Artificial bone implants, or bone grafts, of polymeric composites with bone forming properties

Abstract

The present invention relates to methods for providing polymeric composites with bone forming, such as with osteogenic and/or osteoinductive and/or osteoconductive, properties. The present invention further relates to polymeric composites provided by the present method and the use of thereof for bone implants, or grafts, specifically the use thereof for orbital floor reconstruction. Specifically, the present invention relates to methods for providing a composite with bone forming properties, the method comprises the steps of: a) providing a liquid, or liquefied, polymeric composition of homopolymers or copolymers of 1,3-trimethylene carbonate (TMC); b) adding to said liquid, or liquefied, polymeric composition one or more agents with osteogenic and/or osteoinductive and/or osteoconductive properties thereby providing a dispersion of said agents in said liquid or liquefied polymeric composition; and c) crosslinking the product obtained, thereby providing a composite with bone forming properties.

Claims

1. A method for providing a composite with bone forming properties, said method comprising the steps of: a) providing a liquid, or liquefied, polymeric composition comprising: homopolymers of 1,3-trimethylene carbonate (TMC) with a Mw of more than 250,000 g/mol; or copolymers of TMC with a Mw of more than 250,000 g/mol, wherein said copolymer of TMC is chosen from the group consisting of TMC polymers with polyethylene oxide (PEO), polyethylene glycol (PEG) and e-caprolactone (CL); b) adding to said liquid, or liquefied, polymeric composition one or more agents with bone forming properties thereby providing a dispersion of said agents in said liquid or liquefied polymeric composition, wherein said one or more agents with bone forming properties are one or more compounds selected from the group consisting of calcium phosphates, hydroxyapatite, tricalcium phosphate, Bioglass, calcium sulphate, octacalcium phosphate, and biphasic calcium phosphate and combinations thereof; and c) crosslinking the dispersion, or a solidified form thereof to obtain a composite with bone forming properties.

2. The method according to claim 1, wherein the dispersion of said agents in the liquid or liquefied polymeric composition of step (b) comprises 5 wt % to 95 wt % of said one or more agents with respect to the total weight of the polymer in the dispersion.

3. The method according to claim 1, wherein said homopolymers or copolymers are liquefied by dissolution in a solvent.

4. The method according to claim 1, comprising, after step (b) but before step (c), a step comprising solidifying said dispersion and subsequently moulding said solidified dispersion into a desired shape.

5. The method according to claim 1, wherein step (c) is crosslinking said dispersion, or solidified form thereof, using gamma radiation with an irradiation dose of 10 to 100 kGy thereby providing an elastomeric composite with bone forming properties.

6. The method according claim 3, wherein said solvent is selected from the group consisting of acetone, dichloromethane, chloroform, carbontetrachloride, ethylene carbonate, propylene carbonate, dimethylsulfoxide, toluene, benzene, tetrahydrofuran and 1,4-dioxane.

7. The method according to claim 2, wherein the dispersion of said agents in the liquid or liquefied polymeric composition of step (b) comprises 30 wt % to 70 wt % of said one or more agents with respect to the total weight of the polymer in the dispersion.

8. The method according to claim 4, wherein said solidifying comprises solidifying by precipitation, or solidifying at a temperature below the glass transition temperature of the homopolymers or copolymers of TMC.

9. A method for providing a composite with bone forming properties, said method comprising the steps of: a) providing a liquid, or liquefied, polymeric composition comprising: homopolymers of 1,3-trimethylene carbonate (TMC) with a Mw of more than 250,000 g/mol; or copolymers of 1,3-trimethylene carbonate (TMC) with a Mw of more than 250,000 g/mol; b) adding to said liquid, or liquefied, polymeric composition one or more agents with bone forming properties thereby providing a dispersion of said agents in said liquid or liquefied polymeric composition, wherein said one or more agents with bone forming properties are one or more compounds selected from the group consisting of calcium phosphates, hydroxyapatite, tricalcium phosphate, Bioglass, calcium sulphate, octacalcium phosphate, and biphasic calcium phosphate and combinations thereof; c) solidifying said dispersion and subsequently moulding said solidified dispersion into a desired shape; and d) crosslinking said solidified dispersion using gamma radiation with an irradiation dose of 10 to 100 kGy thereby providing an elastomeric composite with bone forming properties.

