Production of moldable bone substitute

Abstract

Composites and methods of producing a mouldable bone substitute are described. A scaffold for bone growth comprises nanocrystalline hydroxyapatite (HA), a bioresorbable plasticizer, and a biodegradable polymer. Plasticizers of the invention include oleic acid, tocopherol, eugenol, 1,2,3-triacetoxypropane, monoolein, and octyl-beta-D-glucopyranoside. Polymers of the invention include poly(caprolactone), poly(D,L-Lactic acid), and poly(glycolide-co lactide). Methods of regulating porosity, hardening speed, and shapeability are also described. Composites and methods are described using nanocrystalline HA produced with and without amino acids. The scaffold for bone growth described herein displays increased strength and shapeability.

Claims

1. A composite for producing a scaffold for bone growth comprising: calcium phosphate particles having a length of 10-20 nm; a biodegradable polymer; and monoolein, wherein the composite is shapeable by hand for at least 10 minutes after a heating step.

2. The composite of claim 1, further comprising an amphiphilic substance.

3. The composite of claim 1, wherein the scaffold is an implantable scaffold and the shapeable composite is cooled to room temperature to harden the shapeable composite to form the implantable scaffold.

4. The composite of claim 1, wherein the heating step comprises heating the composite to a temperature sufficient to form the composite that is shapeable by hand for a given period of time.

5. The composite of claim 1, wherein the scaffold comprises a biologically active factor selected from the group consisting of an antibiotic, chemotherapeutic, bone cell inducer, bone cell stimulator, tissue promoting factor, tissue decomposition inhibitor, growth factor, and any combination thereof.

6. The composite of claim 1, wherein the calcium phosphate particles are selected from tricalcium phosphate, octacalcium phosphate, tetracalcium phosphate, dicalcium phosphate, hydroxyapatite, or any combination thereof.

7. The composite of claim 1, wherein the composite further comprises 1,2,3-triacetoxypropane and poly-caprolactone.

8. A composite for producing a nanocrystalline hydroxyapatite (HA) scaffold for bone growth comprising: nanocrystalline HA particles having a length of 10-20 nm and a specific surface area of 200 m.sup.2/g or greater; a biodegradable polymer; and monoolein.

9. The composite of claim 8, wherein the nanocrystalline HA particles comprise an amino acid coating.

10. The composite of claim 9, wherein the amino acid coating is removed prior to combining the nanocrystalline HA particles with the biodegradable polymer and monoolein.

11. The composite of claim 9, wherein the amino acid coating comprises L-aspartic acid and L-lysine.

12. The composite of claim 8, wherein the nanocrystalline HA particles, biodegradable polymer and monoolein are combined to form the nanocrystalline HA composite, and the nanocrystalline HA composite is heated and then cooled to room temperature.

13. The composite of claim 12, wherein the composite is heated to a temperature sufficient to make the composite shapeable and the cooling is carried out to harden the shapeable composite to an implantable scaffold.

14. The composite of claim 8, further comprising an amphiphilic substance.

15. The composite of claim 8, wherein the scaffold comprises a biologically active factor selected from the group consisting of an antibiotic, chemotherapeutic, bone cell inducer, bone cell stimulator, tissue promoting factor, tissue decomposition inhibitor, growth factor, and any combination thereof.

16. The composite of claim 8, wherein the scaffold further comprises tricalcium phosphate, octacalcium phosphate, tetracalcium phosphate, dicalcium phosphate, or any combination thereof.

17. The composite of claim 8, wherein the composite further comprises 1,2,3-triacetoxypropane and poly-caprolactone.

18. A nanocrystalline hydroxyapatite (HA) scaffold for bone growth comprising: a plurality of nanocrystalline HA particles; monoolein; 1,2,3-triacetoxypropane; and poly-caprolactone, wherein nanocrystalline HA particles having a length of 10-20 nm, 1,2,3-triacetoxypropane, and poly-caprolactone are combined to form the nanocrystalline hydroxyapatite (HA) scaffold.

19. The nanocrystalline hydroxyapatite (HA) scaffold of claim 18, further comprising an amphiphilic substance.

20. The nanocrystalline hydroxyapatite (HA) scaffold of claim 18, wherein the nanocrystalline HA particles, 1,2,3-triacetoxypropane, and poly -caprolactone are combined to form a composite and the composite is heated to a temperature sufficient to form a shapeable composite.

21. The nanocrystalline hydroxyapatite (HA) scaffold of claim 20, wherein the shapeable composite is shapeable by hand for at least 10 minutes.

22. The nanocrystalline hydroxyapatite (HA) scaffold of claim 18, wherein the composite comprises a biologically active factor selected from the group consisting of an antibiotic, chemotherapeutic, bone cell inducer, bone cell stimulator, tissue promoting factor, tissue decomposition inhibitor, growth factor, and any combination thereof.

23. The composite of claim 8, wherein the composite is shapeable by hand for at least 10 minutes after a heating step.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. XRD diffractogram of the nanosized HA particles, obtained with CuKα-radiation (1.54 Å).

(2) FIG. 2. TEM image of the nanosized HA particles (magnification 1001 k×).

(3) FIG. 3. Graph of compression strength (Pa) versus distance (mm) for pure PCL, PCL/nanosized HA and PCL/microsized HA composites at room temperature.

