Porous polysaccharide scaffold comprising nano-hydroxyapatite and use for bone formation

Abstract

The present invention relate to three dimensional porous polysaccharide matrices able to induce mineralisation of a tissue in osseous site, as well as in non-osseous site, in the absence of stent cells or growth factors.

Claims

1. A method for bone generation at an osseous or a non-osseous site, comprising the step of administering, at the osseous or the non-osseous site, a porous polysaccharide scaffold obtained by i) preparing an alkaline aqueous solution comprising at least one polysaccharide, a cross-linking agent and a porogen agent, ii) transforming the solution into a hydrogel by placing said solution at a temperature from 4° C. to 80° C. for a sufficient time to allow cross-linking of said at least one polysaccharide, iii) submerging said hydrogel into a solvent, and iv) washing the porous polysaccharide scaffold obtained at step iii), wherein the alkaline aqueous solution of step i) further comprises hydroxyapatite, and wherein the step of administering is performed in the absence of cells and growth factors.

2. The method according to claim 1, wherein said porous polysaccharide scaffold is administered to a non-osseous site.

3. The method according to claim 1, wherein the porogen agent is selected from the group consisting of sodium chloride, calcium chloride, ammonium carbonate, ammonium bicarbonate, calcium carbonate, sodium carbonate, sodium bicarbonate and mixtures thereof.

4. The method according to claim 1, wherein a weight ratio of the at least one polysaccharide to the porogen agent is in a range from 1:50 to 50:1.

5. The method according to claim 1, wherein said at least one polysaccharide is selected from the group consisting of dextran, pullulan, agar, alginic acid, starch, hyaluronic acid, inulin, heparin, fucoidan, chitosan and mixtures thereof.

6. The method according to claim 1, wherein said at least one polysaccharide is a mixture of pullulan/dextran in a ratio in a range from 95:5 to 5:95.

7. The method according to claim 1, wherein said at least one polysaccharide is a mixture of pullulan/dextran/fucoidan in a ratio in a range from 70:20:10 to 50:20:30.

8. The method according to claim 7, wherein said ratio is selected from the group consisting of 70:20:10 (w/w), 50:30:20 (w/w), and 73:22:5 (w/w).

9. The method according to claim 1, wherein said cross-linking agent is selected from the group consisting of trisodium trimetaphosphate (STMP), phosphorus oxychloride (POCl.sub.3), epichlorohydrin, formaldehydes, carbodiimides, glutaraldehydes, and mixtures thereof.

10. The method according to claim 1, wherein said hydroxyapatite is nano-hydroxyapatite.

11. The method according to claim 10, wherein said nano-hydroxyapatite is obtained from a solution of phosphoric acid at a concentration between 0.3 to 1M, with a solution of calcium hydroxide at a concentration between 0.5 to 1.5M, through chemical precipitation at room temperature.

12. The method according to claim 11, wherein said phosphoric acid concentration is 0.6M and said calcium hydroxide concentration is 1M.

13. The method according to claim 10, wherein a concentration of nano-hydroxyapatite in the alkaline aqueous solution is between 0.01 and 10% (w/v).

14. The method of claim 13, wherein said nano-hydroxide concentration is between 0.1 and 0.5% (w/v) or between 0.1 and 0.3% (w/v).

15. The method according to claim 1, wherein said porous polysaccharide scaffold contains pores from 1 μm to 500 μm in size and the porosity is from 4% to 75%.

16. The method of claim 15, wherein the pores are from 10 to 200 μm in size and the porosity is from 4% to 50%.

17. The method according to claim 1, wherein said solvent is an aqueous solution.

18. The method according to claim 1, wherein a weight ratio of the at least one polysaccharide to said porogen agent is in a range from 1:30 to 30:1 (w/w).