10. The method according to claim 9, wherein the dispersion of said agents in the liquid or liquefied polymeric composition of step (b) comprises 5 wt % to 95 wt % of said one or more agents with respect to the total weight of the polymer in the dispersion.

11. The method according to claim 10, wherein the dispersion of said agents in the liquid or liquefied polymeric composition of step (b) comprises 30 wt % to 70 wt % of said one or more agents with respect to the total weight of the polymer in the dispersion.

12. The method according to claim 9, wherein said homopolymers or copolymers are liquefied by dissolution in a solvent.

13. The method according to claim 9, wherein said copolymer of TMC is chosen from the group consisting of TMC polymers with lactones cyclic esters, cyclic carbonates, cyclic ethers, cyclic anhydrides, and cyclic depsipeptides morpholine 2,5-dione derivatives.

14. The method according to claim 9, wherein said copolymer of TMC is chosen from the group consisting of TMC polymers with polyethylene oxide (PEO), polyethylene glycol (PEG) and e-caprolactone.

15. The method according to claim 9, wherein said copolymer of TMC is chosen from the group consisting of TMC polymers with 5-valerolacton, 1, 5-dioxepane-2-one, and e-caprolactone.

16. The method according to claim 9, wherein said solidifying comprises solidifying by precipitation, or solidifying at a temperature below the glass transition temperature of the homopolymers or copolymers of TMC.

Description

(1) The present invention will be further detailed in the example below demonstrating the advantageous properties of the present composites in preferred embodiments. In the example, reference is made figures wherein:

(2) FIG. 1: shows light micrographs of intramuscular implantation sites after three months. Figures A-D represent overviews of intramuscular implantations of respectively BCP, the present composite, laminated composite and PTMC. Figures E-H represent magnifications (4) of the overviews. Bone (b) is clearly visible and in close contact with the BCP particles (p) in figures A-C and their corresponding magnifications. The PTMC (.circle-solid.) matrix has resorbed extensively, phagocytosed polymer particles (arrows) can be observed. (.box-tangle-solidup.) designates an area where remnants of disintegrated BCP particles are demonstrated. (.square-solid.) PDLLA polymer;

(3) FIG. 2: shows light micrographs (20-40) of intramuscular implantations after three months of respectively pure BCP particles (A, B), the present composite (C, D) and laminated composite (E, F). BCP particles are surrounded by phagocytic cells. The dust-like aspect at the surface of the particles suggests disintegration. Remaining PTMC particles (arrows) are easily identified. (A, C and E are 20 magnifications, B, D and F 40);

(4) FIG. 3: shows light micrographs of orbital implantations after three (A-F) and nine (G-L) months. Figure A, D, G and J show reconstruction with PTMC sheet. Capsule formation is visible; there is no sign of bone formation. New bone formation is clearly visible and in close contact with the BCP particles after three months in the present composite sheet (B, E) and shows progressive after nine months (H, K). Besides bone formation, also resorption of PTMC and disintegration of BCP particles is demonstrated. The laminated composite sheet also showed bone formation around BCP particles after three months (C, F). After nine months limited amounts of bone formation were found (I, L). (D-F and J-L are 4 magnifications of respectively A-C and G-I; (.circle-solid.) PTMC, (*) maxillary sinus, (b) bone, (.square-solid.) PDLLA polymer, (arrow) residual PTMC polymer particles, (ct) connective tissue and (.box-tangle-solidup.) designates area where remnants of disintegrated BCP particles are demonstrated.)

(5) FIG. 4: shows light micrographs (2.5) representing the transition area of the present composite (A, B) and laminated composite (C, D) implant (and newly formed bone) to host bone in the orbit of the sheep. Figures A and B show excellent osseous integration of newly formed bone with the host bone after respectively 3 and 9 months. Figure C clearly illustrates the PDLLA layer of the laminated composite impeding osseous integration after three months. Figure D shows that after 9 months, although the PDLLA layer has degraded, osseous integration of newly formed bone with the host bone still has not occurred. This could be due to the fibrous capsule which is present between the newly formed bone and host bone.