(4) FIG. 4A. Compression strength (Pa) versus distance (mm) at various times and after being heated to 70° C. for 1 hour and then allowed to cool in room temperature for composites with PCL/tocopherol/nanosized HA with amino acids (29 wt % HA).

(5) FIG. 4B. Compression strength (Pa) versus distance (mm) at various times and after being heated to 70° C. for 1 hour and then allowed to cool in room temperature for composites with PCL/tocopherol/nanosized HA without amino acids (29 wt % HA).

(6) FIG. 4C. Compression strength (Pa) versus distance (mm) at various times and after being heated to 70° C. for 1 hour and then allowed to cool in room temperature for composites with PCL/tocopherol/microsized HA (29 wt % HA).

(7) FIG. 5. Compression strength (Pa) versus distance (mm) at various times for PCL/PEG20000/nanosized HA (31 wt % HA) after being heated to 70° C. for 1 hour and then allowed to cool in room temperature.

(8) FIG. 6. Graph of compression strength (Pa) versus distance (mm) at various times for PCL/tocopherol/eugenol/nanosized HA (29 wt % HA), after being heated to 70° C. for 1 hour and then allowed to cool in room temperature.

(9) FIG. 7A. Compression strength (MPa) versus distance (mm) for composites with PCL/tocopherol/Monoolein/nanosized HA (31 wt % HA) after being heated to 70° C. for 1 hour and then allowed to cool in room temperature.

(10) FIG. 7B. Compression strength (MPa) versus distance (mm) for composites with PCL/tocopherol/Monoolein/nanosized HA (38 wt % HA) after being heated to 70° C. for 1 hour and then allowed to cool in room temperature.

(11) FIG. 8. Compression strength (MPa) versus distance (mm) for ethanol extracted composites measured at both room (r.t.) and body temperature for PCL/tocopherol/nanosized HA (29 wt % HA), PCL/tocopherol/microsized HA (29 wt % HA) and PCL/tocopherol/nanosized HA without amino acids (29 wt % HA).

(12) FIG. 9. SEM image of a PCL/tocopherol/nanosized HA composite after removal of tocopherol via extraction with ethanol (magnification 30 k×).

(13) FIG. 10A. Pore size distributions for PCL/tocopherol/nanosized HA composite, obtained with mercury porosimetry measurements.

(14) FIG. 10B. Pore size distributions for PCL/tocopherol/Monoolein/nanosized HA composite, obtained with mercury porosimetry measurements.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

(15) Reference will now be made in detail to the presently preferred embodiments of this invention.

(16) The present invention comprises a composite of nanocrystalline HA, a plasticizer and a biodegradable polymer. The composite can be shaped to the desired form at room temperature. After a certain period of time, the composite hardens and will have a similar strength as a pure polymer-HA composite. The composite hardening speed is controlled by the addition of a biodegradable plasticizer. When implanted, the plasticizer is resorbed by the body and leaves a porous structure consisting of nanocrystalline HA and polymer. After the removal of the plasticizer, the mechanical strength of the composite will increase considerably. Due to the bioactive properties of the nanosized HA particles, this structure will serve as an excellent scaffold for bone cell growth.

(17) The nanosized HA is synthesized with a method, which involves the mixing of an aqueous dispersion of a calcium compound with a phosphoric acid solution (see Example 1). The resulting HA crystals have a size of 10-20 nm in length, and a specific surface area of above 200 m.sup.2/g. Another method of making nanosized HA involves the mixing of an aqueous dispersion of a calcium compound and amino acids, together with a solution of phosphoric acids and amino acids. A method of producing nanocrystalline HA with amino acids is described in U.S. Patent Publication No. 60/996,561, the disclosure of which is hereby incorporated by reference. A more specific, but not limited, example is created by mixing an aqueous dispersion of a calcium compound and L-aspartic acid, together with a solution of phosphoric acids and L-lysine (see Example 2). The synthesis may also be carried out with other amino acids.

(18) When amino acids are present in a crystallizing solution, the amino acids attach to the surface of the growing calcium phosphate crystals and prevent agglomeration and crystal growth by electrostatic repulsion. The result is a suspension of nanosized HA particles with a size of 10-20 nm in length, coated with amino acids. The amino acids can be used to improve the strength of the composite, but also as anchors to attach other functional groups to the HA crystals, such as carboxylic acids, epoxides, cyanides, aldehydes, esters, alkyl halides, acid halides, acid anhydrides, ketones and phosphates. Optionally, the amino acids may be removed prior to the insertion of the crystals in the composite, by heating to 350° C. or by extensive washing with water.

(19) A powder X-ray diffractogram of the HA powder can be seen in FIG. 1. As the TEM image in FIG. 2 shows, the precipitated crystals are around 10-20 nm long (for preparation of TEM samples, see Example 14).

(20) The plasticizers are fully or partially soluble in the polymer matrix. When the composite is heated, the viscosity decreases due to the increased mobility of the polymer chains. Upon cooling to room temperature, the composite retains its shapeability since the plasticizer prevents rapid aggregation of the polymer chains. After a certain period of time, typically 30 minutes, the polymer chains start to aggregate, leading to a rapid increase in viscosity and strength. Plasticizers, which have the ability to control the hardening process may include biodegradable lipophilic substances, such as oleic acid, tocopherol and eugenol, triglycerides, such as 1,2,3-triacetoxypropane (triacetin) but also amphiphilic substances, such as monoolein and octyl-beta-D-glucopyranoside.

