Macroporous and highly resorbable apatitic calcium-phosphate cement

Abstract

The present invention is directed to a novel cement powder comprising an organic component consisting of one or more biocompatible and bioresorbable polymers and an inorganic component consisting of one or more calcium phosphate compounds. The invention also relates to the apatitic CPC resulting from the mixing of said cement powder with a liquid phase and setting.

Claims

1. An injectable calcium phosphate cement comprising one or more calcium phosphate compounds, microparticles of hydroxypropylmethylcellulose (HPMC) with a diameter between 20 and 300 m, and an aqueous solution of Na.sub.2HPO.sub.4; wherein the one or more calcium phosphate compounds comprises at least 70 % of an -tricalcium phosphate (-TCP); and wherein the calcium phosphate cement is made by forming a power mixture comprising the one or more calcium phosphate compounds, the HPMC in an amount of about 1% to about 3% by weight of the total amount of the cement powder, and the -TCP and then mixing the resulting powder mixture with the aqueous solution of Na2HPO4 to form the injectable calcium phosphate cement.

2. The injectable calcium phosphate cement according to claim 1, wherein said calcium phosphate compounds are selected from the group consisting of hydroxyapatite (HA), amorphous calcium phosphate (ACP), monocalcium phosphate monohydrate (MCPH), dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA), precipitated or calcium-deficient apatite (CDA), -tricalcium phosphate (-TCP), -tricalcium phosphate (-TCP), tetracalcium phosphate (TTCP), and mixtures thereof.

3. The injectable calcium phosphate cement according to claim 2, wherein said calcium phosphate compounds are selected from the group consisting of -TCP, MCPH, DCPD, and mixtures thereof.

4. The injectable calcium phosphate cement according to claim 1, wherein said calcium phosphate compounds consists of -TCP and DCPD.

5. The injectable calcium phosphate cement according to claim 1, wherein said power mixture consists of a mixture of -TCP, DCPD, and HPMC.

6. The injectable calcium phosphate cement according to claim 1, wherein the aqueous solution of Na2HPO4 and powder mixture is in a ratio of about 0.3to about 0.6 mL/g.

7. The injectable calcium phosphate cement according to claim 1, further comprising one or more ingredients selected from the group of antibiotics, anti-inflammatory drugs, anti-cancer drugs, drugs against osteoporosis, and growth factors.

8. A scaffold for tissue engineering comprising the injectable calcium phosphate cement according to claim 1.

9. A dental or bony implant consisting of a molding of the injectable calcium phosphate cement according to claim 1.

Description

(1) FIG. 1: Scanning electron microscopy (SEM) after 24 hours setting of a CPC prepared with a cement powder consisting of -TCP (79%), DCPD (10%), MCPH (10%) and HMPC showing macropores of about 150 m of main diameter.

(2) FIG. 2: X-ray diffraction spectrum of the final product of the reaction of a CPC prepared with a cement powder consisting of -TCP (79%), DCPD (10%), MCPH (10%) and HMPC.

(3) FIG. 3: Scanning electron microscopy (SEM) after 24 hours setting of a CPC prepared with a cement powder consisting of -TCP (88%), HPMC (%), and DCPD (10%) (FIG. 3a) or MCPH (10%) (FIG. 3b).

(4) FIG. 4: Scanning electron microscopy (SEM) of a rabbit femur defect 3 weeks after the implantation of a CPC prepared with a cement powder consisting of -TCP (62%), CaHPO.sub.4 (26%), CaCO.sub.3 (8%) and HPMC (K15M) (4%).

(5) FIG. 5: Scanning electron microscopy (SEM) of a rabbit femur defect 3 weeks after the implantation of a CPC prepared with a cement powder consisting of -TCP (51%), CaHPO.sub.4 (20%), CaCO.sub.3 (4%) and Poly()caprolactone microspheres (25%).

(6) FIG. 6: Scanning electron microscopy (SEM) of a rabbit femur defect 6 weeks after the implantation of a CPC prepared with a cement powder consisting of -TCP (88%), DCPD (5%), MCPM (5%), and E4M (2%).

(7) The following examples illustrate and describe preferred embodiments of the invention.