19. The method according to claim 18, wherein said weight ratio is 75:25 (w/w).

20. A method for bone generation at an osseous or a non-osseous site comprising the step of administering, at the osseous or the non-osseous site, a porous polysaccharide scaffold obtained by: a) preparing an alkaline aqueous solution comprising at least one polysaccharide, and one cross-linking agent, b) freezing the aqueous solution of step a), and c) sublimating the frozen solution of step b), wherein the alkaline aqueous solution of step a) further comprises hydroxyapatite, and wherein step b) is performed before cross-linking of the polysaccharide occurs in the solution of step a), and wherein the step of administering is performed in the absence of cells and growth factors.

Description

FIGURES LEGENDS

(1) FIG. 1A-B: Porous polysaccharide scaffold.

(2) Macroscopic view of hybrid porous discs with n-HA before (FIG. 1A) and after (FIG. 1B) rehydration with phosphate buffer saline (PBS). The scale bar corresponds to 1 mm.

(3) FIG. 2A-B: Electron Microscopy of a freeze-dried polysaccharide scaffold.

(4) The morphology of freeze-dried scaffolds was analyzed by scanning electron microscopy (FIG. 2A). After rehydration in PBS, porosity of hydrated scaffolds was observed with Environmental Scanning Electron Microscopy (ESEM Philips XL 30) (FIG. 2B).

(5) FIG. 3A-C: Healing of critical size defects in nude mice by the polysaccharide- based matrices.

(6) Micro-CT images of calvaria defects filled with polysaccharide matrices without n-HA (FIG. 3A), or with the polysaccharide scaffold (FIG. 3B), loaded (on left side) or not (on right side) with 5×10.sup.5 differentiated adipose derived stromal cells (ADSCs). Imaging on the same animal for each type of scaffold was performed after 15, 30, 60 and 84 days of implantation, and resulting images are respectively referred to as D15, D30, D60, D84. Quantitative analysis of the Tissue Mineral Density (TMD) of implanted polysaccharide scaffold. Calvaria bone was used as a control (FIG. 3C).

(7) FIG. 4A-E: Ectopic mineralized tissue formation in subcutaneous site induced by the polysaccharide scaffold. (A) Micro-CT images at Days 15, 30 and 60 of a mouse implanted with two discs of the polysaccharide scaffold (n-HA/scaffold) (left site) and one disc previously seeded with 5×10.sup.5 differentiated ADSCs (right site). (B) Macroscopic view at D60. (C) Quantitative analysis of the tissue mineral density (TMD). (D) Histological examination of undecalcified (D1; magnification ×10) (stained by Goldner's trichrome) and decalcified (D2; magnification ×2) (D3; magnification ×20) sections (Masson's staining) obtained at Day 60. (E) Von Kossa staining performed on explanted materials at Day 30 and Day 60. Control was performed using the paraffin-embedded composite matrix before implantation (magnification ×2).

(8) FIG. 5A-B: Matrix+n-HA (MATRI+) induces mineralization in ectopic site of mice. (A) Representative micro-CT images of the subcutaneous implantation of the Matrix alone on the left side (indicated by an arrowed doted line) and Matrix+n-HA (MATRI+) on right side (indicated by an arrowed plain line), after 15 (D15), 30 (D30) and 60 days (D60) of implantation in Balb/c mice. (B) Bone Mineral Content (BMC) and Bone Mineral Density (BMD) were measured from reconstructed three-dimensional micro-CT images with Microview Image analyser of the Matrix (white rectangle) and Matrix+n-HA (MATRI+) (black rectangle). Data are presented as means ±standard deviation for n=8. The symbol ** indicates a statistically significant difference compared to the other groups <0.01.

(9) FIG. 6A-B: Matrix+n−HA induces formation of a collagen-based mineralized tissue: histological analyis of the newly formed tissue. (A) Representative histological undecalcified sections of the Matrix and Matrix+n-HA (MATRI+) samples implanted subcutaneously in mice, after 15 days (D15) and 60 days (D60) : Von Kossa staining. (B) Representative histological decalcified sections of Matrix+n−HA (MATRI+) 60 days after implantation: Goldner staining, The images showed a high dense collagen tissue around the implant that colonizes the scaffold, with osteoblast-like cells as indicated by the white arrows, and numerous vessels inside the collagen tissue indicated by the black arrows.

