Clinical Hemorheology and Microcirculation 60 (2015) 3–11 DOI 10.3233/CH-151936 IOS Press
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Preparation and mechanical properties of photo-crosslinked poly(trimethylene carbonate) and nano-hydroxyapatite composites Mike A. Gevena , Davide Barbierib , Huipin Yuanb,c , Joost D. de Bruijna,b and Dirk W. Grijpmaa,d,∗ a
MIRA Institute for Biomedical Technology and Technical Medicine, and Department of Biomaterials Science and Technology, University of Twente, Enschede, The Netherlands b XPand Biotechnology BV, Bilthoven, The Netherlands c MIRA Institute for Biomedical Technology and Technical Medicine, and Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands d University of Groningen, University Medical Center Groningen, W.J. Kolff Institute, Department of Biomedical Engineering, Groningen, The Netherlands Submitted 10 January 2014; accepted 7 March 2014
Abstract. Composite materials of photo-crosslinked poly(trimethylene carbonate) and nanoscale hydroxyapatite were prepared and their mechanical characteristics for application as orbital floor implants were assessed. The composites were prepared by solvent casting poly(trimethylene carbonate) macromers with varying amounts of nano-hydroxyapatite and subsequent photocrosslinking. The incorporation of the nano-hydroxyapatite into the composites was examined by thermogravimetric analysis, scanning electron microscopy and gel content measurements. The mechanical properties were investigated by tensile testing and trouser tearing experiments. Our results show that nano-hydroxyapatite particles can readily be incorporated into photocrosslinked poly(trimethylene carbonate) networks. Compared to the networks without nano-hydroxyapatite, incorporation of 36.3 wt.% of the apatite resulted in an increase of the E modulus, yield strength and tensile strength from 2.2 MPa to 51 MPa, 0.5 to 1.4 N/mm2 and from 1.3 to 3.9 N/mm2 , respectively. We found that composites containing 12.4 wt.% nano-hydroxyapatite had the highest values of strain at break, toughness and average tear propagation strength (376%, 777 N/mm2 and 3.1 N/mm2 , respectively). Keywords: Composites, photo-crosslinked poly(trimethylene carbonate), nano-hydroxyapatite, orbital floor repair
1. Introduction Blow-out fractures of the orbital floor are common injuries in traffic accidents or assaults, caused by excessive force applied to the infraorbital rim [4]. Restoring the original volume of the orbit and ∗
Corresponding author: Prof. Dirk W. Grijpma, MIRA Institute for Biomedical Technology and Technical Medicine, and Department of Biomaterials Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands, Tel.: +31 53 4892966; Fax: +31 53 4892155; E-mail: [email protected]. 1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
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M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
repositioning the globe are very important. In practice, this is done by covering the fracture site with an implant [4]. The current clinically used implants consist of autografts and allografts as well as of alloplastic materials such as hydroxyapatite ceramics, titanium, silicone rubber, ultra high density polyethylene, poly(lactic acid) and hydroxyapatite/polyethylene composites [1]. Ideally, an implant material is biocompatible, mechanically stable enough to support the orbital contents, osteoinductive and bioresorbable with a minimal foreign body response. The limited availability and the possibility for disease transmittance associated with autografts and allografts decrease their appeal as implant materials [1]. Drawbacks of several of the alloplastic materials are sub-optimal mechanical properties, limited potential for bone ingrowth or their non-degradability. For these reasons, composites of synthetic, degradable and biocompatible polymers and calcium phosphates have been investigated for bone tissue engineering and orbital floor repair. Encouraging results have been achieved with composites of poly(L-lactide), poly(L-lactide-co-glycolide) and poly(-caprolactone) with several calcium phosphates such as hydroxyapatite, -tricalcium phosphate (-TCP) and biphasic calcium phosphate [1, 6–14, 19]. Although these polyester-based composites have shown good results, problems may arise from the significant amounts of acidic degradation products that can be formed. Furthermore, the average molar mass of polyesters will decrease during degradation resulting in decreasing mechanical properties [18]. Recently, composites of linear non-crosslinked poly(trimethylene carbonate) (PTMC) and -TCP have been investigated as potential orbital floor implants. PTMC is a flexible, amorphous polymer that degrades enzymatically through surface erosion in presence of macrophages. This surface erosion mechanism produces non-acidic degradation products and the mechanical properties of PTMC are maintained over time as the average molar mass remains constant [21]. It has been shown that composites of PTMC with -TCP had sufficient mechanical properties for the reconstruction of large orbital floor defects (5.5 cm2 ) [15]. Furthermore, implantation of composites of PTMC and biphasic calcium phosphate in sheep orbital floor defects resulted in newly formed bone and excellent osseous integration with the surrounding bone [16]. Although these PTMC composites were shapeable, an exact anatomical fit to the orbital floor defect would be most desirable for the optimal orbital floor reconstruction and repositioning of the orbital contents [1]. Microstereolithography is an additive manufacturing process based on photo-polymerization. It can reach precisions of 5 m and may allow for the fabrication of such precisely fitting implants [17]. For this we have prepared photo-curable three-armed PTMC macromers with methacrylate end-groups (PTMCMA). PTMC-MA based resins with varying amounts of nano-hydroxyapatite were then prepared and photo-crosslinked to obtain PTMC network composites that may have osteoinductive properties [2].
2. Materials and methods 2.1. Materials All materials were used as received unless stated otherwise. ForYou Medical Devices Ltd. provided the trimethylene carbonate (TMC) used for this work. The nano-hydroxyapatite powder consisted of 5 to 25 m sized agglomerates of needle-like hydroxyapatite crystals of 200 to 400 nm long and 20 to 50 nm wide, and was prepared by XPand Biotechnology BV as previously reported [3]. Fig. 1 shows an SEM image of the nano-hydroxyapatite as received, and the size distribution as determined using ImageJ. Tin(II) 2-ethylhexanoate (Sn(Oct)2 ), hydroquinone, triethylamine, methacrylic anhydride and deuterated
M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
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Fig. 1. SEM image (left) and size distribution (right) of the nano-hydroxyapatite particles as were manufactured and received (scale bar: 20 m).
