Terminal Sterilization of BisGMA-TEGDMA Thermoset Materials and

Article pubs.acs.org/Biomac

Terminal Sterilization of BisGMA-TEGDMA Thermoset Materials and Their Bioactive Surfaces by Supercritical CO2 Ivan Donati,*,† Monica Benincasa,† Marie-Pierre Foulc,‡ Gianluca Turco,§ Mila Toppazzini,† Dario Solinas,⊥ Sara Spilimbergo,∥ Ireneo Kikic,⊥ and Sergio Paoletti† †

Department of Life Sciences, University of Trieste, Via Licio Giorgieri 1, I-34127, Trieste, Italy RESCOLL Société de Recherche, allée Geoffroy Saint Hilaire 8, F-33615 Pessac, France § Department of Medical Sciences, University of Trieste, Piazza dell’Ospitale 1, I-34129 Trieste, Italy ∥ Department of Materials Engineering and Industrial Technologies, University of Trento, via Mesiano 77, I-38123 Trento, Italy ⊥ Department of Industrial Engineering and Information Technology, University of Trieste, via Valerio 10, I-34127 Trieste, Italy ‡

ABSTRACT: The development of biomaterials endowed with bioactive features relies on a simultaneous insight into a proper terminal sterilization process. FDA recommendations on sterility of biomaterials are very strict: a sterility assurance level (SAL) of 10−6 must be guaranteed for biomaterials to be used in human implants. In the present work, we have explored the potential of supercritical CO2 (scCO2) in the presence of H2O2 as a low-temperature sterilization process for thermoset materials and their bioactive surfaces. Different conditions allowing for terminal sterilization have been screened and a treatment time−amount of H2O2 relationship proposed. The selected terminal sterilization conditions did not notably modify the mechanical properties of the thermoset nor of their fiber-reinforced composites. This was confirmed by μCT analyses performed prior to and after the treatment. On the contrary, terminal sterilization in the presence of H2O2 induced a slight decrease in the surface hardness. The treatment of the thermoset material with scCO2 led to a reduction in the residual unreacted monomers content, as determined by means of high performance liquid chromatography (HPLC) analyses. Finally, it was found that a thermoset coated with a polysaccharide layer containing silver nanoparticles maintained a very high antimicrobial efficacy even after the scCO2-based terminal sterilization.

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INTRODUCTION Thermoset materials based on bisphenol A glycidyl methacrylate (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) are largely used in the biomedical field, especially in dentistry and orthopedics.1,2 BisGMA/TEGDMA composite materials coated with bioactive glass have been reported to promote healing of critical size calvarian bone and frontal bone defects.3 Moreover, the surface of these thermoset materials can be efficiently altered, through chemical and physical fine-tuning, with a considerable enhancement of in vitro biological performance.4−6 The concept of “biochemical interplay” between biomaterial and host tissue is of fundamental importance in determining the integration of the implant.7−9 Tissue regeneration, integration, and remodeling are guided by the presence on the biomaterial surface of specific biochemical signals (such as proteins,10−12 peptides,13−15 growth factors,16 and polysaccharides17−19) that are able to engage receptors on the cells. Despite the intriguing biotechnological properties of such third generation biomaterials, their widespread medical application is generally hampered by limited resistance to terminal sterilization.20 When medical devices for human use are considered, a sterility assurance level (SAL) of 10−6 is required, © 2012 American Chemical Society

that is, the probability that a product is contaminated is one in a million when starting with an initial bioburden of the bioindicator higher than 106 colony forming units (CFU).21−24 Most common FDA-approved terminal sterilization techniques are performed in conditions causing either degradation or chemical modifications of polymeric and biological materials. Steam sterilization has been reported to damage heat-sensitive and hydrolytically labile materials.25 γ-Radiations are largely used for sterilization of biomaterials, although they have been reported to cause oxidative damage to ultrahigh molecular weight polyethylene (UHMWPE) and to modify shear and tensile strength, elastic modulus, and transparency of several polymers.26,27 Ethylene oxide has very few drawbacks when considered for terminal sterilization, but its flammability and the presence of toxic residuals, which were reported to cause irritation or hemolysis, are a major concern.26 An improvement in the preservation of biomaterial mechanical properties has been accomplished using hydrogen peroxide gas plasma for terminal sterilization purposes; the low temperature of the Received: January 11, 2012 Revised: March 7, 2012 Published: March 27, 2012 1152