(21) Polymers that are suitable to use in combination with the nanocrystalline HA and the plasticizer include, but are not limited to, biodegradable polyesters, such as poly(caprolactone), poly(DL-lactic acid), and poly(glycolide-co-lactide). These polymers may also be used in combination.

(22) In one embodiment, the composite comprises a mixture of nanocrystalline HA, PCL, and tocopherol. This composite is heated to 70° C. and allowed to cool to 37° C. or to room temperature. At 37° C., the composite can be shaped by hand for at least 60 minutes. At room temperature, the composite can be shaped by hand for at least 45 minutes.

(23) In another embodiment, the composite comprises a mixture of nanocrystalline HA, PCL, eugenol, and tocopherol. After melting and cooling in room temperature, this composite can be shaped by hand for at least 120 minutes.

(24) In yet another embodiment, the composite comprises a mixture of nanocrystalline HA, poly(D,L-lactide) and eugenol. After heating and cooling in room temperature, this composite is a viscous paste which does not harden after prolonged storage.

(25) In yet another embodiment, the composite comprises a mixture of nanocrystalline HA, poly(D,L-lactide), tocopherol and eugenol. After heating and cooling in room temperature, this composite has an elastic, rubber-like appearance.

(26) In yet another embodiment, the composite is a mixture of nanocrystalline HA, PCL, poly(D,L-lactide), eugenol and tocopherol. After heating and subsequent cooling in room temperature, this composite can be shaped by hand for approximately 30 minutes.

(27) In yet another embodiment, the composite is a mixture of nanocrystalline HA, PCL, monoolein and 1,2,3 Triacetoxypropane. This composite has similar mechanical properties as the PCL/tocopherol/HA composite.

(28) The advantage of including additional biologically active factors in the bone growth scaffold is apparent to the skilled artisan. Depending on the patient's medical needs, these factors may include, but are not limited to, antibiotics, chemotherapeutics, bone cell inducers, bone cell stimulators, tissue promoting factors, tissue decomposition inhibitors, and growth factors.

(29) The possibility of replacing or mixing the nanocrystalline HA with other calcium phosphates is also apparent to the skilled artisan. Depending on the desired characteristics of the bone scaffold, these calcium phosphates may include, but are not limited to, tricalcium phosphate, octacalcium phosphate, tetracalcium phosphate and dicalcium phosphate.

(30) The mechanical properties of the composite samples were studied by measuring the pressure (Pa) versus distance (mm) at various times (15, 20, 30, 45, 60, 120, 240, 360 and 1440 min) upon cooling after being heated to 70° C. for 1 hour, as can be seen in FIGS. 3-8. It should be noted, that FIGS. 3-8 only exhibit representative examples of the measurements in order to ease the purpose of reading. Each composite was molded into cylinders carrying the dimensions: 7 mm (height)×7 mm (diameter). The molding procedure was executed simply by filling a disposable syringe with composite followed by heating to between 70 and 100° C. for at least 1 h. The cylindrical shape was created by injecting the viscous mass into a teflon mold carrying the dimensions described above. The pressure exerted on the sample was recorded as a function of distance. The values in Tables 1-4 were recorded when the sample had been compressed from an original height of 7 mm to 1 mm. Experiments were performed on a TA-HDi Texture Analyzer (Stable Micro Systems) and employed a compression speed of 0.2 mm/s.

(31) Compression Strength of PCL

(32) As mentioned in the Background section, a melted sample of PCL which is cooled below its melting point will not solidify immediately. The polymer chains will gradually begin to aggregate, creating an increase in viscosity and compression strength. We found that a sample with a compression strength above 4 MPa was hard to shape by hand or to inject with a syringe. As shown in Table 1, for pure PCL this value is reached in less than 15 minutes after the polymer is allowed to cool in room temperature. It can also be seen that after roughly 30 minutes of cooling, PCL reached a maximum strength of 14.8 MPa (see FIG. 3).

(33) Nanosized HA Versus Microsized HA

(34) In the following sections, nanocrystallline HA prepared in the presence of amino acids will be referred to as nanosized HA, whereas nanocrystalline HA synthesized in absence of amino acids will be referred to as HA without amino acids.

(35) A series of compression strength measurements were undertaken in order to compare the effect of nanosized HA to microsized HA. The microsized HA was obtained from Sigma Aldrich, Sweden. The results are shown in Table 1 and FIG. 3. With nanocrystalline HA, (29 wt % HA), the compression strength after 20 minutes was 15.6 MPa, which made the composite impossible to shape by hand after this period of time. The maximum strength was reached after 30 minutes of cooling and was around 17 MPa, thus 15% higher than for pure PCL. The commercially available, microsized HA gave a composite with similar mechanical properties as for pure PCL, with a compression strength of around 15 MPa after 30 min and onwards. Thus, using nanosized HA instead of microsized HA creates a composite with a 15% higher strength. The results also show that blending HA, either nanosized or microsized, with PCL has an effect on the hardening process, mainly on the initial stages, even though the final hardened state has a similar strength. HA accelerates the polymer aggregation process and significantly lowers the time, during which the material can be shaped by hand. The invention includes methods wherein the hardening speed of the composite is increased by adding more nanocrystalline HA.

(36) Addition of Plasticizers

(37) To prolong the time during which the PCL/HA composite could be shaped, a number of different biologically compatible plasticizers were evaluated. The results of these measurements are shown in FIGS. 4a-4c and Table 2.

