapatite composite material of claim 5, wherein the shaped body is a bone substitute material the structure of which has the profile of an osseous body part.

7. The biomimetic collagen-hydroxyapatite composite material of claim 5, wherein the shaped body has a resistance to torque of at least 30 Ncm in the dry state.

8. The biomimetic collagen-hydroxyapatite composite material of claim 1 which is membrane-shaped.

9. A process of preparing the biomimetic collagen-hydroxyapatite composite material of claim 1, the process comprising the steps of: a) immersing an at least partially fibrous collagen scaffold including mature native collagen fibers possessing triple helicity as shown by Circular Dichroism Spectroscopy in a saturated aqueous solution of saturated Ca.sup.2+/H.sub.xPO.sub.4.sup.(3-x), which is a phosphate buffer solution (PBS) containing finely dispersed alpha-TCP, beta-TCP, TTCP, octacalcium phosphate pentahydrate, dicalcium phosphate or dicalcium phosphate dihydrate, to perform the formation process of the composite implant material whereby epitactically grown hydroxyapatite nanocrystals are formed on those mature native collagen fibers, those epitactically grown hydroxyapatite nanocrystals having the same morphology and same size as human bone mineral, namely a length of 30 to 50 nm and a width of 14 to 25 nm and wherein the epitactically grown crystals of nanocrystalline hydroxyapatite form a layer having a thickness of at least 30?15 nm, as determined by X-ray diffraction analysis, b) stopping the formation process of the composite implant material by separating solid material from the aqueous solution, rinsing with water and drying, and c) optionally sterilizing the separated material coming from step b).

10. The process of claim 9 wherein in step a) the pH of the aqueous solution remains within a range of 5.5 to 9.0.

11. The process of claim 9 wherein in step a) the pH of the aqueous solution remains within a range of 6.5 to 8.0.

12. The process claim 9 wherein the temperature in step a) is between 25 and 45? C.

13. The process of claim 9 wherein the temperature in step a) is between 35? C. and 42? C.

14. The biomimetic collagen-hydroxyapatite composite material of claim 1, in the form of an implant or prosthesis for bone formation, bone regeneration, bone repair and/or bone replacement at a defect site in a human subject or an animal.

15. The membrane-shaped biomimetic collagen-hydroxyapatite composite material according to claim 8, in the form of an implant for combined bone and cartilage regeneration.

16. A method of promoting bone formation, bone regeneration and/or bone repair at a defect site in a human subject or an animal by implanting the biomimetic collagen-hydroxyapatite composite material of claim 1 in said human subject or animal.

17. The biomimetic collagen-hydroxyapatite composite material of claim 4, wherein the w/w ratio is between 10:90 and 90:10.

Description

(1) The following description will be better understood by referring to:

(2) FIG. 1 which represents a SEM micrograph of the fibrous side of the membrane-shaped collagen scaffold of Example 1c) coated as described in Example 3 b) with a PBS concentration of 0.2 M for a coating reaction time of 12 hours,

(3) FIG. 2 which represents a SEM micrograph of the fibrous side of the membrane-shaped collagen scaffold of Example 1c) coated as described in Example 3 b) with a PBS concentration of 0.8 M for a coating reaction time of 24 hours, and

(4) FIG. 3 which represents a SEM micrograph of the fibrous side of the membrane-shaped collagen scaffold of Example 1c) coated as described in Example 3 b), which has been colonized with MG63 osteoblast-like cells.

EXAMPLE 1

Preparation of an at Least Partially Fibrous Collagen Scaffold

(5) a) Preparation of a Cylindrical Fibrous Collagen Scaffold Derived from Bio-Gide?

(6) Preparation of Cylindrical Pieces of Dried Compacted Collagen

(7) A Bio-Gide? membrane (Geistlich Pharma A. G., Switzerland) was finely grinded using an ultracentifugal mill and sieved on a 2.0 mm sieve. 0.2 g of the sieved collagen was put into 5 ml of 99.9% ethanol and the collagen mass was put with tweezers into a well of a 24-well plate and compacted with a 2.0 mm diameter Teflon cylinder, then extracted from the well with a 2.0 mm diameter swage tool and dried for 4 hours in a chemistry hood. Those operations were performed in parallel 6 times such as to obtain 6 cylindrical pieces of dried compacted collagen.