chloroform were obtained from Sigma-Aldrich. 1,1,1-tris(hydroxymethyl)propane (trimethylolpropane) was from Fluka analytical and Lucirin® TPO-L was from BASF. Chloroform, propylene carbonate and calcium hydride were from Merck Millipore. Dichloromethane, from VWR chemicals, was dried over calcium hydride and subsequently distilled under dry N2 . 2.2. Methods 2.2.1. Synthesis of PTMC-MA 3-armed PTMC was synthesized by ring-opening polymerization of TMC (Fig. 2). Under dry N2 atmosphere, a three-necked round-bottomed flask was charged with 49.8 g (0.49 mol) TMC and 0.67 g (5.0 mmol) trimethylolpropane as initiator. The polymerization was conducted at 140◦ C for 19 hours, using Sn(Oct)2 as catalyst at a concentration of 0.13 wt.%. After cooling to room temperature, the PTMC oligomer was dissolved in dried dichloromethane. Under dry N2 , 52.7 mg of hydroquinone, 4.5 ml (32 mmol) triethylamine and 4.6 ml (31 mmol) methacrylic anhydride were added and the resulting solution was stirred in the dark for 5 days at room temperature. The solution was then extracted with demineralized water 3 times and the organic phase was precipitated in cold methanol. The precipitate was dried in the dark at ambient conditions overnight and for another 5 days at room temperature in vacuo. The non-functionalized PTMC oligomer and the functionalized precipitated macromer were analyzed by 1 H-NMR (Bruker Ascend 400/Avance III 400 MHz NMR spectrometer) to determine the TMC conversion, the number average molecular mass (Mn ) and the degree of functionalization of PTMC-MA. Deuterated chloroform was used as the solvent.
Fig. 2. Synthesis of 3-armed PTMC and subsequent functionalization using methacrylic anhydride.
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M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
2.2.2. Preparation of photo-crosslinked PTMC networks and composites Photo-crosslinked PTMC networks and composites were prepared with nano-hydroxyapatite contents of 0, 10, 20 and 35 wt.%. An amount of nano-hydroxyapatite powder was dispersed by vigorous stirring in chloroform. PTMC-MA (approximately 0.6 g/ml chloroform) and hydroquinone (0.2 wt.% relative to PTMC-MA) were then dissolved in the dispersion, forming a viscous solution. No sedimentation of nano-hydroxyapatite particles was observed after halting the stirring. Nonetheless, the dispersion was sonicated for 15 minutes to ensure homogeneity, and Lucirin® TPO-L photoinitiator (4 wt.% relative to PTMC-MA) was mixed in. Immediately thereafter, the dispersion was cast on glass plates using a 1 mm casting knife. The formed films were dried to constant weight and subsequently photo-crosslinked under N2 in a UV-crosslinker (Ultra-Lum Electronic Ultraviolet Crosslinker) at 365 nm and 11 mW/cm2 for 30 minutes. The photo-crosslinked films were extracted with a mixture of propylene carbonate/chloroform (7/3 v/v) twice and once with ethanol (no nano-hydroxyapatite particles were seen in the extraction media). The extracted composites were dried at ambient conditions overnight and consequently in vacuo until constant weight was achieved. The resulting films were 300 to 400 m thick. 2.2.3. Characterization of photo-crosslinked PTMC and nano-hydroxyapatite composites The nano-hydroxyapatite content of the composites was determined by thermogravimetric analysis using a TGA 7 from Perkin Elmer. Samples were heated under N2 -flow from 50◦ C to 600◦ C at a heating rate of 20◦ C/min. All measurements were performed in threefold. The morphology of the PTMC networks and nano-hydroxyapatite composites was observed by scanning electron microscopy on an FEI/Philips XL-30 environmental Scanning Electron Microscope. The films were freeze-fractured and the cross-sections were imaged after gold-sputtering. The gel content of the PTMC networks and the unreacted PTMC-MA sol fraction of the composites as synthesized were determined gravimetrically. The gel contents G were determined in triplicate (correcting for the nano-hydroxyapatite content) according to Equation 1.
G=
(1 − x)md ∗100% (1 − x)mi
(1)
The weight fraction of nano-hydroxyapatite in the PTMC network composite is x, the mass of the composite after extraction and drying is md , the mass of the cast films right before extraction is mi . 2.2.4. Mechanical characterization of photo-crosslinked networks and composites For tensile experiments, samples of 100 × 5 mm2 were punched out of the extracted and dried films. A Zwick Z020 tensile tester with a 500 N load cell was used. The initial grip-to-grip separation was 50 mm and the crosshead speed was 50 mm/min. All measurements were conducted in fivefold. From the stress-strain curves, the E modulus, the stress and elongation at break and the yield stress and elongation at yield were determined. The toughness of the materials was calculated from the area under the stress-strain curve. Tear propagation experiments were conducted in analogy to ASTM 1938. Samples measuring 37.5 × 12.5 mm2 with a 25 mm long slit were used. The tearing experiments were conducted at a crosshead speed of 250 mm/min. The average tear propagation strength and the maximum tear propagation strength values were determined in fivefold.
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3. Results and discussion 3.1. Synthesis of PTMC-MA Three-armed PTMC was obtained by ring-opening polymerization of TMC using trimethylolpropane as initiator. The product was subsequently reacted with methacrylic anhydride to yield PTMC-MA. From the 1 H-NMR spectrum of non-functionalized PTMC, Fig. 3, TMC monomer conversion was calculated by comparing the area of the TMC -CO-O-CH2 -CH2 - CH2 -O- peak at 4.46 ppm with that of the PTMC -CO-O-CH2 -CH2 - CH2 -O- peak at 4.24 ppm. A value of approximately 98% was obtained. By comparing the area of the trimethylolpropane initiator -CH3 peak at 0.91 ppm with the area of the PTMC methylene peak at 4.24 ppm, an Mn value of 10.4 kg/mol could be determined. End-group functionalization of PTMC with methacrylic anhydride was confirmed by the appearance of the double bond proton peaks at 5.57 ppm and 6.11 ppm (Fig. 3). The degree of functionalization was determined from the area of these peaks and that of the trimethylolpropane-initiated PTMC –CH3 group. An average degree of functionalization of 80% could be calculated. 3.2. Composites of photo-crosslinked PTMC and nano-hydroxyapatite A series of photo-crosslinked PTMC and nano-hydroxyapatite composites were prepared by photo-crosslinking mixtures of PTMC macromers and nano-hydroxyapatite. The amount of nanohydroxyapatite incorporated into the composites was determined by thermogravimetric analysis (TGA).