(6) FIG. 5: shows light micrographs (20) showing the disintegration of BCP particles in the orbital implantations after three and nine months. Figure A-B represent the present composite implants, C-D represent laminated composite implants. BCP particles are surrounded by phagocytic cells. The dusty aspect of the particles suggests disintegration. Residual PTMC particles (arrows) are easily identified. The disintegration of the BCP particles in the composite and laminated composite tended to be more extensive compared to the disintegration in the intramuscular implantations containing BCP particles only. (*) shows area where BCP is disintegrating, (arrow) phagocytosed PTMC.

(7) FIG. 6: shows epifluorescent confocal micrographs of intramuscularly implanted amounts of BCP (A-B) and the present composite (C-D). E-F and G-H are images of orbital implantations of the present composite after, respectively three and nine months. A, C, E, and G are bright field images, B, D, F, and H are epifluorescent images. Calcein=green, Xylenol Orange=red and Oxytetracycline=blue. It can be seen that bone formation had started after three weeks around the intramuscularly implanted amounts BCP and intramuscularly implanted composites near the edges, where the polymeric PTMC matrix had resorbed. The orbital implantations showed similar results. Bone formation had started after three weeks (E-F) in the three month group. Figures G-H show the process of bone formation being still active at nine months.

(8) FIG. 7: shows assessment of orbital floor position. Figure A shows a lateral view of a postoperative CBCT scan of a sheep. The present composite implant (arrow) has radio-opaque properties and can be clearly identified at the top of the maxillary sinus (*). Figure B shows the superposition of the postoperative scan over the preoperative scan. The region of interest is highlighted. There is a slight negative deformation at the center of the implant when compared to the preoperative situation. Where the implant overlies the defect borders (i.e. is resting on the intact orbital floor borders), the deformation is slightly positive when compared to the preoperative situation of the intact orbital floor.

EXAMPLE

Introduction

(9) Materials and Methods

(10) Materials

(11) Polymerization grade 1,3-trimethylene carbonate (TMC) was obtained from Boehringer Ingelheim, Germany. Stannous octoate (SnOct.sub.2 from Sigma, USA) was used as received. High molecular weight poly(D,L-lactide) (PDLLA, with a 50/50 molar ratio of L- to D-lactide) was obtained from Purac Biochem, the Netherlands, and used as received. Biphasic calcium phosphate ceramic, (205% TCP and 805% HA), which was sintered at 1150 C. and sieved to particle sizes 45-150 m, was obtained from Xpand Biotechnology, the Netherlands. The used solvents were of technical grade and purchased from Biosolve, the Netherlands.

(12) Preparation of Composites and Laminates

(13) Poly(trimethylene carbonate) (PTMC) was prepared by ring opening polymerization of trimethylene carbonate at 130 C. for a period of 3 days. Stannous octoate was used as a catalyst at a concentration of 210.sup.4 mol per mol of monomer. Analysis of the synthesized polymer by proton nuclear magnetic resonance (.sup.1H-NMR), gel permeation chromatography (GPC) and differential scanning calorimetry (DSC) according to standardized procedures indicated that high molecular weight polymer had been synthesized.

(14) GPC measurements showed that Mw=414,000 and Mn=316,000 g/mol, while NMR indicated that the monomer conversion was more than 98%. The glass transition temperature of this amorphous polymer was approximately 17 C., as thermal analysis showed.

(15) The PTMC polymer was purified by dissolving in chloroform and precipitation into an excess of ethanol. Similarly, composites of PTMC with BCP particles were prepared by dissolving PTMC in chloroform at a concentration of 5 g/100 ml, after which the BCP was added and uniformly dispersed in the solution. The dispersion was then precipitated into a five-fold excess of ethanol 100%. The composite was collected and dried under vacuum at room temperature until constant weight was reached. PTMC/BCP composites containing 50 wt % corresponding to 30 vol % of CP were prepared.

(16) After drying, the purified PTMC and the composite precipitate were compression moulded into 1.5 mm thick sheets at 140 C. and a pressure of 3.0 MPa (31 kg/cm.sup.2) using a Carver model 3851-0 laboratory press (Carver, USA). The poly(D,L-lactide) was also of high molecular weight, and had an Mw=234,000 g/mol and an Mn=178,000 g/mol. NMR indicated that the residual monomer content was less than 1%. The glassy polymer was also amorphous, and had a glass transition temperature of approximately 52 C. This polymer was compression moulded into 0.3 mm thick sheets at 140 C.

