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Bioactive glass surface for fiber reinforced composite implants via surface etching by Excimer laser
Medical Engineering and Physics 38 (2016) 664–670
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Medical Engineering and Physics journal homepage: www.elsevier.com/locate/medengphy
Technical note
Bioactive glass surface for fiber reinforced composite implants via surface etching by Excimer laser Julia Kulkova a, Niko Moritz a,∗, Hannu Huhtinen b, Riina Mattila a, Ivan Donati c, Eleonora Marsich d, Sergio Paoletti c, Pekka K Vallittu a a
Turku Clinical Biomaterials Centre (TCBC), Department of Biomaterials Science, Institute of Dentistry, University of Turku and Biocity Turku Biomaterials Research Program and City of Turku Welfare Division, Itäinen pitkäkatu 4B (PharmaCity), FI-20520 Turku, Finland Wihuri Physical Laboratory, Department of Physics and Astronomy, University of Turku, FI-20014, Finland c Department of Life Sciences, University of Trieste, Via Licio Giorgieri 5, I-34127 Trieste, Italy d Department of Medicine, Surgery and Health Sciences, University of Trieste, Piazza dell’Ospitale 1, 34129 Trieste, Italy b
a r t i c l e
i n f o
Article history: Received 22 October 2015 Revised 22 February 2016 Accepted 3 April 2016
Keywords: Fiber-reinforced composites Excimer laser UV laser Laser ablation Bisphenol-A-glycidyldimethacrylate Bioactive glass Implant Intramedullary nail
a b s t r a c t Biostable fiber-reinforced composites (FRC) prepared from bisphenol-A-glycidyldimethacrylate (BisGMA)based thermosets reinforced with E-glass fibers are promising alternatives to metallic implants due to the excellent fatigue resistance and the mechanical properties matching those of bone. Bioactive glass (BG) granules can be incorporated within the polymer matrix to improve the osteointegration of the FRC implants. However, the creation of a viable surface layer using BG granules is technically challenging. In this study, we investigated the potential of Excimer laser ablation to achieve the selective removal of the matrix to expose the surface of BG granules. A UV–vis spectroscopic study was carried out to investigate the differences in the penetration of light in the thermoset matrix and BG. Thereafter, optimal Excimer laser ablation parameters were established. The formation of a calcium phosphate (CaP) layer on the surface of the laser-ablated specimens was verified in simulated body fluid (SBF). In addition, the proliferation of MG63 cells on the surfaces of the laser-ablated specimens was investigated. For the laser-ablated specimens, the pattern of proliferation of MG63 cells was comparable to that in the positive control group (Ti6Al4V). We concluded that Excimer laser ablation has potential for the creation of a bioactive surface on FRC-implants. © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.
1. Introduction Biostable fiber-reinforced composites (FRC) are promising alternatives to metallic implants due to the excellent fatigue resistance and the mechanical properties matching those of bone [1]. FRCs prepared from bisphenol-A-glycidyldimethacrylate (BisGMA)based thermosets reinforced with E-glass fibers have been successfully used in dental reconstructions [2,3]. Clinical use of nonload-bearing FRC implants has already commenced in cranial reconstructions [4]. In addition, preclinical studies demonstrated the potential of BisGMA-based FRC implants in load-bearing orthopaedic applications [5,6]. Bioactive glass (BG) granules can be incorporated into the surface of the FRC implants to improve their
∗ Corresponding author at: Turku Clinical Biomaterials Centre (TCBC), Department of Biomaterials Science, Institute of Dentistry, University of Turku, Itäinen pitkäkatu 4B (PharmaCity), FI-20520 Turku, Finland. Tel.: +358 2 333 8227. E-mail address: niko.moritz@utu.fi (N. Moritz). URL: http://biomaterials.utu.fi (N. Moritz)
http://dx.doi.org/10.1016/j.medengphy.2016.04.003 1350-4533/© 2016 IPEM. Published by Elsevier Ltd. All rights reserved.
osteointegration. The bioactivity of BG is manifested in the formation of direct chemical bond between BG and bone through the cascade of chemical reactions and cellular activity [7–9]. Formation of a calcium phosphate (CaP) surface reaction layer is a crucial aspect of bone bonding. BG granules are certified for clinical use i.e. as bone graft substitutes [10–13]. However, our previous studies revealed technical challenges in the creation of a feasible surface layer using BG granules [5,6]. A large number of unreacted BG granules were trapped inside the polymer matrix, while the surface area of the granules exposed by grinding was insufficient to achieve notable improvement in the osteointegration of the implants [5]. In addition, the mechanical treatment, i.e. grinding, of the implant surface led to the disengagement of the granules either before or after the implantation [5]. Therefore, there is a need for the development of new techniques for the incorporation of BG granules in the surfaces for the FRC implants. Surface ablation of polymers by Excimer lasers was demonstrated in the early 1980s [14,15]. Studies with laser ablation of the composites revealed selective removal of the polymer matrix and
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Table 1 Summary of materials used for the preparation of the specimens. Material
Type of material
Manufacturer
Bisphenol-A-glycidyldimethacrylate (BisGMA) Triethylene glycol dimethacrylate (TEGDMA) 2-(Dimethylamino)ethyl methacrylate (DMAEMA) Camphorquinone Bioactive glass (BG) S53P4, granules: 315–500 μm fraction, oxide composition (wt%): SiO2 53%, Na2 O 23%, CaO 20%, P2 O5 4%
Co-monomer Co-monomer Activator Photoinitiator Osteoconductive surface component
Röhm Chemische Fabrik GmbH, Darmstadt, Germany Aldrich Chemie GmbH, Steinheim, Germany Fluka Chemie GmbH, Buchs, Switzerland Sigma-Aldrich GmbH, Buchs, Switzerland Vivoxid Ltd., Turku, Finland