EXAMPLES

Example 1

Preparation of poly(-caprolactone) Microspheres

(8) 1 g of poly(-caprolactone) (Tone P787, Union Carbide SA, France) has been dissolved in 15 mL of Recaptur dichloromethane (Prolabo, France). This solution has been emulsified in an aqueous solution (1 L) of methylcellulose (Mthocel A15LV premium EP, Colorcon, France) (0.75 g) at 4 C., under constant shaking (550 rpm), for 90 min. The resulting emulsion is then added to 1 litre of distilled water. The resulting suspension is then filtered in vacuum. The microspheres are then washed with 1 litre of distilled water and dried at room temperature for 24 h.

Example 2

Preparation of poly(-caprolactone) Microcapsules Encapsulating Water

(9) The same process as Example 1 is used to produce microcapsules of poly(-caprolactone) encapsulating water except for adding of water in the polymer before the emulsion.

Example 3

Preparation and Characterization of Apatitic Calcium Phosphate Cements According to the Invention

(10) the inorganic component consists of -TCP.

(11) The organic component consists of microspheres or microcapsules of poly(-caprolactone) encapsulating water.

(12) An aqueous solution of Na.sub.2HPO.sub.4 (3%) is used as liquid phase.

(13) Different cements with different liquid/powder ratios (L/P) have been prepared (0.32 mL.g.sup.1<L/P<0.40 mL.g.sup.1) and different percentages of microparticles of poly(-caprolactone) from 0 to 10%.

(14) The inorganic and organic components are mixed with the liquid phase and the mixing is placed in a cylinder-shaped mould. After 15 min, the mould is placed is a 0.9% NaCl solution at 37 C. These conditions simulate the in vivo conditions. The saline solution is changed every three days. The incubation time is one week or one month.

(15) After the incubation period, the cylinders are taken out of moulds and assayed.

(16) Table 1 summarizes the different conditions.

(17) TABLE-US-00001 TABLE 1 L/P -TCP Liquid phase Microparticles Microparticles Incubation No. (mL .Math. g.sup.1) weight (g) Volume (mL) (%) weight (g) time 1 0.32 6.25 2 0 0 1 week 2 0.40 5.00 2 0 0 1 week 3 0.32 6.25 2 0 0 1 week 4 0.40 5.00 2 0 0 1 week 5 0.32 2.94 2 5 0.31 1 week 6 0.40 7.13 3 5 0.38 1 week 7 0.32 5.94 2 5 0.31 1 month 8 0.40 6.75 3 10 0.75 1 month 9 0.32 5.63 2 10 0.63 1 month 10 0.32 2.87 1 10 (encapsulating 0.32 1 week water)
The samples are assayed by mercury porosimetry and the results are summarized in Table 2.

(18) TABLE-US-00002 TABLE 2 No. Porosity (%) Density (g/mL) Diameter in average (m) 1 27 1.85 0.018 2 36 2.5 0.011 5 27 2.20 0.011 6 37 1.98 0.012 7 27 2.34 0.011 8 37 2.10 0.012 9 28 2.19 0.011 10 45 2.74 0.0154

Example 4

-tricalcium Phosphate Preparation

(19) The preparation of -tricalcium phosphate (-TCP) was carried out by reacting in solid state, a stoichiometric mixture (molar ratio=2:1) of CaHPO.sub.4 and CaCO.sub.3, and subsequent cooling (quenching) in air down to room temperature.

(20) The reaction product obtained was -TCP containing impurities of -TCP due to the quenching.

(21) After crushing and milling of the -TCP, a sieved fraction was selected with diameters ranging from 0.1 to 80 wherein about 60% of the particles had an average particle size of 15 m.

(22) This -TCP powder was used as the main part of the inorganic solid phase of the CPCs prepared in all the following experiments.

Example 5

Materiel and Methods of the Preparation of the -TCP Based CPCs Assayed in the Following Examples

(23) The following polymers have been introduced in the -TCP based powder calcium phosphate cements: hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), sodium alginate and poly()caprolactone. HPMC and CMC (Colorcon, Inc.) were used as purchased.

(24) Three types of HPMC (E4M, F4M and K15M) have been used. They have identical chemical structure but differ by their hydroxypropyl, hydroxyethyl or methoxyl content and substitution degrees (Table 3). K15M has a high molecular weight compared to E4M and F4M, which both have slightly different molecular weights.