(17) Laminates of the PTMC/BCP composites and PDLLA were prepared by compression moulding PDLLA sheets onto sheets of the composite material at 140 C. The composite layer was 1.2 mm thick, while the PDLLA layer was 0.3 mm thick.

(18) The prepared sheets were then sealed under vacuum and exposed to 25 kGy gamma irradiation from a .sup.60Co source (Isotron BV, Ede, The Netherlands) for crosslinking.

(19) Experimental Design of the Animal Study

(20) All procedures performed on the animals were done according to international standards on animal welfare as well as being compliant with the Animal Research Committee of the University Medical Center Groningen.

(21) Ten full-grown female Dutch Texel sheep were operated on and (evenly) divided into two groups. The first group had a follow-up of three months, the second a follow-up of nine months. Critical size irregularly shaped circular defects, 2.5-3.0 cm.sup.2 were created in both orbital floors and reconstructed with:

(22) 1) a PTMC sheet,

(23) 2) a composite (PTMC/BCP) sheet or

(24) 3) a laminated composite (PTMC/BCP-PDLLA) sheet. Regarding the latter, the PDLLA layer faced towards the maxillary sinus.

(25) To demonstrate osteoinduction, samples (1.5 mm10 mm ) of the mentioned PTMC, composite and laminated composite sheets as well as an amount of 1 ml of BCP particles were also implanted intramuscularly in the back of the sheep. An overview is provided in Table 1 below.

(26) TABLE-US-00001 TABLE 1 Overview of implantations and implantation sites for the three and nine month group Implantation material 3 months 9 months BCP IM: n = 5 IM: n = 5 PTMC OF: n = 3 OF: n = 3 IM: n = 3 IM: n = 3 Composite OF: n = 4 OF: n = 4 (PTMC/BCP) IM: n = 4 IM: n = 4 Laminated composite OF: n = 3 OF: n = 3 (PTMC/BCP-PDLLA) IM: n = 3 IM: n = 3 OF: orbital floor IM: intramuscular

(27) Furthermore, to assess the position of the reconstructed orbital floor, all sheep were evaluated by cone-beam computer tomography (CBCT) one week before and one week after surgery and at time of termination. To monitor the bone formation over time, fluorochrome markers were administered at nine, six and three weeks prior to the three and nine month termination. Bone formation was evaluated by histology and histomorphometry of non-decalcified sections using epifluorescent confocal and conventional light microscopy.

(28) Surgical Procedure and Fluorochrome Labelling

(29) Ten adult full-grown female Dutch Texel sheep, aged 24-36 months, were acquired and allowed to acclimatize for two weeks. The surgical procedures were performed under general anaesthesia. After the subciliar area was shaved and disinfected, both orbital floors were exposed using an infraorbital approach. The periosteum was elevated and the floor was fractured using a burr and/or chisel. Bone fragments were removed from the defect site. The bony defects created measured 2.5-3.0 cm.sup.2 in size.

(30) Then, the orbital floor was reconstructed using one of the implant materials (PTMC, composite or laminated composite sheet). Care was taken to ensure that the total defect was covered, for this the implant was tailored to size with a scissor. Implants were fixed with one titanium screw (1.53.5 mm, KLS-Martin, Germany) to prevent dislocation.

(31) After reconstruction, the orbital periosteum was incised to mimic a traumatic situation (the incision allowed the orbital fat and musculature to prolapse into the orbit and exert force on the reconstruction material like in a real traumatic situation). The wound was closed in layers with resorbable sutures (Polyglactin 910, Ethicon, USA).

(32) Simultaneously, intramuscular implantation of samples was performed in the paraspinal muscles. The muscle fascia was closed with non-resorbable sutures to mark the different implantation sites in the back (Polypropylene, Ethicon, USA). The other layers with resorbable sutures.

(33) Prior to surgery amoxicilline was administered and continued for six days postoperative. Buprenorphin was administered for peri- and postoperative pain relief.

(34) Fluorochrome markers were administered prior to termination. Calcein Green (10 mg/kg intravenously, Sigma, The Netherlands) was administered at nine weeks, Xylenol Orange (100 mg/kg intravenously, Sigma, The Netherlands) at six weeks and Oxytetracyclin (Engemycine 32 mg/kg intramuscularly, Mycofarm, The Netherlands) at three weeks prior to termination. After three and nine months follow-up, the animals were sacrificed by an overdose of pentobarbital (Organon, The Netherlands) and the implantation areas retrieved and fixed in a 4% phosphate-buffered formalin solution.