(38) The compression strength measurements for PCL/tocopherol/HA composites (see Example 3) with nanosized HA in the presence of amino acids are shown in FIG. 4a and in Table 2. The HA nanoparticles were produced according to Example 2. For this composite, the compressive strength stayed below 4 MPa for 45 minutes, allowing facile moldability during this period of time. After 60 minutes, this composite reached a plateau in compressive strength, with a maximum compressive strength of 7.3 MPa.

(39) For PCL/tocopherol/HA composites (see Example 3) with nanosized HA particles without amino acids prepared according to Example 1, the shapeability was maintained up to approximately 45 minutes with the compressive strength staying below 4 MPa (see FIG. 4b). Maximum compressive strength was reached after 6 h. Substituting nanosized HA with microsized HA gave slightly lower compression strengths, as seen in FIG. 4c and Table 2. Surprisingly, the presence of tocopherol as a plasticizer effectively retards the aggregation of polymer chains, thereby enabling facile shapeability for a longer period of time. Thus, the invention includes methods of decreasing hardening speed of the composite by adding more plasticizer.

(40) A composite with poly(DL-Lactide)/eugenol/HA was prepared according to Example 4. Due to the high solubility of poly(DL-Lactide) in eugenol, this composite was a viscous paste which did not harden even after prolonged storage.

(41) Mixture of Plasticizers

(42) A mixture of two plasticizers can also be employed in order to retard the aggregation process even further. Eugenol, a biologically compatible lipophilic compound, was added to the PCL/tocopherol/HA system. Eugenol is a better solvent for PCL than tocopherol and has a stronger influence on the polymer aggregation process. The hardening speed can also be controlled by the type of plasticizer. Using equal amounts of tocopherol and eugenol (see Example 7) effectively inhibited polymer chain aggregation and made it possible to readily shape the composite for up to 120 minutes at room temperature. This can be compared with a composite with only tocopherol as plasticizer, which enabled facile shapeability. The results from these measurements are shown in FIG. 6 and Table 3. As seen in Table 3, the final strength of the PCL/tocopherol/eugenol/HA composite was around 6.7 MPa, i.e. in the same range as for PCL/tocopherol/HA. The invention includes methods of decreasing the hardening speed of the composite by increasing the ratio of eugenol to tocopherol.

(43) A poly(DL-Lactide)/eugenol/tocopherol/HA composite was prepared according to Example 5. After heating and cooling to room temperature, this composite was possible to shape by hand for 30 minutes. It then solidified to an elastic, rubber-like substance.

(44) Mixture of Polymers

(45) A poly(DL-Lactide)/PCL/eugenol/tocopherol/HA composite was prepared according to Example 6. After heating and cooling, this composite was possible to shape by hand for 120 minutes.

(46) Compression Strength of Porous Composites

(47) As previously described, bioresorbable plasticizers, such as lipophilic and amphiphilic molecules, are successfully used to inhibit the rapid aggregation of polymer chains and thus increase the period of time during which the composite can be freely formed. After implantation of the composite in the body, the plasticizers are resorbed by the human body, leaving a porous structure consisting of nanocrystalline HA and polymer, suitable for bone cell growth in a vertebrate animal. The invention includes methods of inducing bone growth in a bone defect by applying an effective amount of the composite at the site of the bone defect. After the scaffold is implanted in the body, the body resorbs the plasticizers, and bone growth occurs.

(48) In order to mimic the resorption process, the plasticizer was extracted using ethanol (see Example 11) rendering a porous composite (see FIG. 9). Subsequently, compression strength measurements were performed on the obtained porous samples in order to compare the strength after the removal of the plasticizer. This was done for composites with nanosized HA, with and without attached amino acids, and microsized HA. The results of the measurements are shown in FIG. 8 and Table 4. As seen from this table, the compression strength of a composite with nanosized HA was around 6.5 MPa, compared to the composite with microsized HA, which had a strength of 4.0 MPa. Thus, the nanosized HA creates a composite with 70% higher strength than when using microsized HA. As also can be seen from Table 4 and FIG. 8, the extracted composite containing HA without amino acids displayed composite strengths in between that shown by composites containing nanocrystalline and microsized HA. At room temperature the compression strength was measured to 5.7 MPa. At body temperature a slight increase was observed (5.9 MPa). Hence, use of nanocrystalline HA results in a composite with 45% higher compressive strength compared to employing microsized HA. In summary, the use of nanocrystalline HA either in the presence or in the absence of amino acids renders a composite with high compressive strength, which is not affected by a rise in temperature. The compressive strength displayed at room temperature is not decreased at body temperature. This can be compared to the use of microsized HA where a lower compressive strength is observed at body temperature.