dx.doi.org/10.1021/bm300053d | Biomacromolecules 2012, 13, 1152−1160

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process offers considerable advantages over γ-radiations with respect to oxidation,28 but the efficacy of H2O2 plasma-based terminal sterilization has been questioned when applied to complex devices.29 The above-reported terminal sterilization techniques show several limitations when biopolymers are considered. As an example, hyaluronan, a natural glycosaminoglycan involved in cell−cell and cell−ECM interactions,30 is very sensitive to γ-radiations and to autoclave sterilization, as depolymerization and β-elimination reactions might occur, while the treatment with ethylene oxide alters its chemical features.31−33 Due to these considerations, the development of a terminal sterilization method compatible with the use of most biomaterials is of great interest. Supercritical carbon dioxide (scCO2), with critical temperature and pressure values of Tc = 31.1 °C and Pc = 7.38 MPa, respectively, has a liquid-like density (0.9−1.0 × 103 kg m−3),34 gas-like diffusivity (10−7− 10−8 m2 s−1) and viscosity (3−7 × 10−5 N s m−2), and zero surface tension,35 which allows easy penetration through materials. Several reports and patents have dealt with the use of scCO2 for the inactivation of vegetative bacteria and spores.26,36−43 Very recently, scCO2 terminal sterilization did not show any drawbacks to UHMWPE mechanical properties.44 However, a significant decrease in the tensile strength was observed for polymethylmethacrylate (PMMA), polycarbonate (PC), and polyvinyl chloride (PVC)45 upon treatment with scCO2. In the present work, we have explored the potential of scCO2 for the terminal sterilization of BisGMA/TEGDMA thermoset and composite materials. The variation of physical−chemical properties of the material by means of mechanical measurements, surface hardness, and microcomputed tomography (μCT) has been monitored. Moreover, the possibility of removing the unreacted monomers by scCO2 was also assessed. Finally, the terminal sterilization of a coated thermoset material was performed resulting in only a minimal decrease in biological activity.

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pressure control unit (DS Europe AN341). In the terminal sterilization experiments, H2O2 was added to the vessel. Preparation of BisGMA/TEGDMA Thermoset and Fiber Reinforced Composite by Light-Induced Polymerization (lTS and lTS-FRC). BisGMA (70% w/w) and TEGDMA (30% w/w) were mixed under vigorous stirring at 37 °C. CQ (0.7% w/w) and DMAEMA (0.7% w/w) were added, the solution was protected from light and degassed for 12 h in a vacuum oven at 40 °C. It was then poured into a Teflon mold (⌀ =14 mm, h = 2.5 mm for the circular samples; 20 mm length × 2 mm width × 2 mm thickness for bars) and the wells were covered with a PET film. Fiber reinforced composite (FRC) bars of this formulation (lTS-FRC) were prepared by adding 50% (w/w) of longitudinally oriented E-glass fibers in the mold. Circular FRC (lTS-FRC disks) were prepared by embedding S-glass braid in the resin prior to polymerization. Light-Induced Polymerization. The polymerization was light initiated irradiation for 20 s using a hand cure light device (Optilux 501; λ, 400−505 nm; light power, 850 mW/cm2). The curing was performed with a Photopol IR/UV Plus oven (Dentalfarm, Italy) equipped with 8 lamps and 2 spots operating in the wavelength range 320−550 nm with the following procedure: 20 min in light oven (8 lamps) plus 20 min in light oven (8 lamps) on a rotating plate. Postcuring. Samples were postcured with the following procedure: 60 min in light oven (8 lamps) under vacuum plus 7 min in light oven (8 lamps and 2 spots). Preparation of BisGMA/TEGDMA Thermoset and FiberReinforced Composite by Thermal-Induced Polymerization (tTS and tTS-FRC). BPO (1% w/w) was first dissolved by magnetic stirring in TEGDMA (30% w/w) for 15 min at room temperature. Then, BisGMA (70% w/w) was then added and the mixing of the components was performed by manual stirring until complete homogenization. Vit E (0.3% w/w) was finally added. Fiber-reinforced composite thermosets bars of this formulation (tTS-FRC) were prepared by adding 50% (w/w) of longitudinally oriented E-glass fibers in the Teflon mold (20 mm length × 2 mm width × 2 mm thickness for bars) and the wells were covered with a PET film. The polymerization was thermally initiated by exposing the resin for 1 h at 100 °C under vacuum. To optimize the conversion degree, two successive post-treatments under vacuum (2 h at 120 °C followed by 2 h at 150 °C) were performed. Three Points Bending Tests. Mechanical tests were performed on a universal mechanical testing machine (Galdabini Sun 500, Galdabini, Varese, Italy). Three points bending tests were performed on thermoset resin bars (lTS) and on fiber-reinforced composites bars (lTS-FRC and tTS-FRC) according to ISO10477 standard. The probe speed was maintained constant at 5 mm/min. The force−deformation profile was recorded and the flexural modulus (Ef) and flexural strength at break (σf) were calculated. Surface Hardness. Tests were carried out with a LEICA VMHT MOT. Vickers hardness (Hv) of the thermoset (lTS) and composite (lTS-FRC disks) samples was measured by applying a load of 50 gf (F) for 10 s for each measurement, the two diagonals (d) were measured, and the hardness was evaluated according to the following relation