(10) FIG. 7A-B: XRD patterns of matrices before surgery (D0) and 15 days (D15) after subcutaneously implantation in mice. (A) Matrix+n-HA (MATRI+) ; (B) Matrix without n-HA

(11) Specific peaks of hydroxyapatite (HA) are only observed in the XRD patterns after 15 days of implantation of MATRI+. Peaks of Halite (H) due to sample processing, are observed in all spectra. The XRD patterns obtained at day 30 and day 60 are similar than those observed at D15 for both groups (data not shown).

(12) FIG. 8: Matrix+n-HA (MATRI+) retained endogeneous osteoinductive and angiogenic factors.

(13) Measurement by ELISA of BMP2 (A) and VEGF165 (B), retained in the tissue formed within the Matrix (white rectangle) and Matrix+n-HA (MATRI+) (black rectangle) when implanted subcutaneously at D15, D30 and D60. Results are expressed in pg of growth factors retained per μg of proteins quantified by BCA. Data are presented as means ±standard deviation for n=6 samples. The symbols * and ** indicate a statistically significant difference compared to the other groups with p<0.05 and <0.01, respectively.

(14) FIG. 9A-B: Matrix+n−HA (MATRI+) induces a high mineralization of tissue in a critical size bone defect performed in the femoral condyle of rats. (A) Representative micro-CT images of the femoral condyle of rats, 15 days (D15), 30 days (D30) and 90 days (D90) after implantation without scaffold (empty), with Matrix or Matrix+n−HA (MATRI+). (B) Bone Mineral Content (BMC) and Bone Mineral Density (BMD) were measured from reconstructed three-dimensional micro-CT images of the empty group (white rectangle), the Matrix group (grey rectangle) and Matrix+n−HA (MATRI+) (black rectangle). Data are presented as means ±standard deviation for n=4. The symbol ** indicates a statistically significant difference compared to the other groups with p<0.01.

(15) FIG. 10A-B: Matrix+nHA (MATRI+) induces a high mineralized bone tissue in a critical size bone defect performed in the femoral condyle of rats after 90 days of implantation; histological analysis of the newly formed tissue. (A) Representative histological undecalcified sections of Empty, Matrix and Matrix+n−HA (MATRI+) samples implanted in the femoral condyle of rats, after 90 days of implantation: Von Kossa staining. The arrows indicated the position of the bone defect. (B) Representative histological decalcified sections of of Empty, Matrix and Matrix+ nHA samples 90 days after implantation: Goldner staining, A fibrous tissue was formed in the empty bone defect, while bone formation occurred in direct contact of the matrix and was enhanced within the MATRIX+ implant.

EXAMPLE

Example 1

Implantation of the Scaffold of the Invention in Calvaria Site of Athymic Mice

(16) Materials and Methods

(17) Nano-Hydroxyapatite Preparation

(18) Nano-hydroxyapatite (n-HA) was prepared by wet chemical precipitation using a 0.6 M solution of Phosphoric acid (H.sub.3PO.sub.4 Rectapur, Prolabo®, France) and a 1 M solution of calcium hydroxide (CaOH.sub.2 Alfa Aesar, Germany). 100 ml of H.sub.3PO.sub.4 solution were added dropwise in 100 ml of CaOH.sub.2 solution during 30 minutes under vigorous stirring at room temperature. At the end of reaction, pH was adjusted to 9 using 0.4.10.sup.−3 mol of a 0.6 M sodium hydroxide solution, then stirring was continued during 12 hours.