(49) A SEM image of a composite with the plasticizer extracted is shown in FIG. 9 (for procedure, see Example 12). Mercury porosimetry measurements (see Example 13) revealed that after extraction of the tocopherol, the PCL/tocopherol/HA composite generated pores in the size range of roughly 0.1-1 μm (see FIG. 10a). Employing a mixture of equal amounts of tocopherol and monoolein as plasticizers (see Example 8) resulted in pores with small pore volumes in the size range of approximately 0.01-0.1 μm as well as larger pores in the 10 μm region (see FIG. 10b). The overall porosity (ratio of the volume of pores to the total volume, including the solid and void components) was 31% and 14% for PCL/tocopherol/HA and PCL/tocopherol/Monoolein/HA, respectively. After resorption, the absence of plasticizer renders a composite with mechanical strength similar to that observed for a pure polymer/HA composite. Combining tocopherol with the amphiphilic substance monoolein renders composites, which are freely moldable for approximately 30 minutes. Increasing the amount of HA from 31 to 38 wt % increases the strength of the composite as well as affecting the time before complete hardening (see FIG. 7 and Table 3). Conclusively, amphiphilic substances such as monoolein can be used to control the porosity of the implanted bone scaffold material. The porosity can be adjusted by altering the ratio of tocopherol to monolein. Increasing the amount of monoolein decreases the porosity. The compression strength can be increased by increasing the percentage of nanosized HA. As can be seen in Table 3, composites comprising approximately 29-38% by weight of nanosized HA were found to be of suitable strength.

(50) PCL/PEG/HA

(51) As previously mentioned, U.S. Pat. No. 7,186,759 describes a moldable composite based on a three component system consisting of a biodegradable polymer, a water-soluble or hydrolytically degrading polymer, such as poly(ethylene glycol) and a bioactive substance, such as HA. The task assigned to poly(ethylene glycol) is the ability to induce porosity upon hydrolysis. Preparing a composite composed of PCL, polyethylene glycol (PEG20000) and nanocrystalline HA (see Example 10), resulted in a shapeable composite with a final mechanical strength comparable with pure PCL. However, the moldability was restricted to a short period of time, approximately 15 minutes, after which the compression strength levelled out at around 15.5 MPa (see FIG. 5 and Table 2). It can also be seen from Table 2 that around the human body temperature, 37° C., the PCL/PEG/HA composite was impossible to shape by hand. This indicates that poly(ethylene glycol) acts like a hardening agent, which accelerates the hardening process, rather than the opposite effect of that displayed by tocopherol when present in a polymer matrix.

(52) Chemicals Used

(53) The biodegradable polymers poly(caprolactone) and poly(D,L-lactide) were obtained from Sigma Aldrich, Sweden and Polysciences, USA, respectively (see Examples 3-9).

(54) The plasticizers tocopherol, eugenol, monoolein and 1,2,3-triacetoxypropane were all obtained from Sigma Aldrich, Sweden. Similarly, all reagents, such as calcium oxide, L-aspartic acid and L-lysine used in the synthesis of HA nanoparticles were acquired from Sigma Aldrich, Sweden (see Examples 1-10). Phosphoric acid (85 wt %) was obtained from Fluka.

(55) Poly(ethylene glycol) 20000 used in Example 10 was obtained from Fluka.

(56) The commercial HA with a specific surface area of 9.4 m.sup.2/g used in the preparation of composites was obtained from Sigma Aldrich, Sweden.

(57) The features of the present invention will be more clearly understood by reference to the following examples, which are not to be construed as limiting the invention.

EXAMPLES

(58) As was described in U.S. Patent Application No. 60/996,561, the disclosure of which is hereby incorporated by reference, the following examples of the synthesis procedures of mouldable composites, in conjunction with the general and detailed descriptions herein, more fully illustrate the nature and character of the present invention.

Example 1

Synthesis of Nanocrystalline Hydroxyapatite Gel

(59) 2.82 g of CaO was mixed with 150 ml of H.sub.2O in a beaker. The dispersion was allowed to stir for 1 hour. In a separate beaker, 3.45 g of H.sub.3PO.sub.4 (85 wt %) was mixed with 150 ml of H.sub.2O. The contents in the two beakers were mixed at ambient temperature, and the resulting gel was allowed to stir for 12 hours. The mixture was filtered in a grade 4 glass filter and washed extensively with water (2.5 L). A portion of the gel was dried and analyzed with XRD and nitrogen adsorption. The specific surface area, as calculated with the BET method, of this sample was found to be 200 m.sup.2/g.

Example 2

Synthesis of Nanocrystalline Hydroxyapatite Gel with Amino Acids

(60) The nanocrystalline hydroxyapatite was prepared as follows. 6.70 g of L-Aspartic acid was mixed with 150 ml H.sub.2O in a beaker. 2.82 g of CaO was added to this solution, and the mixture was allowed to stir for 1 hour. In a separate beaker, 3.45 g H.sub.3PO.sub.4 (85 wt %), 6.65 g L-Lysine and 150 ml H.sub.2O was mixed. The pH of this solution was 6.46. The contents in the two beakers were mixed at ambient temperature, and the pH was measured to 8.10. The mixture was allowed to stir for 12 hours. The mixture was filtered in a grade 4 glass filter and washed extensively with water (2.5 L) to remove excess amino acids. The pH of the resulting gel was measured to 7.90.

(61) A portion of the gel was dried and analyzed with XRD and nitrogen adsorption. The X-ray diffractogram is shown in FIG. 1. The specific surface area, as calculated with the BET method, of this sample was found to be 210 m.sup.2/g.

Example 3

PCL/Tocopherol/HA

(62) HA gel was prepared according to Example 1 or 2. The gel, consisting of coated hydroxyapatite particles and water, was mixed with 6 grams of poly(caprolactone) with a molecular weight of 80000 g/mol, and 6 grams of tocopherol. The mixture was heated to 70° C. under extensive stirring until complete evaporation of the water had occurred. The yellow colored mixture was removed from the stirring equipment and allowed to cool to room temperature. The composite was readily moldable for approximately 45 minutes, during which the compressive strength was found to be below 4 MPa. The maximum compressive strength of 6.7 MPa was reached after roughly 120 minutes.