Fig. 3. 1 H-NMR spectra of three-armed PTMC (top) and methacrylate end-group functionalized PTMC-MA (bottom).
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M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
Fig. 4. SEM images of cross-sections of photo-crosslinked PTMC (A) and composites containing 12.4 wt.% (B), 20.8 wt.% (C), 36.3 wt.% (D) nano-hydroxyapatite (scale bar: 20 m) (the impurity that can be discerned in Fig. 4B, is likely not a nano-hydroxyapatite agglomerate, given the morphology of the nano-hydroxyapatite particles shown in Fig. 1.).
The PTMC network part thermally decomposed at temperatures from 250 to 450◦ C and the hydroxyapatite ◦ part remained unchanged at temperatures up to 600 C. TGA showed that composites with nanohydroxyapatite contents of 12.4 ± 0.1, 20.8 ± 0.1 and 36.3 ± 0.3 wt.% were prepared. This corresponds well with the amounts added to the macromer resin. To assess the efficiency of the photo-crosslinking process, the gel content of the PTMC composites was determined. The gel content of photo-crosslinked PTMC was 77.0 ± 0.8%. The composites with 12.4, 20.8 and 36.3 wt.% nano-hydroxyapatite had gel contents of 74.6 ± 3.6, 76.2 ± 2.8 and 84.6 ± 0.7%, respectively. This indicates that the presence of large amounts of nano-hydroxyapatite particles does not negatively affect the network formation by photo-crosslinking. Cross-sections of the composite films were imaged using SEM (Fig. 4). The images showed that the cross-sections of the photo-crosslinked PTMC networks that did not contain nano-hydroxyapatite were quite smooth. Images of the composites with apatite showed that the nano-hydroxyapatite particles were well-dispersed. Across the cross-sections of the films no gradient in particle loading could be detected in SEM images (data not shown). This indicates that sedimentation after film casting was minimal. The roughness of the cross-sections increases with increasing nano-hydroxyapatite content. The feature sizes of the rough surfaces is much smaller than the nano-hydroxyapatite particles used. It is likely that the mixing and sonication process in the PTMC macromer solutions leads to disintegration of these nano-hydroxyapatite agglomerates.
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3.3. Mechanical properties of photo-crosslinked PTMC and nano-hydroxyapatite composites Tensile experiments were conducted on composite network films to assess the effect of incorporating nano-hydroxyapatite into photo-crosslinked PTMC networks on their mechanical properties. The data presented in Table 1 shows that adding nano-hydroxyapatite to the PTMC network matrix has a significant effect on the mechanical properties of the composites. Compared to the PTMC network that does not contain nano-hydroxyapatite, incorporation of up to 36.3 wt.% in general increases the E modulus, the yield strength and the tensile strength. The E modulus increased from 2.2 MPa to 51 MPa, the yield strength increased from 0.5 to 1.4 N/mm2 and the strain at yield decreased from 30 to 4.4%. The maximum tensile strength of the composites was increased upon nano-hydroxyapatite incorporation from 1.3 to 3.9 MPa. Interestingly, the table shows that PTMC networks with a nano-hydroxyapatite content of 12.4 wt.% showed extraordinary behavior. Very high values of maximum tensile strength and strain at break of respectively 3.2 MPa and 376% were found. Also the toughness (777 N/mm2 ) of the specimens was exceptionally high. This can be the result of the well-dispersed morphology of the small nano-hydroxyapatite agglomerates in the crosslinked PTMC matrix at this concentration, see Fig. 4B. In addition to their effect on the E modulus, the strain at break and toughness of crosslinked polymer/particle composites can increase significantly if the micro- and nano-sized particles are well-dispersed [5, 20]. Tear propagation experiments were conducted as well. The results in Table 1 show that the tear propagation strength (TPS) was much increased by incorporation of nano-hydroxyapatite into the photocrosslinked PTMC. Here too the highest values were found for the composite with a nano-hydroxyapatite content of 12.4 wt.%. The average and maximum TPS increased from 1.2 to 3.1 N/mm and from 1.4 to 3.5 N/mm, respectively. Currently we are investigating the behavior of photo-crosslinked PTMC and nano-hydroxyapatite composites in biological environments.
Table 1 Mechanical properties of photo-crosslinked PTMC networks and nano-hydroxyapatite composites as determined by tensile testing and trouser tearing experiments Nano-hydroxyapatite content (approximate)
0 wt.%
10 wt.%
20 wt.%
35 wt.%
2.2 ± 0.1
4.9 ± 0.2
9.0 ± 1.2
51 ± 7.4
Yield strength (N/mm )
0.5 ± 0.04
0.7 ± 0.03
0.7 ± 0.1
1.4 ± 0.2
Strain at yield (%)
30 ± 1.7
20 ± 0.5
11 ± 0.4
4.4 ± 0.2
E modulus (MPa) 2
Tensile strength (N/mm2 )
1.3 ± 0.1
3.2 ± 0.4
2.5 ± 0.5
3.9 ± 0.3
Strain at break (%)
172 ± 20
376 ± 38
151 ± 24
100 ± 8.9
Toughness (N/mm2 )
141 ± 14
777 ± 68
204 ± 34
280 ± 37
TPSav (N/mm)a
1.2 ± 0.04
3.1 ± 0.2
1.6 ± 0.2
1.8 ± 0.2
1.4 ± 0.04
3.5 ± 0.3
1.8 ± 0.2
2.1 ± 0.2
TPSmax (N/mm) a
b
Average tear propagation strength, b Maximum tear propagation strength.