(8) Crosslinking of the Cylindrical Pieces of Dried Compacted Collagen

(9) 3.571 ml of an EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)ethanol solution were pipetted into a 100 ml volumetric flask and the volume was completed to 100 ml with 99.9% ethanol, then poured into a 200 ml extraction flask to which the 6 pieces of dried collagen obtained above were added. The ethanol was evaporated during 1 minute under vacuum in a desiccator, then the desiccator was put to atmospheric pressure. The mixture was crosslinked under agitation at 110 rpm for 10 minutes using a horizontal shaker. The EDC solution was decanted and the collagen cylinders were put into a 200 ml beaker to which 100 ml PBS (Phosphate Buffer Solution) were added and eliminated for 1 minute under vacuum. The collagen cylinders were cleaned with 100 ml PBS under agitation at 50 rpm for 5 minutes, the PBS being decanted without evaporating under vacuum. This process was repeated 2 times. The collagen cylinders were cleaned with 100 ml 99.9% ethanol under agitation at 50 rpm for 5 minutes and the ethanol being decanted without evaporation under vacuum, the process being repeated two times. The collagen cylinders were cleaned with 100 ml diethylether without agitation and dried for 14 hours in a chemistry hood.

(10) Other experiments were performed with a different time (up to 240 minutes) for the crosslinking reaction.

(11) The % of mature native collagen fibres possessing triple helicity (as shown by Circular Dichroism spectroscopy) of the cylindrical collagen scaffold, as determined by picture analysis using software Phenom Pro (FEI Phenom Pro Tabletop SEM S/N: 0342; Phenom Pro Suite V.1.1.0.920; Phenom Application System PW-220-001; Phenom Fribremetric PW-210-001, available from Phenom World, Dillenburgstraat 9E, 5652 AM Eindhoven, Netherlands) on SEM (Scanning Electron Microscopy) micrographs of 5000 magnification, was about 90%.

(12) b) Preparation of a Cylindrical Partially Fibrous Spongious Collagen Scaffold Derived from a Sponge of Type I and Type II Collagen

(13) Preparation of a Collagen Sponge:

(14) A resorbable extracellular spongious collagen matrix was prepared from frozen cartilage of freshly slaughtered pigs by defatting followed by basic and acidic treatment as described in Example 1 of EP-B1-810888. That matrix was shown by immunological methods to contain a mixture of type I and type II collagen.

(15) Preparation of Cylindrical Pieces of Dried Compacted Spongious Collagen

(16) The spongeous matrix was finely grinded using an ultracentifugal mill and sieved on a 2.0 mm sieve. 0.2 g of the sieved collagen was put into 5 ml of 99.9% ethanol and the collagen mass was put with tweezers into a well of a 24-well plate and compacted with a 2.0 mm diameter Teflon cylinder, then extracted from the well with a 2.0 mm diameter swage tool and dried for 4 hours in a chemistry hood.

(17) Crosslinking of the Cylindrical Pieces of Dried Compacted Spongious Collagen

(18) The cylindrical pieces of dried compacted spongious collagen obtained above were crosslinked with EDC, cleaned with PBS, ethanol and diethylether, analogously to the procedure described above in a) for the fibrous cylindrical collagen scaffold. The % of mature native fibres possessing triple helicity in the cylindrical partially fibrous spongious collagen scaffold, as determined by picture analysis software Phenom Pro on SEM micrographs of 5000 magnification, was about 5%.

(19) c) Preparation of a Membrane-Shaped Fibrous Collagen Scaffold Derived from Bio-Gide?

(20) A Bio-Gide? membrane (Geistlich Pharma A. G., Switzerland) was cleaned with 100 ml 99.9% ethanol under agitation at 50 rpm for 5 minutes and the ethanol being decanted without evaporation under vacuum, the process being repeated two times. The collagen membrane was then cleaned with 100 ml diethylether without agitation and dried for 14 hours in a chemistry hood.

(21) The % of mature native fibres possessing triple helicity in the fibrous side, as determined by picture analysis software Phenom Pro on SEM micrographs of 5000 magnification, was about 100%.

EXAMPLE 2

Preparation of a Bulk Sintered Material of ?-TCP

(22) For a mixture of 500 g (dry weight), 360 g dicalcium phosphate anhydrous powder, 144 g calcium carbonate powder and 220 ml deionized water were mixed for 7 minutes at 500 rpm using a laboratory stirrer. The slurry from the mixing process was immediately transferred into a high temperature stable platinum cup. The filled platinum cup was placed in a cold furnace. The furnace was heated to 1400? C. by using a heating rate of 60? C. per hour. The heating process was stopped after 72 hours by switching off the furnace. The sample was cooled down to room temperature within the furnace. The bulk sintered material (phase pure ?-Ca.sub.3(PO.sub.4).sub.2) was removed from the furnace and the platinum cup. The bulk product from the sintering process had a weight of 420 g (weight loss 16.7%).

(23) The control of phase purity was performed using powder X-ray diffraction analysis.