Example 4

Poly(D,L-lactide)/Eugenol/HA

(63) a) Synthesis of Nanocrystalline Hydroxyapatite Gel

(64) Prepared as previously described in Example 1 or 2.

(65) b) Production of Mouldable Bone Substitute

(66) The resulting gel, consisting of coated hydroxyapatite particles and water, was mixed with 4 grams of eugenol. The mixture was heated to 70° C. under extensive stirring until complete evaporation of the water had occurred. The paste-like blend was subsequently added to 4 grams of poly(D,L-lactide) dissolved in 4 grams of eugenol. The mixture was then once again heated to 70° C. and stirred until a homogeneous dough-like mixture was obtained. The resulting yellow coloured composite material was removed from the stirring equipment and allowed to cool to room temperature.

Example 5

D,L-Lactide/Tocopherol/Eugenol/HA

(67) a) Synthesis of Nanocrystalline Hydroxyapatite Gel

(68) Prepared as previously described in Example 1 or 2.

(69) b) Production of Moldable Bone Substitute

(70) The resulting gel, consisting of coated hydroxyapatite particles and water, was mixed with 3 grams of tocopherol. The mixture was heated to 70° C. and stirred until a dry and brown colored powder was obtained. In a separate beaker, 3 grams of D,L-lactide was mixed with 3 grams of eugenol followed by heating to 70° C. for approximately 3 h or until a homogenous blend was observed. To the homogenous melt, tocopherol/HA powder was added and the mixture was subsequently kept stirred at 70° C. Gradually, the temperature was increased to 90° C. in order to facilitate blending of the powder with the viscous polymer/oil melt. The temperature was maintained at 90° C. until a homogeneous material was attained, after which the material was removed from the stirring equipment. The reaction mixture was allowed to cool to room temperature, resulting in a paste-like material.

Example 6

PCL/Poly(D,L-Lactide)/Tocopherol/Eugenol/HA

(71) a) Synthesis of Nano Crystalline Hydroxyapatite Gel

(72) Prepared as previously described in Example 1 or 2.

(73) b) Production of Moldable Bone Substitute

(74) Nanocrystalline hydroxyapatite gel was added to 1.5 grams of tocopherol of and 1.5 grams of eugenol. The reaction mixture was heated to 70° C. and stirred until a dry and brown colored powder was obtained. Prior to the addition of the tocopherol/eugenol/HA powder, 4.5 grams of poly(caprolactone) and 1.5 grams of poly(D,L-lactide) were mixed with 1.5 grams of tocopherol and 1.5 grams of eugenol. The viscous mixture was heated to 70° C. for approximately 3 h or until a homogenous blend was observed. To the homogenous melt, tocopherol/eugenol/HA powder was added and the mixture was subsequently stirred and at 70° C. until a homogenous composite material was obtained. The resulting brown coloured composite material was removed from the stirring equipment and allowed to cool to room temperature.

Example 7

PCL/Tocopherol/Eugenol/HA

(75) a.) Synthesis of Nanocrystalline Hydroxyapatite Gel

(76) Prepared as previously described in Example 1 or 2.

(77) b) Production of Moldable Bone Substitute

(78) Prior to the addition of nanocrystalline hydroxyapatite gel, 3 grams of eugenol was mixed with 3 grams of tocopherol and 6 grams of poly(caprolactone) with a molecular weight of 80000 g/mol. The viscous mixture was heated to 70° C. for approximately 2 hours without stirring followed by 2 hours with stirring. To the homogenous and yellow melt, hydroxyapatite, as a gel, was added. The mixture was then heated to 70° C. under extensive stirring until complete evaporation of the water had occurred. The resulting brown colored composite material was removed from the stirring equipment and allowed to cool to room temperature. The combination of tocopherol/eugenol as plasticizer enables the composite to be shapeable for up to 120 minutes. The final compressive strength of the PCL/tocopherol/eugenol/HA composite was around 6.7 MPa.

Example 8

PCL/Tocopherol/Monoolein/HA

(79) a) Synthesis of Nanocrystalline Hydroxyapatite Gel

(80) Prepared as previously described in Example 1 or 2.

(81) b) Production of Moldable Bone Substitute

(82) Prior to the addition of nanocrystalline hydroxyapatite gel, 3 grams of monoolein was mixed with 3 grams of tocopherol and 5 grams of poly(caprolactone) with a molecular weight of 80000 g/mol. The viscous mixture was heated to 70° C. for approximately 12 hours before the hydroxyapatite gel was added. The mixture was subsequently heated to 70° C. under extensive stirring until complete evaporation of the water had occurred. The resulting yellow colored composite material was removed from the stirring equipment and allowed to cool to room temperature. The combination of tocopherol/monoolein as plasticizer renders composites, which are freely moldable for approximately 30 minutes after, and which reach a maximum compression strength of approximately 5.5 MPa.

Example 9

PCL/Monoolein/Triacetin/HA

(83) a) Synthesis of Nanocrystalline Hydroxyapatite Gel

(84) Prepared as previously described in Example 1 or 2.