exposure of the reinforcing glass fibers [16,17]. We hypothesized that with this setup, Excimer laser ablation would allow selective removal of the thermoset matrix and the exposure of the BG granules, which in turn, would enhance the osteoconductivity of the FRC implants without the disengagement of BG granules. 2. Materials and methods 2.1. Methodology of the study The targeted implants are intramedullary nails reported previously [5,22]. The core of the implants is reinforced with long glass fibers while the relatively thick surface layer (∼1 mm) contains unreinforced resin embedded with bioactive glass granules. Therefore, for the sake of simplicity, we excluded the reinforcing phase from consideration and used unreinforced thermosets in all experiments. This study included four experiments. The first experiment, a UV–vis spectroscopic study, was carried out to investigate the differences in the penetration of light in the thermoset matrix and BG. The second experiment was carried out to establish the optimal laser ablation parameters. The third experiment was performed to study CaP formation on the surfaces of the specimens immersed in simulated body fluid (SBF) [18]. The fourth experiment was performed to study MG63 cell proliferation on the surfaces of the specimens to verify the absence of negative effects of laser radiation. The laser ablation parameters used in the third and fourth experiment, were based on the results obtained in the second experiment. 2.2. First experiment: UV–vis spectroscopic study Two groups of specimens, BG and thermosets, were used in the UV–vis spectroscopic study. Materials used for the preparation of the specimens are listed in Table 1. Rectangular-shaped BG specimens (10 × 10 mm) were cut from a 1 mm-thick BG S53P4 plate. This determined the shape of the specimens used in this experiment. The BG specimens were prepared in three different thicknesses: 1.3 mm, 0.5 mm, and 0.2 mm by grinding with silicon carbide paper of 10 0 0, 240 0, and 40 0 0 grit, with further polishing with 0.1 μm alumina paste. Ethanol was used in grinding and polishing to avoid the formation of reaction layers. Subsequently, the specimens were cleaned ultrasonically in ethanol. Impression molds made from dental putty were used to prepare the thermoset specimens. The photopolymerisable resin was prepared by mixing BisGMA (70 wt%) and triethylene glycol dimethacrylate (TEGDMA) (30 wt%) with camphorquinone (CQ) (0.7 wt%) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) (0.7 wt%) [19]. Thereafter, the resin was poured into the molds and briefly pre-cured (40 s) by a hand curing device (Optilux 501, Kerr, Danbury, USA). Next, the thermoset specimens were taken out of the molds and cured in vacuum light oven (Visio Beta vario, 3 M/ESPE, Seefeld, Germany) for 15 min at ambient temperature. Subsequently, in order to reduce the amount of residual
monomers, the specimens were placed in a light oven (Liculite, Dentsply De Trey GmbH, Dreieich, Germany) for 25 min. During the time, the temperature of the curing chamber increased to 90 °C. The thermoset specimens were ground with silicon carbide paper of 10 0 0, 240 0, and 40 0 0 grit, and further polished with 0.1 μm alumina paste. Water was used in grinding. The thermoset specimens were prepared in four different thicknesses: 1.3 mm, 0.5 mm, 0.2 mm and ∼0.05 mm. After the preparation, the specimens were cleaned ultrasonically in water. Light transmittance in the range of 270–340 nm was studied using UV–vis spectrophotometer (Shimadzu UV-1601, Shimadzu Corp. Tokyo, Japan). 2.3. Preparation of the thermoset and thermoset-BG specimens Disc-shaped specimens (Ø8.6 mm, thickness 3 mm) were used in the second, third and fourth experiments. Two experimental groups of specimens were prepared. The specimens in first group, termed “thermoset”, were made of photopolymerisable resin. The specimens in the second group, termed “thermoset-BG”, were made of photopolymerisable resin with a surface layer of BG S53P4 granules entrapped in the resin. In the thermoset group, the preparation method was similar to that used in the first experiment. The specimens were gritpolished (granulometry: 1200). In the thermoset-BG group, the preparation method was modified to incorporate BG granules. After pre-curing (40 s) with a hand curing device, a thin layer of resin was applied to the oxygen inhibited resin surface of the specimens. The specimens were then pressed against BG granules spread over a flat surface. Another thin layer of resin was added and the specimens were once again pressed against BG granules. The excess BG granules were removed by tapping on the back of the specimens. Thereafter, the final resin layer was applied on top of the granules. Subsequently, the thermoset-BG specimens were light-cured using procedures identical to the ones used to cure the specimens in the thermoset group. The thermoset-BG specimens were not grit-polished. Materials used for the preparation of the specimens are listed in Table 1. 2.4. Second experiment: optimization of processing parameters in laser ablation Only thermoset-BG specimens were used in this experiment. Excimer (XeCl) laser (ASX-750, MPB Technologies Inc, Tallinn, Estonia) with a wavelength of 308 nm and a pulse width of 28 ns (FWHM) was used for the ablation of the thermoset resin covering the BG granules. To proceed with the surface ablation, the specimens were mounted on a custom-made computer-controlled stage, which allowed precise spatial positioning. Laser beam was partially focused by a lens. The laser beam remained static during the experiment. In turn, the specimen was moved by the stage. To process a larger area, the specimen was repositioned, without overlapping of the areas affected by the laser beam. A schematic illustration of the impact of the laser beam on the specimen surface is shown in Fig. 1. A series of experiments was carried out to
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Fig. 1. Schematic illustration of Excimer laser ablation of the thermoset-BG specimens. Table 2 Optimization of processing parameters in laser ablation. Energy (mJ)
Affected area (mm2 )
Energy density/pulse (J/cm2 )
Number of pulses
Result
255 255 255 255 175 175 175 175 175
7×4 7×4 7×4 7×4 7×4 7×4 11 × 5 11 × 5 11 × 5
0.91 0.91 0.91 0.91 0.63 0.63 0.32 0.32 0.32
100 50 25 10 25 10 30 25 10
Resin Resin Resin Resin Resin Resin Resin Resin Resin