(35) Histological Preparation

(36) Fixed samples were rinsed with phosphate buffer solution (PBS), dehydrated in a series of ethanol solutions (70%, 80%, 90%, 96%, 100%2) and embedded in methyl methacrylate (LTI, The Netherlands). Using a diamond saw (Leica SP1600, Leica Microsystems, Germany), histological sections (10-20 m thick) were made along the plane perpendicular to the orbital floor for the former and parallel to the long axis of the implants for the latter. Sections for light microscope (Nikon Eclipse E200, Japan) observation were stained with 1% methylene blue (Sigma-Aldrich) and 0.3% basic fuchsin (Sigma-Aldrich) solutions, while unstained sections were made for epifluorescent confocal microscopy (Leica TCS SP2, Leica, Germany) observation.

(37) Epifluorescent data was collected with 20 oil immersion objective, including transmitted light detection. The peak absorption (abs.) and emission (em.) wavelengths where: 351/364 nm abs. and 560 nm em., 543 nm abs. and 580 nm em., 488 nm abs. and 517 nm em., for respectively Tetracycline, Xylenol Orange and Calcein.

(38) Histomorphometry and Statistics

(39) Images of the stained sections for histomorphometric analysis were made using a slide scanner (Dimage Scan Elite 5400 II, Konica Minolta Photo Imaging Inc, Japan).

(40) Histomorphometry was performed using Adobe Photoshop Elements 4.0 software. Briefly, the implant area was selected as the region of interest (ROI) and the corresponding number of pixels registered. Then both BCP particles and mineralized bone were pseudo-colored and the resulting numbers of pixels used to calculate the percentage of bone formation in the available space (available space is defined as the space between the BCP particles where the polymer has resorbed) as:

(41) $\mathrm{Bone}\mathrm{formation}\%=\frac{\mathrm{Bonepixels}}{\mathrm{ROI}-\mathrm{BCPpixels}}100\%$

(42) Averages and standard deviations were calculated for the percentage of bone formation in the available area. A Fisher's Exact Test was used to evaluate the differences in bone formation between the different materials as well as between the individual materials compared for the three and nine month group. The data sets were statistically evaluated using SPSS 17 (Statistical Package for the Social Sciences, SPSS Inc., USA). The null hypothesis (the means of each set are equal) was evaluated with 95% confidence level (=0.05).

(43) Radiologic Examination

(44) Cone-beam computed tomography (CBCT) scanning was performed to assess the position of the preoperative and postoperative (reconstructed) orbital floors, as well as at time of termination. CBCT scanning was carried out under general anaesthesia with propofol, The CBCT images were acquired with I-CAT Scanner with a 0.3 mm voxel size and a 170 mm field of view and stored for further analysis. Each scan was performed with the head of the animal in the same, reproducible position using the laser guide of the scanner as a reference.

(45) Using Mimics Software (Materialise Dental, Belgium) three-dimensional (3D) reconstructions of all individual scans were made employing the same optimal threshold to depict the bone on each dataset.

(46) Next the preoperative (intact) orbital floors and postoperative reconstructed orbital floors (i.e. the orbital floor implants) as well as the reconstructed orbital floors at time of termination were selected as region of interest (ROI).

(47) Using Geomagic Studio Software (Geomagic Gmbh, Stuttgart, Germany) the 3D reconstructed scans were aligned and registered with the preoperative 3D reconstructed scans using an iterative closest point registration algorithm. The preoperative scan thus served as reference. Preoperative and postoperative (or at time of termination) orbital floors were highlighted.

(48) The deviation between the datasets was measured on a sliding colour scale which displayed the distances between the surfaces of the orbital floors (FIG. 6).

(49) The mean negative deviation (i.e. at the level of the defect) for each implant was noted Table 2 below.