(85) b) Production of Moldable Bone Substitute

(86) Prior to the addition of nanocrystalline hydroxyapatite gel, 3 grams of monoolein was mixed with 3 grams of 1,2,3-triacetoxypropane and 6 grams of poly(caprolactone) with a molecular weight of 80000 g/mol. The viscous mixture was heated to 70° C. To the homogenous and slightly opaque melt, hydroxyapatite, as a gel, was added. The mixture was subsequently heated to 70° C. under extensive stirring until complete evaporation of the water had occurred. The obtained off-white composite material was removed from the stirring equipment and allowed to cool to room temperature. The combination of monoolein and 1,2,3-triacetoxypropane as plasticizer results in composites, which are freely moldable for approximately 45 minutes, and with a compression strength of 6.3 MPa, similar to that of PCL/tocopherol/HA composites (see Example 3).

Example 10

PCL/PEG20000/HA

(87) To evaluate the effect of adding a hydrolyzable compound acting as a porogen in combination with a biodegradable polymer and nanosized HA of the invention herein, the mechanical properties and molding times for the composite prepared in U.S. Pat. No. 7,186,759 was compared with a composite described in the invention herein. The composite in U.S. Pat. No. 7,186,759 was prepared as described, however, using nanocrystalline HA particles instead of micrometer sized HA (see description below).

(88) a) Synthesis of Nanocrystalline Hydroxyapatite Gel

(89) Prepared as previously described in Example 1 or 2.

(90) b) Production of Moldable Bone Substitute

(91) Prior to the addition of nanocrystalline hydroxyapatite gel, 5 grams of PEG 20000 was mixed with 6.7 grams of poly(caprolactone) at 80° C. for 2 h followed by mechanical stirring at the same temperature. The mixture was then kept at 80° C. under extensive stirring until complete evaporation of the water had occurred. The resulting opaque composite material was removed from the stirring equipment and allowed to cool to room temperature. The composite reached a maximum compressive strength of approximately 15 MPa after approximately 20-30 minutes.

Example 11

Extraction of Plasticizer

(92) The extraction process took place over a period of 6 days at room temperature with exchange of the ethanolic phase every second day. The compression strength was measured at room temperature and at 37° C., as can be seen from Table 4. For experiments performed at body temperature, the composite material was heated to 37° C. for at least one hour prior to measurement. The compression strength values shown in Table 4 represent an average over at least four experiments.

Example 12

Preparation of Ethanol Extracted Composite Samples for SEM-analysis

(93) Extracted samples were prepared according to the method described in Example 11. Extracted specimens were placed on carbon tape and subsequently sputtered with a thin gold film using a JEOL sputter coater. SEM analysis was performed on a LEO Ultra 55 FEG SEM equipped with an Oxford Inca EDX system, operating at 2-5 kV. A secondary electron detector was used for detection.

Example 13

Preparation of Samples for Hg-porosimetry Measurements

(94) Samples were molded by hand into cubes with the dimensions of approximately 1×1×1 cm. The tocopherol phase was subsequently extracted with ethanol according to the procedure previously described. Prior to analysis, the samples were placed under vacuum at ambient temperature overnight.

Example 14

Preparation of Samples for TEM Analysis

(95) Specimens were prepared by grinding the HA material into a fine powder, dispersing the powder in ethanol and then placing a few drops of the dispersion onto a holey carbon grid followed by drying at room temperature. The analysis was performed on a JEOL 1200 EX II microscope operating at 120 KV.

(96) The invention in its broader aspects is not limited to the specific details shown and described and departures may be made from such details without departing from the principles of the invention and without sacrificing its chief advantages.

REFERENCES

(97) 1. H. A. Lowenstam and S. Weiner, On biomineralization, Oxford University Press, New York, 1989. 2. L. Lidgren and M. Nilsson, 2004. 3. U. Ripamonti, Journal of bone and joint surgery-American Volume, 1991, 73A, 692-703. 4. L. Nair and C. Laurencin, Progress in polymer science, 2007, 32, 762-798. 5. M. C. Azevedo, R. L. Reis, M. B. Claase, D. W. Grijpma and J. Feijen, J. Mater. Sci.: Mat. Med., 2003, 14, 103-107. 6. J.-J. Sun, C.-J. Bae, Y.-H. Koh, H.-E. Kim and H.-W. Kim, J. Mater. Sci.: Mat. Med., 2007, 18. 7. H. Niiranen, T. Pyhalto, O. Rokkanen, M. Kellomaki and P. Tormala, J. Biomed. Mater. Res. A, 2004, 69A, 699-708. 8. M. Wang, Biomaterials, 2003, 24, 2133-2151. 9. C. Agrawal and R. Ray, J. Biomed Mat. Res., 2001, 55, 141-150. 10. R. C. Thomson, M. J. Yaszemski, J. M. Powers and A. G. Mikos, Biomaterials, 1998, 19, 1935-1943. 11. J. DeGroot, H. Kuijper and A. Pennings, J. Mater. Sci.: Mat. Med., 1997, 8, 707-712. 12. S. Lee, B. Kim, S. Kim, S. Choi, S. Jeong, I. Kwon, S. Kang, J. Nikolovski, D. Mooney, Y. Han and Y. Kim, J. Biomed. Mater. Res. A, 2003, 66A, 29-37. 13. K. G. Marra, L. E. Weiss, J. W. Calvert and P. N. Kumta, Carnegie Mellon University, USA, 2000. 14. X. Guo, J. E. Gough, P. Xiao, J. Liu and Z. Shen, J. Biomed Mater. Res. A, 2007, 82A, 1022-1032. 15. L. Meirelles, A. Arvidsson, M. Andersson, P. Kjellin, T. Albrektsson and A. Wennerberg, J. Biomed Mat. Res. A, 2007, 10.1002/jbm.a.31744. 16. J. Wei, Y. Li and K. Lau, Composites part B: engineering, 2007, 38, 301-305. 17. H. Ramay and M. Zhang, Biomaterials, 2003, 24, 3293-3302. 18. J. S. Temenoff and A. G. Mikos, Biomaterials, 2000, 21, 2405-2412. 19. A. A. Deschamps, A. A. v. Apeldoorn, H. Hayen, J. D. d. Bruijn, U. Karst, D. W. Grijpma and J. Feijen, Biomaterials, 2004, 25, 247-258.