select optimal processing parameters; details are given in Table 2. The following parameters were altered: energy (175–255 mJ), affected area (7 × 4 mm–11 × 5 mm) and the number of pulses (10– 50). These parameters affected the energy density delivered to the specimens. The frequency of the pulses was kept constant at 1 Hz. Concurrently, selection of the optimal processing was performed by the examination of the surface of the specimens in scanning electron microscope (SEM) (JEOL JSM-5500, Tokyo Japan). The criteria for the selection of the optimal processing parameters included complete removal of the resin from the surface of BG granules without visible damage to the glass. 2.5. Third experiment: in vitro CaP formation in SBF The SBF test was performed to verify the absence of negative effects of laser radiation on the CaP-forming ability of BG. Thermoset and thermoset-BG specimens were used in the experiment. The thermoset-BG specimens were ablated with the Excimer laser using the optimal processing parameters. The thermoset specimens were not treated with the laser and served as a control group. The ablated thermoset-BG specimens were cleaned with compressed air to remove debris of the ablated material. Five specimens in each group were immersed in SBF for 7 days. The SBF solution was prepared according to Kokubo [18]. The ready SBF was filter-sterilized using a sterile syringe filter unit (VWR, West Chester, PA, USA) with a pore size of 0.2 μm. The total volume of SBF, 6 ml, was calculated according to the surface area of substrates, 58 mm2 . The specimens were placed in standard sterile 6-well flat bottom well-plates (VWR International BVBA, Leuven, Belgium). To prevent the specimens from floating, custom-made holders were prepared from an orthodontic wire. The well-plates were placed in a water bath (Heto Lab Equipment SBD-50 type BIO,
and BG affected/patterned structures and BG affected/patterned structures and BG affected/patterned structures and BG affected/patterned structures affected/patterned structures affected/patterned structures affected, granules well attached: optimal parameters affected moderately affected moderately
50 strokes per minute, amplitude 36 mm) at a constant temperature (37 °C) for 7 days. After incubation, the substrates were dried in a desiccator for 2 days. The specimens were then carbon-coated for analysis by scanning electron microscopy with energy dispersive spectroscopy (SEM–EDS). Thereafter, the specimens were embedded in plastic, cut in cross-section, polished, carbon-coated and studied by SEM–EDS. 2.6. Fourth experiment: cell proliferation test Standard cell proliferation test (Alamar blue assay) was conducted. Three groups of specimens were used in the experiment. The thermoset-BG specimens were ablated with the Excimer laser using the optimal processing parameters. The ablated thermosetBG specimens were cleaned with compressed air to remove debris of the ablated material. The thermoset specimens were not treated with the laser and served as a neutral control group. In addition, Micro-roughened (Ra = 4 μm) disc-shaped specimens made of titanium alloy (Ti6Al4V) (Medacta International SA; Switzerland) served as a positive control group. Six replicates were used for each group of specimens. In addition, in each group, two cell-free specimens were used as blank to subtract the average signal. The specimens were sterilized by UV light for 1 h on both sides. Approximately 20,0 0 0 cells (Osteosarcoma MG-63 cell line, ATCC number: CRL-1427) were suspended in 30 μL of culture medium (Dulbecco’s modified Eagle’s medium, Sigma-Aldrich) supplemented with inactivated fetal bovine serum (10%), penicillin (100 units/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). The cell suspension was placed in the center of the specimens. Thereafter, the specimens were incubated for 4 h at 37 °C at 5% pCO2 . After 4 h of incubation, a total of 0.85 mL of fresh culture medium was added and the specimens were further incubated for
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3.3. Third experiment: in vitro CaP formation in SBF SEM–EDS detected abundant CaP formation on the surfaces of the specimens in the thermoset-BG group. No CaP formation was detected on the surfaces of the specimens in the thermoset group. SEM micrograph of a thermoset-BG specimen after 7 days of immersion in SBF is shown in Fig. 4a. CaP formation was seen on the surface of BG granules as well as the surface of the thermoset resin. SEM micrograph of a specimen cut in cross-section is shown in Fig. 4b. Laser ablation was effective in the selective removal of the thermoset resin matrix and the exposure of the BG granules. SEM–EDS detected the formation of silica rich and calcium CaP reaction layers. The reaction layers formed only adjacent to the exposed surfaces of the BG granules. The surfaces of the granules embedded in the thermoset resin did not form reaction layers; which indicates that the SBF did not penetrate the interface between the BG granules and the thermoset resin.
Fig. 2. Results of the UV–vis spectroscopic study: the transmittance spectra of thermoset and BG specimens of different thickness.
18 h under the same conditions. To stain the cells, 0.1 mL of Alamar blue solution was added and the cells were incubated for an additional 6 h. Subsequently, the media was collected and the fluorescence was measured at the excitation wavelength of 530 nm and at the emission wavelength of 590 nm. Then, the specimens were washed with phosphate buffered saline and 0.9 mL of fresh culture medium was added. The incubation of the specimens was protracted for 16 h and the Alamar blue assay was repeated. The cell proliferation test was performed for four consecutive days.
3. Results 3.1. First experiment: UV–vis spectroscopic study Results of the UV–vis spectroscopic study are shown in Fig. 2. Both thermoset and BG specimens transmitted light at the Excimer laser wavelength (308 nm). For the thermoset specimens, there was a steep gradient in transmittance spectra: the transmittance of the 0.05 mm specimen was around 75% at 340 nm, and effectively zero already for the wavelengths below 295 nm. In addition, the effect of specimen thickness was pronounced. The transmittance of the 0.05 mm specimen was 50% at the laser wavelength (308 nm). The transmittance gradually dropped with the increase in the thickness of the specimens; 23%, for the 0.2 mm specimen, 5% for the 0.5 mm specimen and close to zero for the 1.3 mm specimen. For the BG specimens, the gradient in the transmittance spectra was less steep. The transmittance of the 0.2 mm specimen was around 80% at 340 nm, and around 10% at 270 nm. At the laser wavelength (308 nm), the transmittance was to 55% for the 0.2 mm specimen, 45% for the 0.5 mm specimen and 18% for the 1.3 mm specimen.
3.2. Second experiment: optimization of processing parameters in laser ablation The progression of the optimization of processing parameters in laser ablation of the thermoset specimens are shown in Fig. 3 and Table 2. Excessive ablation resulted in the damage to the resin and BG granules with typical onion- and plate-like patterns (Fig. 3b–d). The optimal processing parameters were: energy of 175 mJ, energy density of 0.32 J/cm2 and 30 pulses (Fig. 3e).