(50) TABLE-US-00002 TABLE 2 deformations of the reconstructed orbital floors are provided for the different materials after 3 and 9 months postoperative. The animal that died after 6 months is evaluated separately. The preoperative scan served as reference. Subsequent the calculated maximum increase in volume of the orbit of the sheep after three and nine months for the different reconstruction materials are provided. For this the most negative deformation of each reconstruction material was used. The (maximum) increase in orbital volume occurred due to deformation of the reconstruction materials. (The defect size was considered to be 3 cm.sup.2.) PTMC Composite Laminated composite 3 months 9 months 3 months 6 months 9 months 3 months 6 months 9 months 0.8 mm 0.7 mm 1.0 mm 1.3 mm 0.6 mm 0.7 mm 0.8 mm 0.9 mm 1.0 mm 1.0 mm 1.1 mm 0.5 mm 0.5 mm 0.4 mm 0.9 mm 1.5 mm 0.6 mm 0.4 mm 0.6 mm 1.3 mm Mean SD 0.77 0.25 1.20 0.36 0.48 0.79 1.1 0.67 0.25 0.80 0.26 0.7 0.70 0.42 (mm) Volume 0.16 0.23 0.19 0.16 0.14 0.15 0.11 0.15 increase (cm.sup.3)

(51) In this way the deformation for the different reconstruction materials at the different time periods was determined. Next, the (overall) mean negative deformation for the different implants was calculated and used to establish the orbital volume increase, using the equation:

(52) $V_{\mathrm{increase}}=\frac{1}{6}h\left(3r^2+h^2\right)$
wherein: V.sub.increase=volume increase of the orbital cavity due to deformation of the reconstruction material (m.sup.3) h=deformation of the disk-shaped implant (m) r=0.0098 (m); this is the radius of a circular orbital floor defect measuring 3.0 cm.sup.2

(53) Changes in orbital volume were used to assess the suitability of the different implants for reconstruction of orbital floor defects in sheep. An increase in orbital volume of +0.7 cm.sup.3 was considered the maximum allowable volume increase. Increases with volumes>0.7 cm.sup.3 can lead to enophthalmos and should therefore be avoided.

(54) Results

(55) During the in vivo experiment, none of the sheep showed signs of infection or adverse tissue reactions. Nine sheep remained in good health, one (otherwise healthy) animal died unexpectedly six months postoperatively. A performed autopsy did not reveal an obvious cause of death. No animals were excluded from this study. The prematurely deceased animal was evaluated as a separate 6 month time point group.

(56) Descriptive Microscopic Observations of Intramuscular Implantations

(57) After three and nine months all intramuscular implants were traced and the implantation sites harvested. Table 3 below provides an overview of the bone incidence for the different implants.

(58) TABLE-US-00003 TABLE 3 Bone incidence in implantations after 3, 6 and 9 months and their consecutive percentages of bone formation in the available area as determined by histomorphometry. Mean standard deviation is presented Bone Bone Bone inci- inci- inci- Implanted dence 3 % dence 6 % dence 9 % material months bone months bone months bone BCP IM: 2/5 2.9 5.9 IM: 1/1 12.8 IM: 2/4 6.4 6.9 particles PTMC OF: 0/3 0 0 NI OF: 0/3 0 0 IM: 0/3 0 0 NI IM: 0/3 0 Composite OF: 3/4 7.7 8.1 OF: 1/1 14.9 OF: 3/3 15.7 14.6 IM: 2/4 0.3 0.6 IM: 0/1 0 IM: 0/3 0 Laminated OF: 3/3 5.3 4.0 OF: 1/1 13.9 OF: 1/2 1.7 2.4 composite IM: 2/3 2.0 1.9 IM: 0/1 0 IM: 0/2 0 OF: orbital floor IM: intramuscular NI: not implanted

(59) Light microscopical evaluation of the stained sections showed that bone formation was present in most of the implantations that contained BCP particles. Implantations of PTMC alone (i.e. not a composite with BCP) did not lead to formation of bone in any of the sheep.

(60) FIG. 1 provides an overview of the intramuscular implantations after three months. Light microscopical observations showed that, when present, bone had formed around the BCP particles and was in close contact with the surface of the particles. Besides bone formation, connective tissue ingrowth was observed. Furthermore it was shown that for the implanted composites the PTMC polymer matrix had resorbed profoundly. Only small amounts of PTMC were found. The PDLLA layer of the laminated composite could still be identified. The PTMC implantations did not show any bone formation. The PTMC polymer could still be identified and was surrounded by a fibrous capsule of dense connective tissue.