(19) Nano-hydroxyapatite (n-HA) has been characterized by transmission electron microscopy (TEM), scanning electron microscopy and by FTIR analysis. TEM revealed n-HA needle-shaped crystals of 50 nm long. FTIR analysis showed specific bands of phosphate ions of at 559 cm.sup.−1, 601 cm.sup.−1, and 1018 cm.sup.−1 and a non-specific carbonate band 1415 cm.sup.'1.

(20) Preparation of Composite Polysaccharide Scaffolds (MATRI+)

(21) Macroporous composite scaffolds (MATRI+) were prepared using a blend of pullulan/dextran 75:25 (pullulan, MW 200,000, Hayashibara Inc, Dextran MW 500,000, Pharmacia), prepared by dissolving 9 g of pullulan and 3 g of dextran into 27 mL of distilled water containing 14 g of NaCl and 13 mL of nano-hydroxyapatite suspension (n-HA, 6.36% w/v). Chemical cross-linking was carried out using trisodium trimetaphosphate STMP (Sigma) under alkaline condition. Briefly, 1 mL of 10 M sodium hydroxide was added to 10 g of the polysaccharide blend, followed by the addition of 1 mL of water containing 300 mg of STMP. After incubation at 50° C. for 15 min, resulting scaffolds were cut into 6 mm diameter discs, neutralized in PBS 10X (pH 7.4) then washed extensively with a 0.025% NaCl solution. After a freeze-drying step, porous composite polysaccharide scaffolds were stored at room temperature until use. Fluorescent scaffolds were prepared by adding 1% of Fluorescein IsoThioCyanate (FITC) dextran (Sigma, St. Louis Mo., USA) to the mixture before cross-linking.

(22) ADSC Cultures and Osteogenic Differentiation

(23) Adipose Derived Stromal Cells (ADSCs) were isolated from human adipose tissue after a digestion with 0.1% (w/v) collagenase type I and cultured as previously described by Gimble et al, 2007. The remaining Stromal Vascular Fraction (SVF) was cultured in a basal medium (DMEM F12 medium (Invitrogen) supplemented with 10% (v/v) Foetal Bovine Serum (FBS) or in an osteogenic medium for inducing osteoblastic differentiation of ADSCs (IMDM medium (Invitrogen), supplemented with 10% (v/v) FBS (Lonza), 10.sup.−8 M dexamethasone (Sigma), 50 mg/ml ascorbic acid (Sigma) and 10 mM □(β-glycerophosphate (Sigma)).

(24) Experimental Models in Nude Mice

(25) Orthotopic new bone formation was assessed on calvaria site of athymic mice. Twelve weeks-old nude mice were anesthetized with an isoflurane/N20 mixture and were subjected to surgery to make a 4 mm diameter full thickness on the left and right parietal bone using a trephine dental burr. Disk-shaped matrices without n-HA (Group 1) and composite polysaccharide scaffold MATRI+ containing n-HA (Group 2) were implanted on top of the periosteum of the parietal bone. Group 3 corresponds to mice implanted with the composite polysaccharide scaffold associated with differentiated ADSCs one week before implantation.

(26) To study ectopic bone formation, polysaccharide-based matrices (Group 1), composite polysaccharide scaffold without cells (Group 2), or matrices previously seeded with differentiated ADSCs (Group 3), were implanted into dorsal, subcutaneous spaces of athymic mice (female, 12 weeks old). Four scaffolds were implanted by mice. Bone formation was followed by a non invasive high resolution X-ray tomography (micro-CT) analysis performed 15, 30 and 60 days after implantation and by histological examination at the end of the experiment (D60).

(27) High Resolution X-ray Tomography (Micro-CT) Analysis

(28) Mice were scanned in an in vivo Explore Locus SP X-Ray micro-computerized tomography (micro-CT) device (General Electric) at an isotropic resolution of 45 μm. Reconstruction of the parietal and subcutaneous region was performed following correction of rotation centre and calibration of mineral density. Bone analysis was performed using the “Advanced Bone Analysis”™ software (GE). Thresholding of grey values was performed using the histogram tool in order to separate mineralized elements from background. The density of mineralized tissue (TMD) was determined in the region of interest (ROI).