3.4. Fourth experiment: cell proliferation test The results of the cell proliferation test are shown in Fig. 5. The results were normalized with respect to day 1 to obtain a relative cell proliferation rate which allows a direct comparison between the specimens. In the thermoset-BG group, the pattern of proliferation of MG63 cells was comparable to that in the in the positive control group (Ti6Al4V). Cell proliferation in the thermoset group was markedly lower. 4. Discussion This paper presents a new method for the creation of BG-based osteoconductive surfaces for FRC implants by Excimer laser ablation. To our knowledge, this is the first study to report the use of this method in relation with the BG-based surfaces. FRC implants allow physiological load-sharing between implant and host bone, which could reduce the risk of adverse peri-implant bone resorption due to the “stress-shielding” [1,20]. In addition, they create fewer artifacts with modern diagnostic imaging, such as CT and MRI and do not interfere with radiotherapy [21]. However, in contrast to metallic implants, more flexible implants, such as FRC implants, may exhibit significant interfacial stresses and micromotion, as demonstrated by the finite element modeling of total hip replacement stems [20,22]. This implies that flexible implants need to be bonded to the endosteal surface of the bone. A firm bond could be achieved either by bone ingrowth (biological fixation) or by a direct chemical bond with bone (bioactive fixation). The biomechanical failure scenario of the unbonded flexible implants is attributed to the difficulties in obtaining adequate press-fit [22]. In addition, material debris is formed due to the friction of the unbonded implant against the endosteal surface. This debris irritates the surrounding tissues and triggers inflammatory response which compromises osteointegration and subsequently leads to implant loosening. Therefore, a firm bond between FRC implants and the host bone is essential for the successful implant performance. Detachment of BG granules from the FRC implants was previously reported for both load-bearing [5,23] and non-loadbearing [26] in vivo conditions. In addition, it was suggested that extensive interfacial sheer forces which arise in the load-bearing of FRC implants may contribute to the detachment of the BG granules [23]. Concerns have been raised over the behavior of the granules in response to a dynamic mechanical load [24]. In conjunction with an intramedullary implant, the granules may act as a rolling surface in the micromotion of the implant [25]. Consequently, the presence of loose granules at the implant interface is objectionable. Therefore, BG granules must be firmly attached to the surface of the implants.
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Fig. 3. Optimization of processing parameters in laser ablation. Original surface of a thermoset-BG specimen imaged before the ablation by Excimer laser; BG granules are covered by the resin (a); excessive ablation (255 mJ, 0.91 J/cm2 , 10 pulses): both resin and BG affected (b); excessive ablation (255 mJ, 0.91 J/cm2 , 10 pulses) (c); resin affected excessively, BG intact (175 mJ, 0.63 J/cm2 , 25 pulses) (d); optimal ablation (175 mJ, 0.32 J/cm2 , 30 pulses): resin affected, BG intact (e); moderate ablation (175 mJ, 0.32 J/cm2 , 25 pulses): resin still affected, BG intact (f).
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Fig. 4. CaP formation on the surfaces of the thermoset-BG specimens after immersion in SBF for 7 days. Overview of the surface of a specimen (a); detailed view of CaP formation on the surface of BG with the spot of EDS analysis encircled (c); results of the EDS analysis (e); cross-sectional micrograph, only exposed BG granules have reacted (b); detail of a BG granule with the typical reaction layers (d); completely reacted BG granule, CaP formation was observed on the surface of resin (arrows) (f).
To embed BG granules within a polymer matrix, typically, BG granules are mixed with the matrix and consequently the matrix is mechanically removed to expose the surface of the granules, or the granules are glued to the surface of the implant. Laser ablation eliminates the need for mechanical treatment of the surface to expose BG granules. No detachment of the granules was noticed in this study, even after immersion in SBF. However, the absence of a chemical bond between the BG granules and the resin matrix could be an issue. Silanation of BG granules provides a chemical attachment of the granules to the matrix in composites [27–29]. In fact, silanation of BG granules could be an attractive option in the case of UV laser ablation, as the silane layer could be removed from the surface of the granules in the ablation process. In this study, however, silanation was not attempted to keep the experimental set-up simple.
The interaction of UV laser beam with organic polymers cannot be explained by a universal model. In general, it is believed that both photothermal and photochemical mechanisms contribute to the laser ablation of polymers [15,30–32]. It was suggested that at low laser fluence, photochemical mechanisms dominate the ablation process while at high fluence the ablation process is governed by the photothermal mechanisms. In the UV laser ablation of polymers, the absence of thermal damage is attributed to the low penetration depth of the beam in the substrate, as well as the low spatial extent of heat diffusion [15]. Upon irradiation with the laser beam, polymeric bonds break and the matter is ejected from the ablation site. The ablation products vary from atoms and diatomics to polyatomic low molecular weight molecules and fragments of the polymer matrix [30]. A part of the ablation products settles back on the ablated surface. Therefore, a possibility of the
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Fig. 5. Results of the of MG63 cell proliferation test (mean ± standard deviation).
toxic effects of the ablation products cannot be excluded. However, the chemical analysis of the ablation products was beyond the scope of this study as we primarily interested in the feasibility of the method. The specimens were cleaned with compressed air and used in the SBF test and cell culture studies. We assumed that the absence of pronounced adverse effects on CaP formation and cell proliferation would signify the potential of UV laser ablation for the creation of a bioactive surface. Chemical analysis and animal experiments are foreseen in the future studies. 5. Conclusion A proof of concept has been established for the creation of an osteoconductive surface by surface etching by Excimer laser of thermoset resin with BG granules embedded. Ethical approval Ethical approval not required. Funding This work was supported financially by European Union׳s Sixth Framework Programme (FP6-NMP) under the Grant agreement of the NEWBONE Project (NMP3-CT-2007–026279). Authors NM and JK acknowledge the support from Prof. Allan Ahos’s fund of the Turku University Foundation. Conflict of interest statement Author PV is member of the board and shareholder of a startup company Skulle Implants Corporation, which is active in commercializing cranial implants based on FRC technology. Author RM is presently an employee and author NM a collaborator for Skulle Implants Corporation. Other authors have nothing to declare. References [1] Evans SL, Gregson PJ. Composite technology in load-bearing orthopaedic implants. Biomaterials 1998;19:1329–42. [2] Vallittu PK. Flexural properties of acrylic resin polymers reinforced with unidirectional and woven glass fibers. J Prosthet Dent 1999;81:318–26.