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M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
4. Conclusions We have prepared photo-crosslinked PTMC and composites thereof with nano-hydroxyapatite, intended for orbital floor reconstruction. The nano-hydroxyapatite particles could readily be incorporated into a PTMC network matrix by mixing the nanoparticles with the photo-reactive PTMC macromer and subsequent photo-crosslinking. The nano-hydroxyapatite particles did not have an adverse effect on the photo-crosslinking. Tensile testing and tear propagation experiments demonstrated that the stiffness, strength and toughness of the composites are much increased by the incorporation of nano-hydroxyapatite. Also the resistance to tearing is considerably increased. Especially the composites with 12.4 wt.% nano-hydroxyapatite had outstanding mechanical properties. The attractive mechanical properties of photo-crosslinked PTMC and nano-hydroxyapatite composites make them interesting materials for use in the preparation of orbital floor implants. Precisely fitting implants can then be manufactured by microstereolithography. Acknowledgments This work was funded by the EU-China grant of the Seventh Framework Program of the European Union, Rapidos project number 604517. References [1] F. Baino, Biomaterials and implants for orbital floor repair, Acta Biomater 7(9) (2011), 3248. DOI: 10.1016/j.actbio.2011.05.016 [2] D. Barbieri, A.J. Renard, J.D. de Bruijn and H. Yuan, Heterotopic bone formation by nano-apatite containing poly(D,Llactide) composites, Eur Cell Mater 19 (2010), 252. [3] D. Barbieri, H.P. Yuan, X.M. Luo, S. Fare, D.W. Grijpma and J.D. de Bruijn, Influence of polymer molecular weight in osteoinductive composites for bone tissue regeneration, Acta Biomater 9(12) (2013), 9401. DOI: 10.1016/j.actbio.2013.07.026 [4] M.W. Betz, J.F. Caccamese, D.P. Coletti, J.J. Sauk and J.P. Fisher, Challenges associated with regeneration of orbital floor bone. Tissue Eng Pt B - Rev 16(5) (2010), 541. DOI: 10.1089/ten.teb.2009.0393 [5] P.H.C. Camargo, K.G. Satyanarayana and F. Wypych, Nanocomposites: synthesis, structure, properties and new application opportunities, Mater Res - Ibero-Am J 12(1) (2009), 1. [6] G. Ciapetti, L. Ambrosio, L. Savarino, D. Granchi, E. Cenni and N. Baldini, Osteoblast growth and function in porous poly epsilon -caprolactone matrices for bone repair: A preliminary study, Biomaterials 24(21) (2003), 3815. DOI: 10.1016/S0142-9612(03)00263-1. [7] J.E. Davies, R. Matta, V.C. Mendes and P.S. Perri de Carvalho, Development, characterization and clinical use of a biodegradable composite scaffold for bone engineering in oro-maxillo-facial surgery, Organogenesis 6(3) (2010), 161. DOI: 10.4161/org.6.3.12392 [8] D.W. Hutmacher, J.T. Schantz, C.X. Lam, K.C. Tan and T.C. Lim, State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective, J Tissue Eng Regen Med 1(4) (2007), 245. DOI: 10.1002/term.24 [9] A. Kolk, J. Handschel, W. Drescher, D. Rothamel, F. Kloss and M. Blessmann et al., Current trends and future perspectives of bone substitute materials - from space holders to innovative biomaterials, J Carniomaxillofac Surg 40(8) (2012), 706. DOI: 10.1016/j.jcms.2012.01.002 [10] C. Landes, A. Ballon, S. Ghanaati, A. Tran and R. Sader, Treatment of malar and midfacial fractures with osteoconductive forged unsintered hydroxyapatite and poly-l-lactide composite internal fixation devices, J Oral Maxillofac Surg (2014), Article in Press. DOI: 10.1016/j.joms.2014.02.027
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[11] D. Lickorish, L. Guan and J.E. Davies, A three-phase, fully resorbable, polyester/calcium phosphate scaffold for bone tissue engineering: Evolution of scaffold design, Biomaterials 28(8) (2007), 1495. DOI: 10.1016/j.biomaterials.2006.11.025 [12] P.X. Ma, R.Y. Zhang, G.Z. Xiao and R. Franceschi, Engineering new bone tissue in vitro on highly porous poly(alpha-hydroxyl acids)/hydroxyapatite composite scaffolds, J Biomed Mater Res 54(2) (2001), 284. DOI: 10.1002/1097-4636(200102)54:23.0.Co;2-W [13] L.M. Mathieu, T.L. Mueller, P.E. Bourban, D.P. Pioletti, R. Muller and J.A. Manson, Architecture and properties of anisotropic polymer composite scaffolds for bone tissue engineering, Biomaterials 27(6) (2006), 905. DOI: 10.1016/j.biomaterials.2005.07.015 [14] A.J. McManus, R.H. Doremus, R.W. Siegel and R. Bizios, Evaluation of cytocompatibility and bending modulus of nanoceramic/polymer composites, J Biomed Mater Res 72(1) (2005), 98. DOI: 10.1002/jbm.a.30204 [15] A.C. Van Leeuwen, R.R. Bos and D.W. Grijpma, Composite materials based on poly(trimethylene carbonate) and betatricalcium phosphate for orbital floor and wall reconstruction, J Biomed Mater Res B Appl Biomater 100(6) (2012), 1610. DOI: 10.1002/jbm.b.32729 [16] A.C. Van Leeuwen, H. Yuan, G. Passanisi, J.W. van der Meer, J.D. de Bruijn and TG van Kooten, et al., Poly(trimethylene carbonate) and biphasic calcium phosphate composites for orbital floor reconstruction: a feasibility study in sheep, Eur Cell Mater 27 (2014), 81. [17] B. Wendel, D. Rietzel, F. Kuhnlein, R. Feulner, G. Hulder and E. Schmachtenberg, additive processing of polymers, Macromol Mater Eng 293(10) (2008), 799. DOI: 10.1002/mame.200800121 [18] L. Wu and J. Ding, In vitro degradation of three-dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering, Biomaterials 25(27) (2004), 5821. DOI: 10.1016/j.biomaterials.2004.01.038 [19] J.C. Zhang, H.Y. Lu, G.Y. Lv, A.C. Mo, Y.G. Yan and C. Huang, The repair of critical-size defects with porous hydroxyapatite/polyamide nanocomposite: An experimental study in rabbit mandibles, Int J Oral Maxillofac Surg 39(5) (2010), 469. DOI: 10.1016/j.ijom.2010.01.013 [20] J. Zhang, B. Han, N.L. Zhou, J. Fang, J.A. Wu, Z.M. Ma et al., Preparation and Characterization of Nano/Micro-Calcium Carbonate Particles/Polypropylene Composites, J Appl Polym Sci 119(6) (2011), 3560. DOI: 10.1002/App.33037 [21] Z. Zhang, R. Kuijer, S.K. Bulstra, D.W. Grijpma and J. Feijen, The in vivo and in vitro degradation behavior of poly(trimethylene carbonate), Biomaterials 27(9) (2006), 1741. DOI: 10.1016/j.biomaterials.2005.09.017
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Preparation and mechanical properties of photo-crosslinked poly(trimethylene carbonate) and nano-hydroxyapatite composites Mike A. Gevena , Davide Barbierib , Huipin Yuanb,c , Joost D. de Bruijna,b and Dirk W. Grijpmaa,d,∗ a