(98) TABLE-US-00001 TABLE 1 PCL/HA (29 wt % PCL/HA (29 wt % PCL nanosized HA).sup.[a] microsized HA).sup.[b] Com- Com- Com- pression pression pression Time Temp. strength Temp. strength Temp. strength (min) (° C.) (MPa) (° C.) (MPa) (° C.) (MPa) 0 70.0 — 70.5 — 70.0 — 10 48.5 — 49.0 — 47.5 — 15 41.5 5.65 41.5 11.9 40.5 11.0 20 39.5 9.65 40.0 15.6 40.0 10.8 30 31.5 14.8 31.0 16.9 32.0 14.9 45 25.5 15.7 26.5 15.2 27.0 13.3 60 23.5 14.1 25.0 16.5 24.5 14.1 120 22.5 13.2 24.0 15.8 23.0 13.8 240 22.0 14.8 24.0 16.1 23.0 13.5 360 22.0 15.1 24.0 16.6 23.0 15.6 1440 22.0 15.0 22.0 17.2 22.0 14.5 .sup.[a]The nanosized HA was prepared according to Example 2. .sup.[b]The microsized HA was purchased from Sigma Aldrich. The specific surface area was determined to 18 m.sup.2/g.

(99) TABLE-US-00002 TABLE 2 PCL/TOC/HA PCL/TOC/HA PCL/TOC/HA PCL/PEG/HA (29 wt % nanosized (29 wt % nano- (29 wt % microsized (31 wt % nanosized HA, without sized HA).sup.[a] HA).sup.[b] HA).sup.[c] amino acids).sup.[d] Compression Compression Compression Compression Time Temp. strength Temp. strength Temp. strength Temp. strength (min) (° C.) (MPa) (° C.) (MPa) (° C.) (MPa) (° C.) (MPa) 0 69.5 — 69.0 — 70.5 — 72.5 — 10 43.5 — 47.0 — 47.5 — 52 — 15 37.0 2.60 40.5 — 42.0 11.8 46 — 20 34.5 1.89 35.0 2.21 41.0 42.0 40 — 30 30.5 3.14 30.5 3.03 32.0 15.9 32 2.79 45 27.5 5.39 26.0 4.13 27.5 15.4 32 3.56 60 25.0 6.40 24.0 5.23 25.0 15.8 25.5 5.01 120 23.5 6.75 23.0 6.13 22.5 14.9 23.5 4.68 240 23.5 6.27 23.0 6.47 22.5 15.5 22 4.91 360 23.5 7.32 23.0 6.32 23.0 15.1 23 4.62 1440 22.0 6.65 22.0 6.24 22.0 15.6 22 5.45 .sup.[a]The HA used was prepared according to Example 2. .sup.[b]The HA used was purchased from Sigma Aldrich. The specific surface area was determined to 18 m.sup.2/g .sup.[c]PEG20000 was used. .sup.[d]The HA used was prepared according to Example 1.

(100) TABLE-US-00003 TABLE 3 PCL/TOC/ Eugenol/HA PCL/TOC/MO/HA PCL/TOC/MO/HA (29 wt % nanosized (31 wt % nanosized (38 wt % nanosized HA).sup.[a] HA).sup.[a] HA).sup.[a] Compression Compression Compression Time Temp strength Temp strength Temp strength (min) (° C.) (MPa) (° C.) (MPa) (° C.) (MPa) 0 69.0 — 70 — 70.0 — 10 50.0 — 47.5 — 45.0 — 15 43.5 0.92 41 3.91 39.0 — 20 36.5 0.94 37 3.63 36.5 4.71 30 30.0 1.12 31 5.40 31.0 5.95 45 26.0 1.10 — — 26.5 6.12 60 24.5 1.57 24.5 5.52 24.5 6.11 120 23.0 5.75 22.5 5.64 23.5 6.09 240 22.0 6.58 22.5 5.41 23.5 10.9 360 21.5 7.21 22.5 5.55 24.5 10.5 1440 22 6.44 — — 22 9.6 .sup.[a]The HA used was prepared according to Example 2.

(101) TABLE-US-00004 TABLE 4 PCL/ PCL/HA (29 wt % PCL/HA (29 wt % HA (29 wt % nanosized HA Temperature nanosized microsized HA, without amino (° C.) HA, MPa).sup.[a] MPa).sup.[b] acids, MPa) Room temp. 6.52 4.45 5.67 (20-22) 37 6.88 4.09 5.92 .sup.[a]The composite was prepared by extracting PCL/TOC/HA (29 wt % nanosized HA) with EtOH (see Example 11 for further details). .sup.[b]The composite was prepared by extracting PCL/TOC/HA (29 wt % microsized HA) with EtOH (see Example 11 for further details).