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Medical Engineering and Physics journal homepage: www.elsevier.com/locate/medengphy
Technical note
Bioactive glass surface for fiber reinforced composite implants via surface etching by Excimer laser Julia Kulkova a, Niko Moritz a,∗, Hannu Huhtinen b, Riina Mattila a, Ivan Donati c, Eleonora Marsich d, Sergio Paoletti c, Pekka K Vallittu a a
Turku Clinical Biomaterials Centre (TCBC), Department of Biomaterials Science, Institute of Dentistry, University of Turku and Biocity Turku Biomaterials Research Program and City of Turku Welfare Division, Itäinen pitkäkatu 4B (PharmaCity), FI-20520 Turku, Finland Wihuri Physical Laboratory, Department of Physics and Astronomy, University of Turku, FI-20014, Finland c Department of Life Sciences, University of Trieste, Via Licio Giorgieri 5, I-34127 Trieste, Italy d Department of Medicine, Surgery and Health Sciences, University of Trieste, Piazza dell’Ospitale 1, 34129 Trieste, Italy b
a r t i c l e
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Article history: Received 22 October 2015 Revised 22 February 2016 Accepted 3 April 2016
Keywords: Fiber-reinforced composites Excimer laser UV laser Laser ablation Bisphenol-A-glycidyldimethacrylate Bioactive glass Implant Intramedullary nail
a b s t r a c t Biostable fiber-reinforced composites (FRC) prepared from bisphenol-A-glycidyldimethacrylate (BisGMA)based thermosets reinforced with E-glass fibers are promising alternatives to metallic implants due to the excellent fatigue resistance and the mechanical properties matching those of bone. Bioactive glass (BG) granules can be incorporated within the polymer matrix to improve the osteointegration of the FRC implants. However, the creation of a viable surface layer using BG granules is technically challenging. In this study, we investigated the potential of Excimer laser ablation to achieve the selective removal of the matrix to expose the surface of BG granules. A UV–vis spectroscopic study was carried out to investigate the differences in the penetration of light in the thermoset matrix and BG. Thereafter, optimal Excimer laser ablation parameters were established. The formation of a calcium phosphate (CaP) layer on the surface of the laser-ablated specimens was verified in simulated body fluid (SBF). In addition, the proliferation of MG63 cells on the surfaces of the laser-ablated specimens was investigated. For the laser-ablated specimens, the pattern of proliferation of MG63 cells was comparable to that in the positive control group (Ti6Al4V). We concluded that Excimer laser ablation has potential for the creation of a bioactive surface on FRC-implants. © 2016 IPEM. Published by Elsevier Ltd. All rights reserved.
1. Introduction Biostable fiber-reinforced composites (FRC) are promising alternatives to metallic implants due to the excellent fatigue resistance and the mechanical properties matching those of bone [1]. FRCs prepared from bisphenol-A-glycidyldimethacrylate (BisGMA)based thermosets reinforced with E-glass fibers have been successfully used in dental reconstructions [2,3]. Clinical use of nonload-bearing FRC implants has already commenced in cranial reconstructions [4]. In addition, preclinical studies demonstrated the potential of BisGMA-based FRC implants in load-bearing orthopaedic applications [5,6]. Bioactive glass (BG) granules can be incorporated into the surface of the FRC implants to improve their
∗ Corresponding author at: Turku Clinical Biomaterials Centre (TCBC), Department of Biomaterials Science, Institute of Dentistry, University of Turku, Itäinen pitkäkatu 4B (PharmaCity), FI-20520 Turku, Finland. Tel.: +358 2 333 8227. E-mail address: niko.moritz@utu.fi (N. Moritz). URL: http://biomaterials.utu.fi (N. Moritz)
http://dx.doi.org/10.1016/j.medengphy.2016.04.003 1350-4533/© 2016 IPEM. Published by Elsevier Ltd. All rights reserved.
osteointegration. The bioactivity of BG is manifested in the formation of direct chemical bond between BG and bone through the cascade of chemical reactions and cellular activity [7–9]. Formation of a calcium phosphate (CaP) surface reaction layer is a crucial aspect of bone bonding. BG granules are certified for clinical use i.e. as bone graft substitutes [10–13]. However, our previous studies revealed technical challenges in the creation of a feasible surface layer using BG granules [5,6]. A large number of unreacted BG granules were trapped inside the polymer matrix, while the surface area of the granules exposed by grinding was insufficient to achieve notable improvement in the osteointegration of the implants [5]. In addition, the mechanical treatment, i.e. grinding, of the implant surface led to the disengagement of the granules either before or after the implantation [5]. Therefore, there is a need for the development of new techniques for the incorporation of BG granules in the surfaces for the FRC implants. Surface ablation of polymers by Excimer lasers was demonstrated in the early 1980s [14,15]. Studies with laser ablation of the composites revealed selective removal of the polymer matrix and
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Table 1 Summary of materials used for the preparation of the specimens. Material
Type of material
Manufacturer
Bisphenol-A-glycidyldimethacrylate (BisGMA) Triethylene glycol dimethacrylate (TEGDMA) 2-(Dimethylamino)ethyl methacrylate (DMAEMA) Camphorquinone Bioactive glass (BG) S53P4, granules: 315–500 μm fraction, oxide composition (wt%): SiO2 53%, Na2 O 23%, CaO 20%, P2 O5 4%
Co-monomer Co-monomer Activator Photoinitiator Osteoconductive surface component
Röhm Chemische Fabrik GmbH, Darmstadt, Germany Aldrich Chemie GmbH, Steinheim, Germany Fluka Chemie GmbH, Buchs, Switzerland Sigma-Aldrich GmbH, Buchs, Switzerland Vivoxid Ltd., Turku, Finland