MIRA Institute for Biomedical Technology and Technical Medicine, and Department of Biomaterials Science and Technology, University of Twente, Enschede, The Netherlands b XPand Biotechnology BV, Bilthoven, The Netherlands c MIRA Institute for Biomedical Technology and Technical Medicine, and Department of Tissue Regeneration, University of Twente, Enschede, The Netherlands d University of Groningen, University Medical Center Groningen, W.J. Kolff Institute, Department of Biomedical Engineering, Groningen, The Netherlands Submitted 10 January 2014; accepted 7 March 2014
Abstract. Composite materials of photo-crosslinked poly(trimethylene carbonate) and nanoscale hydroxyapatite were prepared and their mechanical characteristics for application as orbital floor implants were assessed. The composites were prepared by solvent casting poly(trimethylene carbonate) macromers with varying amounts of nano-hydroxyapatite and subsequent photocrosslinking. The incorporation of the nano-hydroxyapatite into the composites was examined by thermogravimetric analysis, scanning electron microscopy and gel content measurements. The mechanical properties were investigated by tensile testing and trouser tearing experiments. Our results show that nano-hydroxyapatite particles can readily be incorporated into photocrosslinked poly(trimethylene carbonate) networks. Compared to the networks without nano-hydroxyapatite, incorporation of 36.3 wt.% of the apatite resulted in an increase of the E modulus, yield strength and tensile strength from 2.2 MPa to 51 MPa, 0.5 to 1.4 N/mm2 and from 1.3 to 3.9 N/mm2 , respectively. We found that composites containing 12.4 wt.% nano-hydroxyapatite had the highest values of strain at break, toughness and average tear propagation strength (376%, 777 N/mm2 and 3.1 N/mm2 , respectively). Keywords: Composites, photo-crosslinked poly(trimethylene carbonate), nano-hydroxyapatite, orbital floor repair
1. Introduction Blow-out fractures of the orbital floor are common injuries in traffic accidents or assaults, caused by excessive force applied to the infraorbital rim [4]. Restoring the original volume of the orbit and ∗
Corresponding author: Prof. Dirk W. Grijpma, MIRA Institute for Biomedical Technology and Technical Medicine, and Department of Biomaterials Science and Technology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands, Tel.: +31 53 4892966; Fax: +31 53 4892155; E-mail: [email protected]. 1386-0291/15/$35.00 © 2015 – IOS Press and the authors. All rights reserved
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M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
repositioning the globe are very important. In practice, this is done by covering the fracture site with an implant [4]. The current clinically used implants consist of autografts and allografts as well as of alloplastic materials such as hydroxyapatite ceramics, titanium, silicone rubber, ultra high density polyethylene, poly(lactic acid) and hydroxyapatite/polyethylene composites [1]. Ideally, an implant material is biocompatible, mechanically stable enough to support the orbital contents, osteoinductive and bioresorbable with a minimal foreign body response. The limited availability and the possibility for disease transmittance associated with autografts and allografts decrease their appeal as implant materials [1]. Drawbacks of several of the alloplastic materials are sub-optimal mechanical properties, limited potential for bone ingrowth or their non-degradability. For these reasons, composites of synthetic, degradable and biocompatible polymers and calcium phosphates have been investigated for bone tissue engineering and orbital floor repair. Encouraging results have been achieved with composites of poly(L-lactide), poly(L-lactide-co-glycolide) and poly(-caprolactone) with several calcium phosphates such as hydroxyapatite, -tricalcium phosphate (-TCP) and biphasic calcium phosphate [1, 6–14, 19]. Although these polyester-based composites have shown good results, problems may arise from the significant amounts of acidic degradation products that can be formed. Furthermore, the average molar mass of polyesters will decrease during degradation resulting in decreasing mechanical properties [18]. Recently, composites of linear non-crosslinked poly(trimethylene carbonate) (PTMC) and -TCP have been investigated as potential orbital floor implants. PTMC is a flexible, amorphous polymer that degrades enzymatically through surface erosion in presence of macrophages. This surface erosion mechanism produces non-acidic degradation products and the mechanical properties of PTMC are maintained over time as the average molar mass remains constant [21]. It has been shown that composites of PTMC with -TCP had sufficient mechanical properties for the reconstruction of large orbital floor defects (5.5 cm2 ) [15]. Furthermore, implantation of composites of PTMC and biphasic calcium phosphate in sheep orbital floor defects resulted in newly formed bone and excellent osseous integration with the surrounding bone [16]. Although these PTMC composites were shapeable, an exact anatomical fit to the orbital floor defect would be most desirable for the optimal orbital floor reconstruction and repositioning of the orbital contents [1]. Microstereolithography is an additive manufacturing process based on photo-polymerization. It can reach precisions of 5 m and may allow for the fabrication of such precisely fitting implants [17]. For this we have prepared photo-curable three-armed PTMC macromers with methacrylate end-groups (PTMCMA). PTMC-MA based resins with varying amounts of nano-hydroxyapatite were then prepared and photo-crosslinked to obtain PTMC network composites that may have osteoinductive properties [2].
2. Materials and methods 2.1. Materials All materials were used as received unless stated otherwise. ForYou Medical Devices Ltd. provided the trimethylene carbonate (TMC) used for this work. The nano-hydroxyapatite powder consisted of 5 to 25 m sized agglomerates of needle-like hydroxyapatite crystals of 200 to 400 nm long and 20 to 50 nm wide, and was prepared by XPand Biotechnology BV as previously reported [3]. Fig. 1 shows an SEM image of the nano-hydroxyapatite as received, and the size distribution as determined using ImageJ. Tin(II) 2-ethylhexanoate (Sn(Oct)2 ), hydroquinone, triethylamine, methacrylic anhydride and deuterated
M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
5
Fig. 1. SEM image (left) and size distribution (right) of the nano-hydroxyapatite particles as were manufactured and received (scale bar: 20 m).