(29) Histological Evaluation

(30) At the end of the experimental periods, mice were euthanized and samples were dissected out and fixed in 3.7% (v/v) paraformaldehyde in PBS 0.1 M pH 7.4. One part of the samples were decalcified and embedded in paraffin. Permanent sections of 7 micron were stained with hematoxylin and eosin and Masson trichrome dye. The other part of the samples were embedded in methylmethacrylate as described by Schenk et al, 1984. Longitudinal sections (15 μm thick) were prepared using a Leica microtome and tungsten carbide blades. Sections were stained with Goldner's trichrome, Von Kossa, and observed using a Nikon Eclipse 80i microscope. Pictures were generated using a DXM 1200 C (Nikon) CCD camera.

(31) Results

(32) 3D porous matrices (FIG. 1) were obtained according to the methods disclosed in the PCT patent applications WO2009/047346 and WO2009/047347, with n-HA included in the starting formulation. n-HA in suspension (6.36% (w/v)) allowed an homogeneous dispersion of the HA nanoparticles in the resulting 3D matrices. The n-HA matrices contained in the dry state, 2.8 +/−0.1% (w/w) of HA. The use of n-HA in the dry form instead of a n-HA suspension, induced large aggregates inside the matrices. The 3D matrices in the presence of n-HA are porous (FIG. 2) with pore sizes controlled by the patented process.

(33) Discs of 4 mm in diameter of 3D porous matrices with or without n-HA (composite scaffold) and previously seeded or not with human adipose derived mesenchymal stem cells (ADSCs) were then evaluated in two mice models.

(34) Orthotopic new bone formation on calvariae site of athymic mice revealed that only the polysaccharide-based matrices associated with n-HA (composite scaffold) induced formation of a mineralized tissue in nude mice. The porous matrices without n-HA do not induce any mineralization within 60 days. The orthotopic new bone formation was observed with composite matrices in absence of human mesenchymal stem cells, and even if the scaffold moved out of the bone defect (FIG. 3B). The mineralization occurred four weeks after implantation and increased with time (FIG. 3C). Histological examination (Goldner's trichrome staining) revealed a fibrous tissue formed when polysaccharide-based matrices without n-HA were implanted, whereas the composite polysaccharide scaffold provides an efficient scaffold for local production of collagen network within the matrices.

(35) Since the n-HA matrix (composite scaffold) was found to induce mineralization outside the bone defect, the inventors next examined its potency to stimulate ectopic bone formation. They observed that implantation of matrices without n-HA did not form any mineralized tissue at day 60. In contrast, implantation of n-HA matrices (composite polysaccharide scaffold of the invention) in subcutaneous site lead to the formation of a dense mineralized tissue (FIGS. 4A and 4B) four weeks after implantation and without ADSCs seeding. The mineralization increased with time. Quantification indicated that the TMD of the calcified tissue was about 420 mg/cm.sup.3 and close to the density of the implanted composite matrix in orthotopic site (FIG. 4C) 60 days after implantation. Histological analysis on undecalcified (FIG. 4D.sub.1) and decalcified (FIG. 4D.sub.2) sections of the ectopically induced mineralized tissue revealed that n-HA matrices (composite polysaccharide scaffold MATRI+) stimulated a dense collagen network and blood vessel formation as well as the recruitment of osteoblast-like cells (FIG. 4D.sub.3). To visualize the level of calcification in the newly formed tissue, sections of n-HA/scaffold were stained according to Von Kossa technique at day 30 and day 60 (FIG. 4E). Controls were performed on the paraffin-embedded composite polysaccharide. This staining showed a well-calcified tissue of n-HA/scaffold that increases with time of implantation. To the knowledge of the inventors, no material so far in the absence of stem cells or growth factors, was able to give this effect.