EXAMPLE 3

Coating of a Fibrous Collagen Scaffold with Epitactically Grown Crystals of Nanocrystalline Hydroxyapatite in a PBS Solution in the Presence of a Dispersion of Fine Particles of ?-TCP

(24) a) Coating of the Cylindrical Pieces of Fibrous Collagen Obtained in Example 1 a) Preparation of a 0.5 M PBS Solution:

(25) 100 ml of a 0.5 M NaH.sub.2PO.sub.4.H.sub.2O solution (solution A) were prepared by dissolving 6.9 g of NaH.sub.2PO.sub.4.H.sub.2O in sterile deionized water at room temperature under agitation at 250 rpm for 30 minutes and at 600 rpm for 4 hours. 100 ml of a 0.5 M Na.sub.2HPO.sub.4.2H.sub.2O solution (solution B) were prepared by dissolving 8.9 g of Na.sub.2HPO.sub.4.2H.sub.2O in sterile deionized water at room temperature under agitation at 250 rpm for 30 minutes and at 600 rpm for 4 hours.

(26) 19 ml of solution A were mixed with 81 ml of solution B such as to give a 0.5 M PBS solution having a pH between 7.3 and 7.4.

(27) Crushing of ?-TCP into Fine Particles:

(28) The bulk product from Example 2 was crushed by using a jaw crusher (slot size 4 mm) The course granules were sieved by using a sieving machine and sieve inserts with mesh aperture 2 mm and 0.25 mm. The sieved granules were furthermore milled using a planet mill to a final size of less than 10 ?m.

(29) Coating of the Fibrous Collagen Cylinders Obtained in Example 1 a) with Epitactically Grown Crystals of Nanocrystalline Hydroxyapatite:

(30) 5 g of fine particles of ?-TCP and 100 ml of a 0.5 M PBS solution obtained as described above and the fibrous collagen cylinders obtained in Example 1 a) were added to a glass weighing bottle which was put into a desiccator under vacuum for 5 minutes, then at atmospheric pressure. The coating reaction was performed under agitation at 37? C. during 3 days by putting the bottle on a horizontal shaker operated at 5-50 rpm in a thermostatic compartment.

(31) Visual observation showed that the collagen scaffold retained its cylindrical shape but was covered with a white crystalline substance.

(32) Other Experiments of Coating the Fibrous Collagen Cylinders Obtained in Example 1 a)

(33) Other experiments of coating of the fibrous collagen cylinders obtained in Example 1 at the end of a) were performed varying the concentration of the PBS solution from 0.2 M to 0.8 M and the coating reaction time from 12 hours to 4 days.

(34) Visual observation showed that the collagen scaffold retained its cylindrical shape but was covered with a white crystalline substance.

(35) SEM analysis showed that crystal growth as well as the size, morphology and habitus of the hydroxyapatite crystal assemblies could be controlled by varying the concentration of the PBS solution and the coating reaction time.

(36) In the above experiments the w/w ratio of the fibrous collagen scaffold to the epitactically grown crystals of nanocrystalline hydroxyapatite in the coated fibrous collagen cylinders was from 90/10 to 30/70.

(37) b) Coating of the Cylindrical Partially Fibrous Spongeous Collagen Scaffold Obtained in Example 1) b) and the Membrane Shaped Fibrous Collagen Scaffold Obtained in Example 1 c) with Epitactically Grown Crystals of Nanocrystalline Hydroxyapatite:

(38) Experiments of coating the cylindrical partially fibrous collagen sponge cylinders obtained in Example 1 b) or coating the fibrous side of the membrane-shaped fibrous collagen scaffold of Example 1c), were performed varying the concentration of the PBS solution from 0.2 M to 0.8 M and the coating reaction time from 12 hours to 4 days.

(39) Visual observation showed that the collagen scaffold retained its shape but was covered with a white crystalline substance.

(40) SEM analysis showed that crystal growth as well as the size, morphology and habitus of the hydroxyapatite crystal assemblies can be controlled by varying the concentration of the PBS solution and the coating reaction time.

(41) In the above experiments the w/w ratio of the fibrous collagen scaffold to the epitactically grown crystals of nanocrystalline hydroxyapatite was from 90/10 to 30/70 for the coated partially fibrous collagen sponge cylinders obtained in Example 1b) and from 90/10 to 50/50 for the coated membrane-shaped collagen scaffold.

EXAMPLE 4

Properties of the at Least Partially Fibrous Collagen Scaffold Coated with Epitactically Grown Crystals of Nanocrystalline Hydroxyapatite

(42) a) Physicochemical Properties:

(43) The measured porosity (pore volume) was 96 v/v % for the cylindrical pieces of fibrous collagen obtained in Example 1a) (varying the crosslinking conditions) and 85 to 95% v/v for the hydroxyapatite coated cylindrical pieces obtained in Example 3)a).

(44) The specific surface measured by mercury porosimetry was 1.5 to 2.5 m.sup.2/g for the cylindrical pieces of fibrous collagen obtained in Example 1a) (varying the crosslinking conditions) and from 20 to 60 m.sup.2/g for the hydroxyapatite coated cylindrical pieces obtained in Example 3)a).