chloroform were obtained from Sigma-Aldrich. 1,1,1-tris(hydroxymethyl)propane (trimethylolpropane) was from Fluka analytical and Lucirin® TPO-L was from BASF. Chloroform, propylene carbonate and calcium hydride were from Merck Millipore. Dichloromethane, from VWR chemicals, was dried over calcium hydride and subsequently distilled under dry N2 . 2.2. Methods 2.2.1. Synthesis of PTMC-MA 3-armed PTMC was synthesized by ring-opening polymerization of TMC (Fig. 2). Under dry N2 atmosphere, a three-necked round-bottomed flask was charged with 49.8 g (0.49 mol) TMC and 0.67 g (5.0 mmol) trimethylolpropane as initiator. The polymerization was conducted at 140◦ C for 19 hours, using Sn(Oct)2 as catalyst at a concentration of 0.13 wt.%. After cooling to room temperature, the PTMC oligomer was dissolved in dried dichloromethane. Under dry N2 , 52.7 mg of hydroquinone, 4.5 ml (32 mmol) triethylamine and 4.6 ml (31 mmol) methacrylic anhydride were added and the resulting solution was stirred in the dark for 5 days at room temperature. The solution was then extracted with demineralized water 3 times and the organic phase was precipitated in cold methanol. The precipitate was dried in the dark at ambient conditions overnight and for another 5 days at room temperature in vacuo. The non-functionalized PTMC oligomer and the functionalized precipitated macromer were analyzed by 1 H-NMR (Bruker Ascend 400/Avance III 400 MHz NMR spectrometer) to determine the TMC conversion, the number average molecular mass (Mn ) and the degree of functionalization of PTMC-MA. Deuterated chloroform was used as the solvent.
Fig. 2. Synthesis of 3-armed PTMC and subsequent functionalization using methacrylic anhydride.
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M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
2.2.2. Preparation of photo-crosslinked PTMC networks and composites Photo-crosslinked PTMC networks and composites were prepared with nano-hydroxyapatite contents of 0, 10, 20 and 35 wt.%. An amount of nano-hydroxyapatite powder was dispersed by vigorous stirring in chloroform. PTMC-MA (approximately 0.6 g/ml chloroform) and hydroquinone (0.2 wt.% relative to PTMC-MA) were then dissolved in the dispersion, forming a viscous solution. No sedimentation of nano-hydroxyapatite particles was observed after halting the stirring. Nonetheless, the dispersion was sonicated for 15 minutes to ensure homogeneity, and Lucirin® TPO-L photoinitiator (4 wt.% relative to PTMC-MA) was mixed in. Immediately thereafter, the dispersion was cast on glass plates using a 1 mm casting knife. The formed films were dried to constant weight and subsequently photo-crosslinked under N2 in a UV-crosslinker (Ultra-Lum Electronic Ultraviolet Crosslinker) at 365 nm and 11 mW/cm2 for 30 minutes. The photo-crosslinked films were extracted with a mixture of propylene carbonate/chloroform (7/3 v/v) twice and once with ethanol (no nano-hydroxyapatite particles were seen in the extraction media). The extracted composites were dried at ambient conditions overnight and consequently in vacuo until constant weight was achieved. The resulting films were 300 to 400 m thick. 2.2.3. Characterization of photo-crosslinked PTMC and nano-hydroxyapatite composites The nano-hydroxyapatite content of the composites was determined by thermogravimetric analysis using a TGA 7 from Perkin Elmer. Samples were heated under N2 -flow from 50◦ C to 600◦ C at a heating rate of 20◦ C/min. All measurements were performed in threefold. The morphology of the PTMC networks and nano-hydroxyapatite composites was observed by scanning electron microscopy on an FEI/Philips XL-30 environmental Scanning Electron Microscope. The films were freeze-fractured and the cross-sections were imaged after gold-sputtering. The gel content of the PTMC networks and the unreacted PTMC-MA sol fraction of the composites as synthesized were determined gravimetrically. The gel contents G were determined in triplicate (correcting for the nano-hydroxyapatite content) according to Equation 1.
G=
(1 − x)md ∗100% (1 − x)mi
(1)
The weight fraction of nano-hydroxyapatite in the PTMC network composite is x, the mass of the composite after extraction and drying is md , the mass of the cast films right before extraction is mi . 2.2.4. Mechanical characterization of photo-crosslinked networks and composites For tensile experiments, samples of 100 × 5 mm2 were punched out of the extracted and dried films. A Zwick Z020 tensile tester with a 500 N load cell was used. The initial grip-to-grip separation was 50 mm and the crosshead speed was 50 mm/min. All measurements were conducted in fivefold. From the stress-strain curves, the E modulus, the stress and elongation at break and the yield stress and elongation at yield were determined. The toughness of the materials was calculated from the area under the stress-strain curve. Tear propagation experiments were conducted in analogy to ASTM 1938. Samples measuring 37.5 × 12.5 mm2 with a 25 mm long slit were used. The tearing experiments were conducted at a crosshead speed of 250 mm/min. The average tear propagation strength and the maximum tear propagation strength values were determined in fivefold.
M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
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3. Results and discussion 3.1. Synthesis of PTMC-MA Three-armed PTMC was obtained by ring-opening polymerization of TMC using trimethylolpropane as initiator. The product was subsequently reacted with methacrylic anhydride to yield PTMC-MA. From the 1 H-NMR spectrum of non-functionalized PTMC, Fig. 3, TMC monomer conversion was calculated by comparing the area of the TMC -CO-O-CH2 -CH2 - CH2 -O- peak at 4.46 ppm with that of the PTMC -CO-O-CH2 -CH2 - CH2 -O- peak at 4.24 ppm. A value of approximately 98% was obtained. By comparing the area of the trimethylolpropane initiator -CH3 peak at 0.91 ppm with the area of the PTMC methylene peak at 4.24 ppm, an Mn value of 10.4 kg/mol could be determined. End-group functionalization of PTMC with methacrylic anhydride was confirmed by the appearance of the double bond proton peaks at 5.57 ppm and 6.11 ppm (Fig. 3). The degree of functionalization was determined from the area of these peaks and that of the trimethylolpropane-initiated PTMC –CH3 group. An average degree of functionalization of 80% could be calculated. 3.2. Composites of photo-crosslinked PTMC and nano-hydroxyapatite A series of photo-crosslinked PTMC and nano-hydroxyapatite composites were prepared by photo-crosslinking mixtures of PTMC macromers and nano-hydroxyapatite. The amount of nanohydroxyapatite incorporated into the composites was determined by thermogravimetric analysis (TGA).
Fig. 3. 1 H-NMR spectra of three-armed PTMC (top) and methacrylate end-group functionalized PTMC-MA (bottom).