exposure of the reinforcing glass fibers [16,17]. We hypothesized that with this setup, Excimer laser ablation would allow selective removal of the thermoset matrix and the exposure of the BG granules, which in turn, would enhance the osteoconductivity of the FRC implants without the disengagement of BG granules. 2. Materials and methods 2.1. Methodology of the study The targeted implants are intramedullary nails reported previously [5,22]. The core of the implants is reinforced with long glass fibers while the relatively thick surface layer (∼1 mm) contains unreinforced resin embedded with bioactive glass granules. Therefore, for the sake of simplicity, we excluded the reinforcing phase from consideration and used unreinforced thermosets in all experiments. This study included four experiments. The first experiment, a UV–vis spectroscopic study, was carried out to investigate the differences in the penetration of light in the thermoset matrix and BG. The second experiment was carried out to establish the optimal laser ablation parameters. The third experiment was performed to study CaP formation on the surfaces of the specimens immersed in simulated body fluid (SBF) [18]. The fourth experiment was performed to study MG63 cell proliferation on the surfaces of the specimens to verify the absence of negative effects of laser radiation. The laser ablation parameters used in the third and fourth experiment, were based on the results obtained in the second experiment. 2.2. First experiment: UV–vis spectroscopic study Two groups of specimens, BG and thermosets, were used in the UV–vis spectroscopic study. Materials used for the preparation of the specimens are listed in Table 1. Rectangular-shaped BG specimens (10 × 10 mm) were cut from a 1 mm-thick BG S53P4 plate. This determined the shape of the specimens used in this experiment. The BG specimens were prepared in three different thicknesses: 1.3 mm, 0.5 mm, and 0.2 mm by grinding with silicon carbide paper of 10 0 0, 240 0, and 40 0 0 grit, with further polishing with 0.1 μm alumina paste. Ethanol was used in grinding and polishing to avoid the formation of reaction layers. Subsequently, the specimens were cleaned ultrasonically in ethanol. Impression molds made from dental putty were used to prepare the thermoset specimens. The photopolymerisable resin was prepared by mixing BisGMA (70 wt%) and triethylene glycol dimethacrylate (TEGDMA) (30 wt%) with camphorquinone (CQ) (0.7 wt%) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) (0.7 wt%) [19]. Thereafter, the resin was poured into the molds and briefly pre-cured (40 s) by a hand curing device (Optilux 501, Kerr, Danbury, USA). Next, the thermoset specimens were taken out of the molds and cured in vacuum light oven (Visio Beta vario, 3 M/ESPE, Seefeld, Germany) for 15 min at ambient temperature. Subsequently, in order to reduce the amount of residual
monomers, the specimens were placed in a light oven (Liculite, Dentsply De Trey GmbH, Dreieich, Germany) for 25 min. During the time, the temperature of the curing chamber increased to 90 °C. The thermoset specimens were ground with silicon carbide paper of 10 0 0, 240 0, and 40 0 0 grit, and further polished with 0.1 μm alumina paste. Water was used in grinding. The thermoset specimens were prepared in four different thicknesses: 1.3 mm, 0.5 mm, 0.2 mm and ∼0.05 mm. After the preparation, the specimens were cleaned ultrasonically in water. Light transmittance in the range of 270–340 nm was studied using UV–vis spectrophotometer (Shimadzu UV-1601, Shimadzu Corp. Tokyo, Japan). 2.3. Preparation of the thermoset and thermoset-BG specimens Disc-shaped specimens (Ø8.6 mm, thickness 3 mm) were used in the second, third and fourth experiments. Two experimental groups of specimens were prepared. The specimens in first group, termed “thermoset”, were made of photopolymerisable resin. The specimens in the second group, termed “thermoset-BG”, were made of photopolymerisable resin with a surface layer of BG S53P4 granules entrapped in the resin. In the thermoset group, the preparation method was similar to that used in the first experiment. The specimens were gritpolished (granulometry: 1200). In the thermoset-BG group, the preparation method was modified to incorporate BG granules. After pre-curing (40 s) with a hand curing device, a thin layer of resin was applied to the oxygen inhibited resin surface of the specimens. The specimens were then pressed against BG granules spread over a flat surface. Another thin layer of resin was added and the specimens were once again pressed against BG granules. The excess BG granules were removed by tapping on the back of the specimens. Thereafter, the final resin layer was applied on top of the granules. Subsequently, the thermoset-BG specimens were light-cured using procedures identical to the ones used to cure the specimens in the thermoset group. The thermoset-BG specimens were not grit-polished. Materials used for the preparation of the specimens are listed in Table 1. 2.4. Second experiment: optimization of processing parameters in laser ablation Only thermoset-BG specimens were used in this experiment. Excimer (XeCl) laser (ASX-750, MPB Technologies Inc, Tallinn, Estonia) with a wavelength of 308 nm and a pulse width of 28 ns (FWHM) was used for the ablation of the thermoset resin covering the BG granules. To proceed with the surface ablation, the specimens were mounted on a custom-made computer-controlled stage, which allowed precise spatial positioning. Laser beam was partially focused by a lens. The laser beam remained static during the experiment. In turn, the specimen was moved by the stage. To process a larger area, the specimen was repositioned, without overlapping of the areas affected by the laser beam. A schematic illustration of the impact of the laser beam on the specimen surface is shown in Fig. 1. A series of experiments was carried out to
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Fig. 1. Schematic illustration of Excimer laser ablation of the thermoset-BG specimens. Table 2 Optimization of processing parameters in laser ablation. Energy (mJ)
Affected area (mm2 )
Energy density/pulse (J/cm2 )
Number of pulses
Result
255 255 255 255 175 175 175 175 175
7×4 7×4 7×4 7×4 7×4 7×4 11 × 5 11 × 5 11 × 5
0.91 0.91 0.91 0.91 0.63 0.63 0.32 0.32 0.32
100 50 25 10 25 10 30 25 10
Resin Resin Resin Resin Resin Resin Resin Resin Resin