MATERIALS AND METHODS

Chitlac (lactose-modified chitosan, CAS registry number 85941-43-1) was prepared according to the procedure reported elsewhere46 starting from a highly deacetylated chitosan (residual degree of acetylation ≈ 16%). The molecular weight of chitlac was estimated to be approximately 1.5 × 106. Silver nitrate, ascorbic acid, hydrogen peroxide, tetrahydrofuran, bisphenol A glycidylmethacrylate (BisGMA), triethyleneglycol dimethacrylate (TEGDMA), vitamin E (vit E), and benzoyl peroxide (BPO) were obtained from Sigma-Aldrich (St. Louis, MO). Camphorquinone (CQ) and 2-dimethylamino ethylmethacrylate (DMAEMA) were purchased from Fluka. Longitudinal E-glass fibers were from Ahlström (Karhula, Finland). S-glass braid (braiding angle 45°) was from Eurocarbon. Mueller-Hinton (MH) was from Difco Microbiology (Sparks, MD). Chitlac-silver nanoparticle (chitlac-nAg) suspension was prepared as reported elsewere.47 Thermosets coated with chitlac-silver nanoparticles (chitlac-nAg) were prepared as reported by Travan et al.5 Supercritical Carbon Dioxide (scCO2) Experiments. Samples were exposed to scCO2 under controlled conditions (temperature, pressure, and time) in a 100 mL stainless steel reactor (NWA, Lörrach, Germany). The high pressure vessel was disinfected with sodium hypochlorite prior to use. A standard CO2 cylinder was used and the gas was pressurized by a high-pressure syringe pump (NWA, PM-101) equipped with a EUROTHERM 2216E heating unit. The reactor temperature was controlled by a digital GTH 1150 thermometer (Greiser Electronic). The internal pressure of the reactor was controlled by a transducer (DS Europe LP632) connected to a

Hv = 1854.4 F /d2 Differential Scanning Calorimetry (DSC). The glass transition temperature of tTS samples exposed to scCO2 in different conditions was obtained from DSC analyses performed by means of a DSC QT100 apparatus (TA Instruments) according to ISO 11357 standard. Samples were heated from −50 to 250 °C at a heating rate of 10 °C/ min under a 50 mL/min nitrogen flow. High Performance Liquid Chromatography (HPLC). Residual monomer analysis was performed on lTS (light polymerized and postcured, respectively) prior to and after scCO2 terminal sterilization (27 MPa, 40 °C, 4 h, 200 ppm H2O2). Samples (0.5 to 1 g of thermoset) were immersed in 5 mL of tetrahydrofuran for 72 h under continuous stirring. After filtration and evaporation of tetrahydrofuran, the solid residue was dissolved in 10 mL of methanol. The filtered solutions were then injected into a 1090 HPLC series II Liquid Chromatograph (Agilent Technologies) equipped with a Synergi 4u 1153


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Figure 1. (a) Exposure time dependence of the flexural modulus (Ef, ■, left-y scale), and of the flexural strength at break (σf, □, internal right-y scale) for tTS-FRC bars and glass transition temperature (Tg, ●, external right-y scale) for tTS. Conditions: p = 20 MPa, T = 40 °C. The graph reports the mean ± s.d. for both Ef and σf (n = 10). (b) Flexural modulus (Ef, dense bars, left-y scale) and flexural strength at break (σf, open bars, right-y scale) for lTS-FRC bars prior to and after treatment with scCO2 (p = 20 MPa, 40 °C, 4 h). The graph reports the means value ± s.d. (n = 10). Hydro-RP 80 Å column (Phenomenex, 250 × 4.60 mm). The mobile phase was acetonitrile (Lichrosolv hypergrade for LC-MS, Merck) and deionized water in a 55/45 ratio and a 1.0 mL/min flux was maintained. A total of 50 μL of the extract from the lTS disks were injected and the unreacted monomers were detected with a UV detector operating at 227 nm. BisGMA and TEGDMA residual monomers were identified on the chromatogram and quantified by means of a calibration curve. Standards were dissolved in a mixture of water/methanol (90:10). The peak identification was performed with a HPLC-UV system (Thermo-Finnigan) equipped with a Synergi 4u Hydro-RP 80 Å column (Phenomenex, 250 × 2.00 mm) coupled with a ion trap mass spectrometer DecaXP (Thermo-Finnigan) using the same mobile phase. Bacterial Inactivation by scCO2. A midlog phase culture in Mueller-Hinton (MH) broth of S. aureus (ATCC 25923) was pelleted and resuspended in phosphate buffer saline (PBS) at an optical density of 0.5 (∼1 × 108 CFU/mL). lTS thermoset disks were dipped into the bacterial suspension and incubated for 3 h at 37 °C under stirring and, at the end of the incubation time, treated with scCO2 (20 MPa, 4 h, 40 °C). After the treatment, the coated thermoset disks were placed in a high salt solution (10 mM Na-phosphate, 400 mM NaCl, 10 mM MgCl2) to favor the detachment of bacterial cells from the disk, and vortexed vigorously for 30 s. The bacterial suspensions were serially diluted in PBS and plated on MH agar to allow the viable colony counts (CFU) after overnight incubation at 37 °C. Thermoset samples without scCO2 treatment were used as a control. scCO2 treated and untreated lTS disks were then placed in MH broth and incubated overnight at 37 °C to verify the presence of viable bacterial cells. The values reported are the mean ± standard deviation of three independent experiments. Spore Inactivation by Means of scCO2. Four spore strips (106 CFU/strip, Raven Laboratories) loaded with Geobacillus stearothermophilus (ATCC 7953) were placed in the vessel and treated with scCO2 (27 MPa, 40 °C) for different time intervals and with different additive (H2O2) concentrations. At the end of the CO2 treatment, the processed strips were aseptically transferred into tubes containing tryptic soy broth with bromocresol purple indicator and incubated at 55−60 °C for 7 days as reported by the manufacturer protocol. The tubes were observed daily for cell growth, which is detected as a change in medium color (red-yellow change) and turbidity. Antimicrobial Efficacy Test on Silver-Coated lTS Thermoset. The antimicrobial efficacy was evaluated by means of a slightly modified protocol of the Japanese Industrial Standard method (JIS Z 2801:2000). A midlog phase culture in Mueller Hinton (MH) of S. aureus ATCC25923 was centrifuged at 1000g for 5 min and resuspended in PBS (final concentration: 1−5 × 108 CFU/mL). A total of 10 μL of bacterial suspension was deposited on chitlac-nAg-