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M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
Fig. 4. SEM images of cross-sections of photo-crosslinked PTMC (A) and composites containing 12.4 wt.% (B), 20.8 wt.% (C), 36.3 wt.% (D) nano-hydroxyapatite (scale bar: 20 m) (the impurity that can be discerned in Fig. 4B, is likely not a nano-hydroxyapatite agglomerate, given the morphology of the nano-hydroxyapatite particles shown in Fig. 1.).
The PTMC network part thermally decomposed at temperatures from 250 to 450◦ C and the hydroxyapatite ◦ part remained unchanged at temperatures up to 600 C. TGA showed that composites with nanohydroxyapatite contents of 12.4 ± 0.1, 20.8 ± 0.1 and 36.3 ± 0.3 wt.% were prepared. This corresponds well with the amounts added to the macromer resin. To assess the efficiency of the photo-crosslinking process, the gel content of the PTMC composites was determined. The gel content of photo-crosslinked PTMC was 77.0 ± 0.8%. The composites with 12.4, 20.8 and 36.3 wt.% nano-hydroxyapatite had gel contents of 74.6 ± 3.6, 76.2 ± 2.8 and 84.6 ± 0.7%, respectively. This indicates that the presence of large amounts of nano-hydroxyapatite particles does not negatively affect the network formation by photo-crosslinking. Cross-sections of the composite films were imaged using SEM (Fig. 4). The images showed that the cross-sections of the photo-crosslinked PTMC networks that did not contain nano-hydroxyapatite were quite smooth. Images of the composites with apatite showed that the nano-hydroxyapatite particles were well-dispersed. Across the cross-sections of the films no gradient in particle loading could be detected in SEM images (data not shown). This indicates that sedimentation after film casting was minimal. The roughness of the cross-sections increases with increasing nano-hydroxyapatite content. The feature sizes of the rough surfaces is much smaller than the nano-hydroxyapatite particles used. It is likely that the mixing and sonication process in the PTMC macromer solutions leads to disintegration of these nano-hydroxyapatite agglomerates.
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3.3. Mechanical properties of photo-crosslinked PTMC and nano-hydroxyapatite composites Tensile experiments were conducted on composite network films to assess the effect of incorporating nano-hydroxyapatite into photo-crosslinked PTMC networks on their mechanical properties. The data presented in Table 1 shows that adding nano-hydroxyapatite to the PTMC network matrix has a significant effect on the mechanical properties of the composites. Compared to the PTMC network that does not contain nano-hydroxyapatite, incorporation of up to 36.3 wt.% in general increases the E modulus, the yield strength and the tensile strength. The E modulus increased from 2.2 MPa to 51 MPa, the yield strength increased from 0.5 to 1.4 N/mm2 and the strain at yield decreased from 30 to 4.4%. The maximum tensile strength of the composites was increased upon nano-hydroxyapatite incorporation from 1.3 to 3.9 MPa. Interestingly, the table shows that PTMC networks with a nano-hydroxyapatite content of 12.4 wt.% showed extraordinary behavior. Very high values of maximum tensile strength and strain at break of respectively 3.2 MPa and 376% were found. Also the toughness (777 N/mm2 ) of the specimens was exceptionally high. This can be the result of the well-dispersed morphology of the small nano-hydroxyapatite agglomerates in the crosslinked PTMC matrix at this concentration, see Fig. 4B. In addition to their effect on the E modulus, the strain at break and toughness of crosslinked polymer/particle composites can increase significantly if the micro- and nano-sized particles are well-dispersed [5, 20]. Tear propagation experiments were conducted as well. The results in Table 1 show that the tear propagation strength (TPS) was much increased by incorporation of nano-hydroxyapatite into the photocrosslinked PTMC. Here too the highest values were found for the composite with a nano-hydroxyapatite content of 12.4 wt.%. The average and maximum TPS increased from 1.2 to 3.1 N/mm and from 1.4 to 3.5 N/mm, respectively. Currently we are investigating the behavior of photo-crosslinked PTMC and nano-hydroxyapatite composites in biological environments.
Table 1 Mechanical properties of photo-crosslinked PTMC networks and nano-hydroxyapatite composites as determined by tensile testing and trouser tearing experiments Nano-hydroxyapatite content (approximate)
0 wt.%
10 wt.%
20 wt.%
35 wt.%
2.2 ± 0.1
4.9 ± 0.2
9.0 ± 1.2
51 ± 7.4
Yield strength (N/mm )
0.5 ± 0.04
0.7 ± 0.03
0.7 ± 0.1
1.4 ± 0.2
Strain at yield (%)
30 ± 1.7
20 ± 0.5
11 ± 0.4
4.4 ± 0.2
E modulus (MPa) 2
Tensile strength (N/mm2 )
1.3 ± 0.1
3.2 ± 0.4
2.5 ± 0.5
3.9 ± 0.3
Strain at break (%)
172 ± 20
376 ± 38
151 ± 24
100 ± 8.9
Toughness (N/mm2 )
141 ± 14
777 ± 68
204 ± 34
280 ± 37
TPSav (N/mm)a
1.2 ± 0.04
3.1 ± 0.2
1.6 ± 0.2
1.8 ± 0.2
1.4 ± 0.04
3.5 ± 0.3
1.8 ± 0.2
2.1 ± 0.2
TPSmax (N/mm) a
b
Average tear propagation strength, b Maximum tear propagation strength.