(61) Besides signs of the degradation of the polymers, also disintegration of the BCP particles was observed. In FIG. 2, the degradation of the PTMC polymeric matrix and the disintegration of the BCP particles is shown at higher magnifications. Closer observations showed that recruitment of phagocytic and multi-nucleated giant cells had occurred. These cells surrounded and adhered to the remnants of the PTMC polymeric matrix as well as to the surface of the BCP particles.

(62) After nine months the implantations of pure BCP particles showed progressive bone formation, while none of the intramuscular implanted (laminated) composites showed bone formation. The polymeric PTMC matrix of the composite had resorbed almost completely, only few phagocytosed PTMC particles were observed. Signs of disintegration of the BCP particles were also observed after nine months in all implantations containing BCP particles.

(63) The intramuscularly implanted samples of PTMC were still identified, although signs of degradation were progressive. Implants were still surrounded by a fibrous capsule consisting of dense connective tissue.

(64) Descriptive Microscopic Observations of Orbital Implantations

(65) The results for the orbital implantations are shown in Table 3 and FIG. 3. After three months, the composite and laminated composite implants clearly showed bone formation. Most of the polymeric PTMC matrix had resorbed, only small remnants of PTMC were observed. The newly formed bone was in close contact with the BCP particles. Moreover, the newly formed bone in the composite implants showed osseous integration with the host bone at places (i.e. the bone of the animal) where the composite implants were in contact with the host bone (i.e. at the orbital floor defect borders) (FIG. 4).

(66) At the level of the defect, where the composite implants were consequently not in contact with the host bone, several layers of dense connective tissue covered the implants. The laminated composites, by contrast, did not show this osseous integration of newly formed bone with host bone and were completely surrounded by a fibrous capsule composed of dense connective tissue (FIG. 4).

(67) After nine months the bone formation appeared to be progressive for the composite implants. Both the composite implants and laminated composite implants showed almost complete resorption of the polymeric PTMC matrix. Only phagocytised PTMC polymer was observed. The PDLLA layer seemed to have been resorbed completely at this time point. Whereas the newly formed bone in the composite implants still showed integration with the host bone, the laminated composites were still surrounded by the fibrous capsule and subsequently did not.

(68) Signs of disintegration of the BCP particles were also found in the orbital implants. FIG. 5 shows a composite implant and laminated composite implant after three and nine months at higher magnifications. Recruitment of phagocytic cells was also observed here. These cells adhered to the BCP particles as well as to remnants of the PTMC.

(69) The histological findings for the animal that died after six months were comparable to the observations found for the other animals after nine months. In this animal the orbital composite implant showed new bone formation that had integrated with the host bone. Although the laminated composite implant placed in the other orbit did show bone formation, the newly formed bone (again) had not integrated with the host bone. A fibrous capsule surrounded the laminated composite implant. Besides the implanted amounts of pure BCP particles, none of the intramuscular implantations demonstrated bone formation (Table 3).

(70) The degradation and resorption of the polymer matrix and PDLLA layer showed to be progressive compared to the specimens after three months, but was not as advanced as in the nine month group. Remnants of the PDLLA layer were still identified. Disintegration of the BCP particles was also observed in all implantations containing BCP particles.

(71) Fluorescence Microscopy

(72) Epifluorescent confocal microscopy of the sequential fluorochrome labels revealed that upon three weeks after implantation formation of bone had started in the intramuscular implantations of pure BCP particles and (laminated) composites. Similar observations were found for the composite and laminated composite implantations in the orbital implantations (FIG. 6).

(73) Analysis of the fluorochrome labels indicated that the bone formation started at the surface of the BCP particles and progressed toward the periphery. After nine months the fluorochrome labelling showed that the process of bone formation and remodelling was still active in the orbital floor implants. None of the intramuscular implanted (laminated) composite samples showed fluorescent labelling after nine months.

(74) Histomorphometry

(75) The results of the histomorphometrical analysis for the intramuscular and orbital implantations are shown in Table 3. The mean percentages and standard deviations are presented for the bone formation in the available area: defined as the space between the BCP particles where the polymer has resorbed. Besides the fact that not all animals showed bone formation in every implantation, large variations in the amounts of formed bone (when present) in and between the individual animals were found.