(45) The measured porosity (pore volume) was about 96% v/v % for the collagen sponge cylindrical scaffold prepared in Example 1 b) and from 88 to 92% for the hydroxyapatite coated collagen sponge cylinders obtained in Example 3 b).

(46) The specific surface measured by mercury porosimetry was 2 m.sup.2/g for the collagen sponge cylindrical scaffold prepared in Example 1 b) and from 25 to 50 m.sup.2/g for the coated collagen sponge cylinders obtained in Example 3 b).

(47) b) Mechanical Properties:

(48) b1) Compressive Strength

(49) The compressive strength (resistance to pressure), i.e. the maximum pressure to be applied for a compression of the cylinders to 50% of their original height, was measured using a mechanical compression test machine (Proline Z010 manufactured by Zwick/Roell).

(50) The measured compressive strength in the wet state was from 0.3 to 0.7 MPa for the cylindrical pieces of fibrous collagen obtained in Example 1a) (varying the crosslinking conditions) and from 1.1 to 3.5 Mpa for the hydroxyapatite coated cylindrical pieces obtained in Example 3)a), the compressive strength increasing with the % of hydroxyapatite present in the hydroxyapatite coated cylindrical pieces.

(51) b2) Resilience

(52) The resilience, i.e. the % of original height recovered after compression to 50% or original height, was measured using a mechanical compression test machine (Proline Z010 manufactured by Zwick/Roell).

(53) The measured resilience in the wet state was from 95 to 99% for the cylindrical pieces of fibrous collagen obtained in Example 1a) (varying the crosslinking conditions) and 92 to 100% for the hydroxyapatite coated cylindrical pieces obtained in Example 3)a), the % of hydroxyapatite present in those hydroxyapatite coated pieces appearing not to influence the resilience.

(54) b3) Resistance to Torque

(55) A protocol similar to the Straumann? Bone Block Fixation Method (cf. http://www.straumann.ch/ch-index/products/products-biologics/products-bone-block-fixation.htm) was used.

(56) Briefly, using a driller to bore a 0.9 mm hole into a Teflon cylinder (having mechanical properties comparable to those of pig mandible bone) and into some the dried cylindrical pieces of fibrous collagen obtained in Example 1a) (varying the crosslinking conditions) and some of the dried hydroxyapatite coated cylindrical pieces obtained in Example 3)a), 2, and a magnetic screw driver (Klinge fTi Mikro Schr Kreuzschl, Ref. 75.23.19 available from Medicon) comprising a 1.5?12 mm screw, the maximum torque at which the cylindrical pieces could be screwed to the Teflon cylinder without breaking was measured.

(57) All the tested uncoated cylindrical pieces of fibrous collagen obtained in Example 1a showed a resistance to torque of about 20 Ncm, whereas all of the tested hydroxyapatite coated cylindrical pieces obtained in Example 3)a) showed a resistance to torque of more than about 60 Ncm, the % of hydroxyapatite present in those hydroxyapatite coated pieces appearing not to influence the resistance to torque. A resistance to torque of about 30 Ncm is generally considered in the art as sufficient for screwing a piece to an osseous body part.

(58) The dramatic increase in the resistance to torque is due to the strong epitactic binding between hydroxyapatite and the collagen scaffold.

(59) Indeed, in comparative experiments performed on those crosslinked collagen scaffolds where according to conditions of the prior art hydroxyapatite was precipitated on or inside the collagen scaffold and thus weakly linked to the latter by adsorption, the resistance to torque was not significantly increased.

(60) c) Hydroxyapatite Crystal Assembly Morphology as Determined by SEM

(61) FIGS. 1 and 2 represent SEM micrographs of the fibrous side of the membrane-shaped collagen scaffold of Example 1c) coated as described in Example 3 b) with a PBS concentration of 0.2 M for a coating reaction time of 12 hours and O,8 M collagen for a coating time of 24 hours.

(62) One can observe on FIG. 1 small hydroxyapatite crystal assemblies of finely distributed nano-sized crystal platelets with hexagonal symmetry closely interconnected with collagen fibrils and on FIG. 2 small micron-sized rosette-like aggregates of hexagonal hydroxyapatite crystal assemblies which completely covers the collagen fibre structure.

(63) d) Assays of Colonization by Bone Forming Cells

(64) It was shown on that the human MG63 osteoblast-like cells colonize with a high proliferation rate all sites of the coated membrane-shaped collagen scaffold obtained in Example 3 b). See FIG. 3.

(65) In cytotoxicity tests the coated membrane-shaped collagen scaffold obtained in Example 3 b) showed results comparable to those obtained with the Bio-Gide? membrane.