(25) TABLE-US-00003 TABLE 3 Different HPMC used as powders HPMC DS Methoxyl Methoxyl % MS Hydroxypropyl Hydroxypropyl % METHOCEL E 1.9 29 0.23 8.5 (E4M) METHOCEL F 1.8 28 0.13 5.0 (F4M) METHOCEL K 1.4 22 0.21 8.1 (K15M)

(26) Poly()caprolactone was prepared by the method of LeRay A M et al (Biomaterials. 2001 Oct.; 22(20):2785-94) and a sieve fraction of 80-200 m were used as microspheres.

(27) Sodium alginate was used as purchased.

(28) The liquid-to-powder ratios (L/P) used for the experiments were 0.40 and 0.50 ml/g. The liquids used as liquid phases of the cements were 3% solution of Na.sub.2HPO.sub.4 in distilled water, 3% solution of Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 (pH 7.4) in distilled water and saline solution (0.9% NaCl).

(29) The different CPCs were moulded in cylinders, 12 mm high and 6 mm in diameter, which were stored for 24 h and 36 h in saline solution at 37 C. The hardened cylinders were removed from the moulds prior to determination of the compressive strength and porosity measurements.

(30) The initial and final setting times were determined at room temperature (20 C.1) according to ASTM C266-8 standard by means of Gilmore needles. The compressive strength was determined using a Texture Analyser. The final reaction product was determined by means of X-ray diffractometry.

(31) The study of the morphology evolution of the crystalline structures formed during the cement setting process was carried out by examining the fractured surfaces of samples by scanning electron microscopy.

Example 6

Comparison of Different Liquid Phases and Different Concentrations of HPMC

(32) The inorganic component of the cement powders was composed of -TCP (79%), dicalcium phosphate dihydrate (DCPD; CaHPO.sub.4.2H.sub.2O) (10%) and monocalcium phosphate monohydrate (MCPH; Ca(H.sub.2PO.sub.4).H.sub.2O) (10%).

(33) Different combinations with HMPC (E4M) and liquid phases were assayed. To prepare the cement samples, the cement powder was mixed with the liquid phase for 30 seconds at a L/P ratio of 0.40 ml/g. The following Table 4 summarizes the results of setting times, compressive strengths and morphologies of the set samples.

(34) TABLE-US-00004 TABLE 4 Initial Compressive -TCP DCPD MCPH HPMC setting strength (%) (%) (%) (%) Liquid phase time (min) (MPa) 79 10 10 E4M Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 34 12 1% NaCl (0.9%) 25 11 Na.sub.2HPO.sub.4 16 11 78 10 10 E4M Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 45 10 2% NaCl (0.9%) 28 12 Na.sub.2HPO.sub.4 25 10

(35) The nature of the liquid phase and the polymer concentration influence the setting time of the cement, meanwhile the compressive strength is only slightly affected by these parameters.

(36) Scanning electron microscopy (SEM) showed an open morphology (FIG. 1) and presence of macroporosity after 24 hours setting with macropores of about 150 m of main diameter.

(37) The final product of the reaction was a calcium deficient apatite as determined by X-ray diffraction (FIG. 2)

Example 7

Comparison of Different Types of HPMC

(38) Cement powder samples were prepared with -TCP (84%), DCPD (5%) and MCPH (10%) combined with different HPMC samples (E4M, F4M and K15M) at 1% in weight.

(39) The cement pastes were prepared with a 3% solution of Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 (pH 7.4) and a L/P ratio of 0.40 ml/g.

(40) After mixing the liquid phase and the cement powder during 30 seconds in a mortar, the resulting pastes had initial setting times respectively of 17 min, 25 min and 27 min for the cements prepared with K15M, F4M and E4M.

(41) The results showed that the methoxyl content of HPMC is a parameter which influences the setting time of the reaction. On the contrary, the molecular weight and the hydroxypropyl content have a lower impact on the setting time.

(42) The final product of the setting reaction for all samples was a calcium deficient apatite.

Example 8

Comparison of a combination -TCP/DCPD/HPMC with a Combination -TCP/MCPH/HPMC

(43) Cement powder samples were prepared with -TCP (88%) and DCPD (10%) or MCPH (10%) combined HPMC (E4M) at 2% in weight.

(44) The cement pastes were prepared with different liquid phases: 3% solution of Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 (pH 7.4) in distilled water or 3% solution of Na.sub.2HPO.sub.4 in distilled water or 0.9% solution of NaCl. The L/P ratio was 0.40 ml/g.