(36) The inventors further investigated for comparison the role of n-HA alone on non-osseous site. For this purpose, they proceed to the implantation of n-HA alone in subcutaneous site. After 15 days and 30 days, they only observed a classical reaction to a foreign body. Indeed, the histological examination of undecalcified section (Cyanine Solochrome staining) of non-osseous site implanted with n-HA alone did not show the presence of any mineralized tissue. Implantation of n-HA alone hence did not lead to the formation of a mineralized tissue.

(37) The inventors have thus shown that the porous composite polysaccharide scaffold of the invention provides unexpected results by stimulating mineralized tissue formation in osseous site, as well as in non-osseous site, in the absence of stem cells or growth factors.

Example 2

Implantation of the Scaffold According to the Invention in a Non Osseous Site in Mice and Osseous Site in Rat

(38) Materials and Methods

(39) Nanohydroxyapatite and scaffold according to the invention were prepare as described in Example 1. The inventors assessed the implantation of said scaffold in animal. Both the procedure and the animal treatment complied with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research. The studies were carried out in accredited animal facilities at the University of Bordeaux Segalen, under authorization (NO: 3300048 of the Ministere de l'Agriculture, France) and were approved by the Animal Research Committee of Bordeaux University.

(40) Non-osseous Implantation in Mice: Ectopic Bone Formation Analysis

(41) The two different formulations of scaffolds: disk-shaped matrices without n-HA (Group 1) and the composite scaffold containing n-HA (MATRI+) (Group 2) (cylinders of 4 mm diameter and 6 mm depth) were inserted into subcutaneous pockets created in the dorsum of the 12-week-old Balb/c mice weighing 25-30 g (Charles River Laboratories, France). Samples were retrieved after 15, 30 and 60 days of implantation and treated for micro-CT and histological analysis. Eight samples were used for histological observation and micro-CT in each group.

(42) Osseous Implantation in Rats: Orthotopic New Bone Formation Analysis

(43) Medial holes, 5 mm diameter and 6 mm depth were created in both left and right femoral condyles of Wistar rats weighing 150-200 g (Charles River Laboratories, France) using trephine dental burr. Bone pieces were removed from the bone defect, the hole was rinsed with physiological solution (NaCl 0.9% (w/v) before introducing the scaffold within the defect. The two different scaffold formulations (matrices without n-HA and composite scaffold containing n-HA) were implanted into each bone defect. A control experiment without scaffold was also conducted. Implants were retrieved 15, 30, 60 and 90 days after surgery and treated for micro-CT and histological analysis. Six samples were used for micro-CT and histological observation in each group.

(44) Histological Procedure

(45) At the end of each implantation period, animals were euthanized by injecting an overdose of pentobarbital sodium (Nembutal®) Immediately afterwards, the implants and surrounding tissue were retrieved, fixed with 4% (w/v) paraformaldehyde in a 0.1 M phosphate buffer and scanned with micro-CT before histology. The samples were then prepared for histological analysis. One part was decalcified, dehydrated and embedded in paraffin. Thin sections (7 μm in thickness) were prepared and stained with hematoxylin and eosin and with Goldner's Trichrome for osteoid staining. The other part were dehydrated in a graded series of ethanol, and then embedded with methylmethacrylate, which was subsequently polymerized. Ten to 15 μm transverse sections were made using a modified diamond blade microtome (Leica Microsystems SP1600, Rijswijk, The Netherlands), with four sections obtained from each implant. Sections were stained with Goldner's trichrome, Von Kossa, and observed using a Nikon Eclipse 80i microscope. Pictures were generated using a DXM 1200 C (Nikon) CCD camera.