select optimal processing parameters; details are given in Table 2. The following parameters were altered: energy (175–255 mJ), affected area (7 × 4 mm–11 × 5 mm) and the number of pulses (10– 50). These parameters affected the energy density delivered to the specimens. The frequency of the pulses was kept constant at 1 Hz. Concurrently, selection of the optimal processing was performed by the examination of the surface of the specimens in scanning electron microscope (SEM) (JEOL JSM-5500, Tokyo Japan). The criteria for the selection of the optimal processing parameters included complete removal of the resin from the surface of BG granules without visible damage to the glass. 2.5. Third experiment: in vitro CaP formation in SBF The SBF test was performed to verify the absence of negative effects of laser radiation on the CaP-forming ability of BG. Thermoset and thermoset-BG specimens were used in the experiment. The thermoset-BG specimens were ablated with the Excimer laser using the optimal processing parameters. The thermoset specimens were not treated with the laser and served as a control group. The ablated thermoset-BG specimens were cleaned with compressed air to remove debris of the ablated material. Five specimens in each group were immersed in SBF for 7 days. The SBF solution was prepared according to Kokubo [18]. The ready SBF was filter-sterilized using a sterile syringe filter unit (VWR, West Chester, PA, USA) with a pore size of 0.2 μm. The total volume of SBF, 6 ml, was calculated according to the surface area of substrates, 58 mm2 . The specimens were placed in standard sterile 6-well flat bottom well-plates (VWR International BVBA, Leuven, Belgium). To prevent the specimens from floating, custom-made holders were prepared from an orthodontic wire. The well-plates were placed in a water bath (Heto Lab Equipment SBD-50 type BIO,
and BG affected/patterned structures and BG affected/patterned structures and BG affected/patterned structures and BG affected/patterned structures affected/patterned structures affected/patterned structures affected, granules well attached: optimal parameters affected moderately affected moderately
50 strokes per minute, amplitude 36 mm) at a constant temperature (37 °C) for 7 days. After incubation, the substrates were dried in a desiccator for 2 days. The specimens were then carbon-coated for analysis by scanning electron microscopy with energy dispersive spectroscopy (SEM–EDS). Thereafter, the specimens were embedded in plastic, cut in cross-section, polished, carbon-coated and studied by SEM–EDS. 2.6. Fourth experiment: cell proliferation test Standard cell proliferation test (Alamar blue assay) was conducted. Three groups of specimens were used in the experiment. The thermoset-BG specimens were ablated with the Excimer laser using the optimal processing parameters. The ablated thermosetBG specimens were cleaned with compressed air to remove debris of the ablated material. The thermoset specimens were not treated with the laser and served as a neutral control group. In addition, Micro-roughened (Ra = 4 μm) disc-shaped specimens made of titanium alloy (Ti6Al4V) (Medacta International SA; Switzerland) served as a positive control group. Six replicates were used for each group of specimens. In addition, in each group, two cell-free specimens were used as blank to subtract the average signal. The specimens were sterilized by UV light for 1 h on both sides. Approximately 20,0 0 0 cells (Osteosarcoma MG-63 cell line, ATCC number: CRL-1427) were suspended in 30 μL of culture medium (Dulbecco’s modified Eagle’s medium, Sigma-Aldrich) supplemented with inactivated fetal bovine serum (10%), penicillin (100 units/mL), streptomycin (100 μg/mL), and L-glutamine (2 mM). The cell suspension was placed in the center of the specimens. Thereafter, the specimens were incubated for 4 h at 37 °C at 5% pCO2 . After 4 h of incubation, a total of 0.85 mL of fresh culture medium was added and the specimens were further incubated for
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3.3. Third experiment: in vitro CaP formation in SBF SEM–EDS detected abundant CaP formation on the surfaces of the specimens in the thermoset-BG group. No CaP formation was detected on the surfaces of the specimens in the thermoset group. SEM micrograph of a thermoset-BG specimen after 7 days of immersion in SBF is shown in Fig. 4a. CaP formation was seen on the surface of BG granules as well as the surface of the thermoset resin. SEM micrograph of a specimen cut in cross-section is shown in Fig. 4b. Laser ablation was effective in the selective removal of the thermoset resin matrix and the exposure of the BG granules. SEM–EDS detected the formation of silica rich and calcium CaP reaction layers. The reaction layers formed only adjacent to the exposed surfaces of the BG granules. The surfaces of the granules embedded in the thermoset resin did not form reaction layers; which indicates that the SBF did not penetrate the interface between the BG granules and the thermoset resin.
Fig. 2. Results of the UV–vis spectroscopic study: the transmittance spectra of thermoset and BG specimens of different thickness.
18 h under the same conditions. To stain the cells, 0.1 mL of Alamar blue solution was added and the cells were incubated for an additional 6 h. Subsequently, the media was collected and the fluorescence was measured at the excitation wavelength of 530 nm and at the emission wavelength of 590 nm. Then, the specimens were washed with phosphate buffered saline and 0.9 mL of fresh culture medium was added. The incubation of the specimens was protracted for 16 h and the Alamar blue assay was repeated. The cell proliferation test was performed for four consecutive days.
3. Results 3.1. First experiment: UV–vis spectroscopic study Results of the UV–vis spectroscopic study are shown in Fig. 2. Both thermoset and BG specimens transmitted light at the Excimer laser wavelength (308 nm). For the thermoset specimens, there was a steep gradient in transmittance spectra: the transmittance of the 0.05 mm specimen was around 75% at 340 nm, and effectively zero already for the wavelengths below 295 nm. In addition, the effect of specimen thickness was pronounced. The transmittance of the 0.05 mm specimen was 50% at the laser wavelength (308 nm). The transmittance gradually dropped with the increase in the thickness of the specimens; 23%, for the 0.2 mm specimen, 5% for the 0.5 mm specimen and close to zero for the 1.3 mm specimen. For the BG specimens, the gradient in the transmittance spectra was less steep. The transmittance of the 0.2 mm specimen was around 80% at 340 nm, and around 10% at 270 nm. At the laser wavelength (308 nm), the transmittance was to 55% for the 0.2 mm specimen, 45% for the 0.5 mm specimen and 18% for the 1.3 mm specimen.
3.2. Second experiment: optimization of processing parameters in laser ablation The progression of the optimization of processing parameters in laser ablation of the thermoset specimens are shown in Fig. 3 and Table 2. Excessive ablation resulted in the damage to the resin and BG granules with typical onion- and plate-like patterns (Fig. 3b–d). The optimal processing parameters were: energy of 175 mJ, energy density of 0.32 J/cm2 and 30 pulses (Fig. 3e).