(45) After mixing the liquid phase and the cement powder during 30 seconds in a mortar, the resulting pastes showed the following results (Table 5).

(46) The samples prepared with -TCP and DCPD showed a longer setting time compared to that prepared with -TCP and MCPH.

(47) After setting, the final product of reaction was a calcium deficient apatite, and evident macroporosity was observed after 24 hours setting for all the cement samples.

(48) The pores created by the combination -TCP (88%), DCPD (10%) (FIG. 3a) were greater that those created by the combination -TCP (88%), MCPH (10%) (FIG. 3b).

(49) TABLE-US-00005 TABLE 5 Initial Compressive -TCP DCPD MCPH HPMC setting strength (%) (%) (%) (%) Liquid time (min) (MPa) 88 10 0 E4M Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 >60 2% NaCl (0.9%) >60 Na.sub.2HPO.sub.4 >60 14 88 0 10 E4M Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 33 2% NaCl (0.9%) 28 Na.sub.2HPO.sub.4 20 8

Example 9

Preparation of CPCs with CMC and Sodium Alginate

(50) Cement powder samples were prepared with -TCP (86%), and DCPD (10%) combined sodium alginate or CMC at 4% in weight.

(51) The cements were prepared with the liquid phase NaCl (0.9%) and a L/P ratio of 0.40 ml/g.

(52) After mixing the liquid phase and the cement powder during 30 seconds in a mortar, the resulting pastes showed the following results (Table 6).

(53) The samples prepared with sodium alginate showed a drastically retarded setting time (>120 minutes). After 24 h of setting, the presence of DCPD was still evident, the hydration of -TCP and its precipitation into apatite was not complete.

(54) The cement samples prepared with CMC showed an evident open structure with macroporosity. The CMC allowed the transformation of -TCP to calcium deficient apatite.

(55) TABLE-US-00006 TABLE 6 -TCP DCPD Polymer Compressive (%) (%) (%) Liquid strength (MPa) 86 10 Sodium alginate NaCl (0.9%) 4 (24 h) (4%) 6 (36 h) 86 10 CMC (Blanose NaCl (0.9%) 10 (24 h) 7HXF) (4%) 13 (36 h)

Example 10

In Vivo Implantation of -TCP Based CPCs with HPMC or Poly()caprolactone Microspheres for 3 Weeks

(56) Two cement powder formulations were assayed for animal studies: (a) -TCP (62%), CaHPO.sub.4 (26%), CaCO.sub.3 (8%) and HPMC (K15M) (4%), and (b) -TCP (51%), CaHPO.sub.4 (20%), CaCO.sub.3 (4%) and Poly(c)caprolactone microspheres (25%).

(57) Cement pastes were prepared by mixing the sterilized cement powder and a sterilized solution of NaCl (0.9%). The L/P ratio was 0.40 ml/g.

(58) Both cements showed a compressive strength of 25 MPa after 48 h setting.

(59) The cement pastes were injected into a surgically created bone defect (6 mm diameter) in a rabbit femur. Implantations were performed under general anaesthesia. The rabbits were sacrificed after 3 weeks of implantation.

(60) The results showed that the new bone formed with composition (a) had a good quality and was comparable to the host bone. The new bone was observed directly in contact with the implant without an intervening layer. After 3 weeks, an open structure and porosity was observed in the set cement (FIG. 4)

(61) The composition (b) showed a good distribution of the poly()caprolactone microspheres in the cement matrix. After degradation, they allowed to create an open structure with macropores ranging from 80 to 200 m (FIG. 5).

Example 11

In Vivo Implantation of -TCP Based CPCs with HPMC for 6 Weeks

(62) A formulation -TCP (88%), DCPD (5%), MCPM (5%), and E4M (2%) was assayed for animal studies for 6 weeks. The cement pastes were prepared by mixing sterilized cement powder and liquid phase.

(63) The L/P ratio was 0.50 ml/g. The liquid phase was a 3% solution of Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 (pH 7.4) in distilled water.

(64) The cement pastes were injected into a surgically created bone defect (6 mm diameter) in a rabbit femur. Implantations were performed under general anaesthesia. The rabbits were sacrificed after 6 weeks of implantation.

(65) The new bone was observed directly in contact with the implant without an intervening layer. After 6 weeks, new bone was formed surrounding the implant, and the bone growth has started with a great dissolution of the implant from the periphery (contact with host bone) to the core of the implant (FIG. 6).