(46) Micro-computed Tomography (Micro-CT)

(47) Micro-CT was used to develop three-dimensional images of the implants and surrounding tissue; these models were used to quantify the bone formation at each implant site. An ex vivo General Electric (GE) micro-CT (Explore LP Locus, General Electric), with a source voltage of 80 kV, a current of 60 μA, and 15 μm resolution, was used to acquire X-ray radiographs. In vivo micro-CT (General Electric) was performed with a source voltage of 150 mV, a current of 450 μA, and 45 μm resolution. After scanning, cross-sectional slices were reconstructed and 3D analyses were performed using Microview software. Each scan result was reconstructed using the same threshold values to distinguish bone and air. Bone Mineral Content (BMC) and Bone Mineral density (BMD) volume were measured for each group and statistically analyzed using the Student's t-test.

(48) Protein Extraction from Subcutaneous Implants and ELISA Analysis of Osteogenic and Angiogenic Growth Factors Retained within the Implants.

(49) Subcutaneous implants retrieved after 2, 15, 30 and 60 days of implantation were crushed on ice with an electric crusher in PBS containing a cocktail of protease inhibitors (10 μg/ml Aprotinine (Sigma), 10 μg/ml Leupeptin (Sigma) and 1 mM (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) (Fluka). The lysates were then centrifuged at 16 000 rpm and 4° C. for 20 min. The supernatant was collected and then frozen at −80° C. for ELISA analysis. Quantification of the protein was performed using bicinchoninic acid (BCA) protein assay kit (Thermoscientific) described by Smith PK et al. (1985). Absorbance was read at 550 nm. There were eight matrices without n-HA (Group 1) and composite scaffold MATRI+ containing n-HA samples (Group 2), respectively for each time of implantation. The amounts of VEGF.sub.165 and BMP2 retained within the two different formulations of implants were quantified with the mouse VEGF immunoassay kit (MMV00, Quantikine®, R&D systems), and BMP-2 immunoassay kit (DBP200, Quantikine®, R&D systems), respectively.

(50) X-ray Diffraction Analysis

(51) Subcutaneous implants of matrices without n-HA and composite scaffold MATRI+ containing n-HA were retrieved after 15, 30 and 60 days of implantation. In order to obtain a fine powder without any organic tissues, they were treated with bleach for 2 hours at room temperature and then centrifuged to keep only the pellet. Structural properties were explored by X-ray diffraction (XRD) using PANalytical X'pert MPD diffractometer (Bragg Brentano t-t geometry) equipped with a secondary monochromator and uses a copper radiation (mean λ=1,5418 A°), the working tension and intensity were 40 kV and 40 mA, respectively.

(52) Samples were placed on a single-crystalline wafer sample holder made of silicium. Diffractograms were all measured with the same parameters: angular range from 8 to 80° (2t), step: 0.02°, measure time: one hour; Following X-ray diffraction (XRD) analysis of the material, phase identification through JCPDS-ICDD data (Diffract-Plus Eva Software, Bruker©) was compatible with a carbonated hydroxyapatite [Ca10(PO4)3(CO3)0.01(OH)1.3], displaying hexagonal lattice parameters (a=9.3892 A°; c=6.9019 A°; α=β=90° and g=120°; space group: P63/m(176)).

(53) Statistical Analysis

(54) All data were expressed as means ±standard deviation (SD) and were analyzed using standard analysis of Student's t-test. Differences were considered significant when p≦0.05 (a) or p≦0.01 (b).

(55) Results

(56) Two different scaffolds, matrices without n-HA (Group 1) and the composite scaffold MATRI+ containing n-HA (Group 2), were implanted in Balb/c mice for 15, 30 and 60 days. Micro-CT, quantification of mineralization (BMC and BMD analysis) and histological studies were performed for both groups. Implantation of matrices without n-HA did not form any mineralized tissue from day 15 to day 60, as showed by micro-CT (FIG. 5A) and BMC and BMD quantification (FIG. 5B). In contrast, implantation in subcutaneous site of matrices containing n-HA (without any cells and growth factors) lead to the formation of a dense mineralized tissue (FIG. 5A) as quantified by BMC (Bone Mineral Content) and BMD (Bone Mineral Density) measured at each time (FIG. 1B). The mineralization process starts at day 15 from the periphery of the scaffold (FIG. 1A) and lead to a high and dense mineralized tissue after 60 days of implantation.