3.4. Fourth experiment: cell proliferation test The results of the cell proliferation test are shown in Fig. 5. The results were normalized with respect to day 1 to obtain a relative cell proliferation rate which allows a direct comparison between the specimens. In the thermoset-BG group, the pattern of proliferation of MG63 cells was comparable to that in the in the positive control group (Ti6Al4V). Cell proliferation in the thermoset group was markedly lower. 4. Discussion This paper presents a new method for the creation of BG-based osteoconductive surfaces for FRC implants by Excimer laser ablation. To our knowledge, this is the first study to report the use of this method in relation with the BG-based surfaces. FRC implants allow physiological load-sharing between implant and host bone, which could reduce the risk of adverse peri-implant bone resorption due to the “stress-shielding” [1,20]. In addition, they create fewer artifacts with modern diagnostic imaging, such as CT and MRI and do not interfere with radiotherapy [21]. However, in contrast to metallic implants, more flexible implants, such as FRC implants, may exhibit significant interfacial stresses and micromotion, as demonstrated by the finite element modeling of total hip replacement stems [20,22]. This implies that flexible implants need to be bonded to the endosteal surface of the bone. A firm bond could be achieved either by bone ingrowth (biological fixation) or by a direct chemical bond with bone (bioactive fixation). The biomechanical failure scenario of the unbonded flexible implants is attributed to the difficulties in obtaining adequate press-fit [22]. In addition, material debris is formed due to the friction of the unbonded implant against the endosteal surface. This debris irritates the surrounding tissues and triggers inflammatory response which compromises osteointegration and subsequently leads to implant loosening. Therefore, a firm bond between FRC implants and the host bone is essential for the successful implant performance. Detachment of BG granules from the FRC implants was previously reported for both load-bearing [5,23] and non-loadbearing [26] in vivo conditions. In addition, it was suggested that extensive interfacial sheer forces which arise in the load-bearing of FRC implants may contribute to the detachment of the BG granules [23]. Concerns have been raised over the behavior of the granules in response to a dynamic mechanical load [24]. In conjunction with an intramedullary implant, the granules may act as a rolling surface in the micromotion of the implant [25]. Consequently, the presence of loose granules at the implant interface is objectionable. Therefore, BG granules must be firmly attached to the surface of the implants.
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Fig. 3. Optimization of processing parameters in laser ablation. Original surface of a thermoset-BG specimen imaged before the ablation by Excimer laser; BG granules are covered by the resin (a); excessive ablation (255 mJ, 0.91 J/cm2 , 10 pulses): both resin and BG affected (b); excessive ablation (255 mJ, 0.91 J/cm2 , 10 pulses) (c); resin affected excessively, BG intact (175 mJ, 0.63 J/cm2 , 25 pulses) (d); optimal ablation (175 mJ, 0.32 J/cm2 , 30 pulses): resin affected, BG intact (e); moderate ablation (175 mJ, 0.32 J/cm2 , 25 pulses): resin still affected, BG intact (f).
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Fig. 4. CaP formation on the surfaces of the thermoset-BG specimens after immersion in SBF for 7 days. Overview of the surface of a specimen (a); detailed view of CaP formation on the surface of BG with the spot of EDS analysis encircled (c); results of the EDS analysis (e); cross-sectional micrograph, only exposed BG granules have reacted (b); detail of a BG granule with the typical reaction layers (d); completely reacted BG granule, CaP formation was observed on the surface of resin (arrows) (f).
To embed BG granules within a polymer matrix, typically, BG granules are mixed with the matrix and consequently the matrix is mechanically removed to expose the surface of the granules, or the granules are glued to the surface of the implant. Laser ablation eliminates the need for mechanical treatment of the surface to expose BG granules. No detachment of the granules was noticed in this study, even after immersion in SBF. However, the absence of a chemical bond between the BG granules and the resin matrix could be an issue. Silanation of BG granules provides a chemical attachment of the granules to the matrix in composites [27–29]. In fact, silanation of BG granules could be an attractive option in the case of UV laser ablation, as the silane layer could be removed from the surface of the granules in the ablation process. In this study, however, silanation was not attempted to keep the experimental set-up simple.
The interaction of UV laser beam with organic polymers cannot be explained by a universal model. In general, it is believed that both photothermal and photochemical mechanisms contribute to the laser ablation of polymers [15,30–32]. It was suggested that at low laser fluence, photochemical mechanisms dominate the ablation process while at high fluence the ablation process is governed by the photothermal mechanisms. In the UV laser ablation of polymers, the absence of thermal damage is attributed to the low penetration depth of the beam in the substrate, as well as the low spatial extent of heat diffusion [15]. Upon irradiation with the laser beam, polymeric bonds break and the matter is ejected from the ablation site. The ablation products vary from atoms and diatomics to polyatomic low molecular weight molecules and fragments of the polymer matrix [30]. A part of the ablation products settles back on the ablated surface. Therefore, a possibility of the
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Fig. 5. Results of the of MG63 cell proliferation test (mean ± standard deviation).
toxic effects of the ablation products cannot be excluded. However, the chemical analysis of the ablation products was beyond the scope of this study as we primarily interested in the feasibility of the method. The specimens were cleaned with compressed air and used in the SBF test and cell culture studies. We assumed that the absence of pronounced adverse effects on CaP formation and cell proliferation would signify the potential of UV laser ablation for the creation of a bioactive surface. Chemical analysis and animal experiments are foreseen in the future studies. 5. Conclusion A proof of concept has been established for the creation of an osteoconductive surface by surface etching by Excimer laser of thermoset resin with BG granules embedded. Ethical approval Ethical approval not required. Funding This work was supported financially by European Union׳s Sixth Framework Programme (FP6-NMP) under the Grant agreement of the NEWBONE Project (NMP3-CT-2007–026279). Authors NM and JK acknowledge the support from Prof. Allan Ahos’s fund of the Turku University Foundation. Conflict of interest statement Author PV is member of the board and shareholder of a startup company Skulle Implants Corporation, which is active in commercializing cranial implants based on FRC technology. Author RM is presently an employee and author NM a collaborator for Skulle Implants Corporation. Other authors have nothing to declare. References [1] Evans SL, Gregson PJ. Composite technology in load-bearing orthopaedic implants. Biomaterials 1998;19:1329–42. [2] Vallittu PK. Flexural properties of acrylic resin polymers reinforced with unidirectional and woven glass fibers. J Prosthet Dent 1999;81:318–26.
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