(76) After three months measurements showed 7.78.1% (meanSD) of bone had formed in the composite orbital floor implantations. After nine months the percentage of bone had increased to 15.512.0%. The laminated composite orbital floor implants showed 5.34.0% and 1.72.4% of bone formation, respectively after three and nine months. The intramuscular implantations showed limited bone formation.

(77) The intramuscularly placed composite samples demonstrated 0.30.6% and the laminated composite 2.01.9% of bone formed after three months. The intramuscularly placed amounts of pure BCP particles showed 2.95.9% bone formation, which progressed to 6.46.9% of bone, after nine months.

(78) The prematurely deceased animal showed respectively 13.9% and 14.9% of bone formation for the laminated composite and composite implantations in the orbits. The intramuscular implanted pure BCP particles measured 12.8% of bone formation.

(79) Evaluation by CBCT

(80) FIG. 7 graphically presents the evaluation process of the reconstruction of the orbital floors and illustrates the performance of the implants. It can be seen that the radio-opaque composite and laminated composite sheets were easily identified. As stated, the colour mapping shows the changes in deviation of the reconstructed orbital floor compared to the preoperative intact orbital floor.

(81) The results for the deformation and maximum calculated changes in orbital volume due to deformation of the implants for the different time periods are summarized in Table 2.

(82) It can be seen that the increase of the orbital volume in animals treated with the PTMC implants ranged from +0.16 to +0.23 cm.sup.3, respectively after three and nine months. The animals treated with the composite implants showed an orbital volume increase ranging from +0.14 to +0.19 cm.sup.3, while the animals treated with the laminated composite implants showed volume increases ranging from +0.11 to +0.15

(83) Discussion

(84) The present example describes the evaluation of the osteoinductive properties of composite materials composed of PTMC and microstructured BCP. The composite materials were evaluated both in an orthotopic (orbit) as well as in an ectopic (intramuscular) site in sheep. Simultaneously with the evaluation of the osteoinductive properties of the composite materials, the suitability of the composite materials to serve as a load bearing material was assessed.

(85) It was shown that the PTMC/BCP composite materials and the PDLLA-laminated PTMC/BCP composite materials have osteoinductive properties. Moreover, fluorochrome labelling indicated that the osteoinductive potential of the composites remained active at nine months.

(86) During the surgical procedures, it became clear that regarding the shape-ability the composite and the laminated composite, as well as the PTMC implants could be cut easily into the desired shape. Most importantly, the present results showed that the composite as well as the laminated composite implants exerted osteoinductive properties. Moreover, the composite implants, in contrast with the laminates, showed excellent osseous integration of the newly formed bone with the host bone.

(87) The histological observations of the degradation of the polymeric PTMC matrix with simultaneous formation of bone supported the hypothesis that a resorbable polymeric matrix could enhance the mechanical properties of calcium phosphate ceramics, without negatively affecting the osteoinductive properties of the calcium phosphate particles.

(88) For the laminated composites, however, a negative effect on bone formation as well as osseous integration of newly formed bone with host bone was observed.

(89) The present composites, besides not showing hindrance to regeneration of normal/local tissue (excellent osseous integration), can also be expected to have a more favourable surface-to-volume ratio, since the degradation process can continue along the exposed BCP particles, thereby increasing the surface-to-volume ratio.

(90) The relatively large variations of induced amounts of bone that that were observed in and between the individual animals are not uncommon with research concerning osteoinductive materials. It is known that besides animal-specific factors have an effect on the amount of induced bone, implantation site-specific factors also play a role.

(91) Orthotopic locations tend to give larger amounts of induced bone compared to ectopic locations. Also, it is suggested that the intrinsic ability of individual animals to form new bone in osteopromotive environments could vary because of genetic factors leading to different responses to exogenous cells (e.g. exogenous bone morphogenetic proteins (BMPs) as well as different actions of endogenous cells involved in the process of osteoinduction.

(92) The evaluation by CBCT showed that all the reconstructed orbital floors were adequately positioned compared to the preoperative anatomical situation. None of the calculated volume increases, due to deformation of the implants, of the orbits were above the aforementioned critical value of 0.7 cm.sup.3 (Table 2).

CONCLUSIONS

(93) The present example describes the preparation and evaluation of osteoinductive composites composed of PTMC and microstructured BCP particles. From the results it can be concluded that the composite materials are shapeable, exert osteoinductive and osteogenic properties and show integration with, or attachment to, the surrounding host bone.