(57) From histological data, the porous n-HA matrices exhibited favorable mineralized tissue responses at D15 and D60, as demonstrated by von Kossa staining of undecalcified sections of MATRI+ (FIG. 6A), compared to matrix without n-HA. Von kossa staining is high after 60 days of implantation of MATRI+, compared to the same scaffold at day 15. The n-HA matrices before implantation stained with von kossa revealed a slight staining, due to the presence of the nanohydroxyapatite within the scaffold (not shown). However, the staining is much lower than that observed after 30 and 60 days of implantation.

(58) Moreover, Goldner staining performed 60 days after implantation on decalcified sections of MATRI+ (FIG. 6B), revealed, a dense fibrous collagen tissue, mainly around the implant. Some collagen tissue penetrate within the scaffold, exhibiting some lining osteoblast-like cells indicated by white arrows, in contact with the scaffold and numerous vessels marked by black arrows on the histological picture. No inflammatory event was detectable with both scaffolds, whatever the time of implantation.

(59) The XRD patterns of powder of n-HA matrices before implantation (D0) or retrieved at day 15 (D15) revealed specific peaks of hydroxyapatite at D15 on the spectrum (FIG. 7A). Peaks of Halite (H), probably due to the treatment of the samples with bleach, were observed in all spectra. The XRD patterns obtained at day 30 and day 60 were similar than those observed at D15 for both groups (data not shown).

(60) The inventors also explored whether the n-HA matrices compared to matrices without n-HA could interact with endogeneous osteogenic and angiogenic growth factors. They have tested two major growth factors that play a fundamental role in angiogenesis and osteogenesis, the isoform VEGF165 and BMP2, an osteoinductive factor that could, by itself, induces mineralization and bone formation. Two days of implantation, corresponding to the inflammatory phase observed following material implantation, both samples retained the two growth factors but to a different extent. Strikingly, the amount of BMP2 retained on MATRI+ is 1.41 pg/μg protein extracted from the samples, while the matrix without n-HA retained only 0.12 pg/μg protein. For VEGF165, the amount retained in MATRI+ and matrix without n-HA are 0.089 pg/μg protein and 0.055 pg/μg protein, respectively. With time of implantation, and during the formation of the dense mineralized tissue, the concentration of BMP2 (FIG. 8A) and VEGF165 (FIG. 8B) decreased in both groups, compared to data obtained after 2 days, but remains significantly higher in the MATRI+ group after 30 and 60 days of implantation, compared to matrix without n-HA.

(61) The scaffolds, matrices without n-HA (Group 1) and the composite scaffold MATRI+ containing n-HA (Group 2), were implanted in a critical size bone defect of 5 mm diameter and 6 mm depth in the femoral condyle of rats, for 15, 30 and 90 days. Micro-CT, quantification of mineralization (BMC and BMD analysis) and histological analysis were performed for both groups. As showed by micro-CT, matrices with n-HA (MATRI+) (FIG. 9A) formed within the bone defect, a highly dense mineralized tissue, compared to matrix without n-HA. Mineralization increases with time of implantation as shown by quantification analysis of the BMD and BMC (FIG. 9B) from day 15 to day 90 of implantation. BMC and BMD in the control group (empty) remain lower than in the other groups, whatever the time of implantation.

(62) Histological data after 90 days of implantation confirmed, a high staining by von Kossa of the matrices with n-HA (MATRI+) compared with the matrix alone without n-HA or the empty group (FIG. 10A). Goldner staining evidenced a fibrous tissue in the empty bone defect, while bone formation was enhanced within the MATRI+ implant after 90 days of implantation and occurred in direct contact of the MATRI+ implant (FIG. 10B).