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M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
4. Conclusions We have prepared photo-crosslinked PTMC and composites thereof with nano-hydroxyapatite, intended for orbital floor reconstruction. The nano-hydroxyapatite particles could readily be incorporated into a PTMC network matrix by mixing the nanoparticles with the photo-reactive PTMC macromer and subsequent photo-crosslinking. The nano-hydroxyapatite particles did not have an adverse effect on the photo-crosslinking. Tensile testing and tear propagation experiments demonstrated that the stiffness, strength and toughness of the composites are much increased by the incorporation of nano-hydroxyapatite. Also the resistance to tearing is considerably increased. Especially the composites with 12.4 wt.% nano-hydroxyapatite had outstanding mechanical properties. The attractive mechanical properties of photo-crosslinked PTMC and nano-hydroxyapatite composites make them interesting materials for use in the preparation of orbital floor implants. Precisely fitting implants can then be manufactured by microstereolithography. Acknowledgments This work was funded by the EU-China grant of the Seventh Framework Program of the European Union, Rapidos project number 604517. References [1] F. Baino, Biomaterials and implants for orbital floor repair, Acta Biomater 7(9) (2011), 3248. DOI: 10.1016/j.actbio.2011.05.016 [2] D. Barbieri, A.J. Renard, J.D. de Bruijn and H. Yuan, Heterotopic bone formation by nano-apatite containing poly(D,Llactide) composites, Eur Cell Mater 19 (2010), 252. [3] D. Barbieri, H.P. Yuan, X.M. Luo, S. Fare, D.W. Grijpma and J.D. de Bruijn, Influence of polymer molecular weight in osteoinductive composites for bone tissue regeneration, Acta Biomater 9(12) (2013), 9401. DOI: 10.1016/j.actbio.2013.07.026 [4] M.W. Betz, J.F. Caccamese, D.P. Coletti, J.J. Sauk and J.P. Fisher, Challenges associated with regeneration of orbital floor bone. Tissue Eng Pt B - Rev 16(5) (2010), 541. DOI: 10.1089/ten.teb.2009.0393 [5] P.H.C. Camargo, K.G. Satyanarayana and F. Wypych, Nanocomposites: synthesis, structure, properties and new application opportunities, Mater Res - Ibero-Am J 12(1) (2009), 1. [6] G. Ciapetti, L. Ambrosio, L. Savarino, D. Granchi, E. Cenni and N. Baldini, Osteoblast growth and function in porous poly epsilon -caprolactone matrices for bone repair: A preliminary study, Biomaterials 24(21) (2003), 3815. DOI: 10.1016/S0142-9612(03)00263-1. [7] J.E. Davies, R. Matta, V.C. Mendes and P.S. Perri de Carvalho, Development, characterization and clinical use of a biodegradable composite scaffold for bone engineering in oro-maxillo-facial surgery, Organogenesis 6(3) (2010), 161. DOI: 10.4161/org.6.3.12392 [8] D.W. Hutmacher, J.T. Schantz, C.X. Lam, K.C. Tan and T.C. Lim, State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective, J Tissue Eng Regen Med 1(4) (2007), 245. DOI: 10.1002/term.24 [9] A. Kolk, J. Handschel, W. Drescher, D. Rothamel, F. Kloss and M. Blessmann et al., Current trends and future perspectives of bone substitute materials - from space holders to innovative biomaterials, J Carniomaxillofac Surg 40(8) (2012), 706. DOI: 10.1016/j.jcms.2012.01.002 [10] C. Landes, A. Ballon, S. Ghanaati, A. Tran and R. Sader, Treatment of malar and midfacial fractures with osteoconductive forged unsintered hydroxyapatite and poly-l-lactide composite internal fixation devices, J Oral Maxillofac Surg (2014), Article in Press. DOI: 10.1016/j.joms.2014.02.027
M.A. Geven et al. / Photo-crosslinked PTMC and nano-hydroxyapatite composites
11
[11] D. Lickorish, L. Guan and J.E. Davies, A three-phase, fully resorbable, polyester/calcium phosphate scaffold for bone tissue engineering: Evolution of scaffold design, Biomaterials 28(8) (2007), 1495. DOI: 10.1016/j.biomaterials.2006.11.025 [12] P.X. Ma, R.Y. Zhang, G.Z. Xiao and R. Franceschi, Engineering new bone tissue in vitro on highly porous poly(alpha-hydroxyl acids)/hydroxyapatite composite scaffolds, J Biomed Mater Res 54(2) (2001), 284. DOI: 10.1002/1097-4636(200102)54:23.0.Co;2-W [13] L.M. Mathieu, T.L. Mueller, P.E. Bourban, D.P. Pioletti, R. Muller and J.A. Manson, Architecture and properties of anisotropic polymer composite scaffolds for bone tissue engineering, Biomaterials 27(6) (2006), 905. DOI: 10.1016/j.biomaterials.2005.07.015 [14] A.J. McManus, R.H. Doremus, R.W. Siegel and R. Bizios, Evaluation of cytocompatibility and bending modulus of nanoceramic/polymer composites, J Biomed Mater Res 72(1) (2005), 98. DOI: 10.1002/jbm.a.30204 [15] A.C. Van Leeuwen, R.R. Bos and D.W. Grijpma, Composite materials based on poly(trimethylene carbonate) and betatricalcium phosphate for orbital floor and wall reconstruction, J Biomed Mater Res B Appl Biomater 100(6) (2012), 1610. DOI: 10.1002/jbm.b.32729 [16] A.C. Van Leeuwen, H. Yuan, G. Passanisi, J.W. van der Meer, J.D. de Bruijn and TG van Kooten, et al., Poly(trimethylene carbonate) and biphasic calcium phosphate composites for orbital floor reconstruction: a feasibility study in sheep, Eur Cell Mater 27 (2014), 81. [17] B. Wendel, D. Rietzel, F. Kuhnlein, R. Feulner, G. Hulder and E. Schmachtenberg, additive processing of polymers, Macromol Mater Eng 293(10) (2008), 799. DOI: 10.1002/mame.200800121 [18] L. Wu and J. Ding, In vitro degradation of three-dimensional porous poly(D,L-lactide-co-glycolide) scaffolds for tissue engineering, Biomaterials 25(27) (2004), 5821. DOI: 10.1016/j.biomaterials.2004.01.038 [19] J.C. Zhang, H.Y. Lu, G.Y. Lv, A.C. Mo, Y.G. Yan and C. Huang, The repair of critical-size defects with porous hydroxyapatite/polyamide nanocomposite: An experimental study in rabbit mandibles, Int J Oral Maxillofac Surg 39(5) (2010), 469. DOI: 10.1016/j.ijom.2010.01.013 [20] J. Zhang, B. Han, N.L. Zhou, J. Fang, J.A. Wu, Z.M. Ma et al., Preparation and Characterization of Nano/Micro-Calcium Carbonate Particles/Polypropylene Composites, J Appl Polym Sci 119(6) (2011), 3560. DOI: 10.1002/App.33037 [21] Z. Zhang, R. Kuijer, S.K. Bulstra, D.W. Grijpma and J. Feijen, The in vivo and in vitro degradation behavior of poly(trimethylene carbonate), Biomaterials 27(9) (2006), 1741. DOI: 10.1016/j.biomaterials.2005.09.017