coated samples terminally sterilized (27 MPa, 40 °C, 4 h, 200 ppm H2O2) using untreated coated samples and uncoated samples as controls. Samples were covered with UV sterilized plastic sheets (14 × 14 mm; from Wako Pure Chemical Industries, Ltd., Cat. No. 16008893). The “sandwich” was incubated for 3 h at 37 °C at saturation humidity. At the end of incubation, the samples were immersed in 2 mL of high salt solution (10 mM Na-phosphate, 400 mM NaCl and 10 mM MgCl2) and vigorously vortexed for 30 s to allow the detachment of bacteria from the support. After proper serial dilutions in PBS, the bacterial suspensions were plated on MH agar and incubated overnight at 37 °C to allow the viable colony counts.

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RESULTS Effect of scCO2 on Light and Heat Polymerized Thermoset. The effect of CO2 in supercritical conditions on heat polymerized thermoset samples was evaluated from the variation of Tg with exposure time. Figure 1a shows that a pressure of 20 MPa did not induce detectable modifications in the glass transition temperature of the sample for treatment of up to four hours. The same conclusion holds when looking at the flexural modulus and flexural strength at break of tTS-FRC. In fact, given the standard deviation associated with the measurements, the tTS-FRC sample treated with scCO2 for 4 h (p = 20 MPa, 40 °C) does not show significant variations in both Ef, and σf (Figure 1a) with respect to the unmodified sample. The same analysis was performed on light polymerized and postcured composites (lTS-FRC) treated with scCO2 (20 MPa, 40 °C) for 4 h (Figure 1b). No variation of the mechanical properties was detected in this case either, emphasizing nonadverse effects of the supercritical fluid at these experimental conditions on the bulk properties of the resin-based composite. Screening of scCO2 Conditions for Bacterial Inactivation and Terminal Sterilization. S. aureus was loaded onto the surface of a lTS disk at a concentration of approximately 106 CFU/mL and the system was treated with scCO2 (20 MPa, 40 °C, 4 h; Figure 2). After the treatment, the bacterial viability was evaluated with respect to an untreated lTS disk. Although no additives were present in the scCO2 vessel, a 6-log decrease in bacterial population was verified. The result was also confirmed by the absence of any bacterial growth when the treated disk was immersed in cellular medium (Figure 2, inset). To assess the potential of CO2 in supercritical conditions for terminal sterilization, paper strips loaded with G. stearothermo1154


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terminal sterilization for a treatment as short as 90 min, while a further reduction to 60 min causes only the sterilization of some (but not all) strips. The condition of terminal sterilization, for a treatment time of 60 min, can be regained by doubling the concentration of the additive (i.e., 400 ppm). It must be noted that terminal sterilization of the spore strips can be achieved with only 30 min of scCO2 treatment when the concentration of H2O2 is increased to 600 ppm. The same result was obtained in the presence of an equal amount of hydrogen peroxide after a 2 h treatment at 40 °C, with the pressure in the vessel decreased to 16 MPa. This latter finding is in contrast with the results reported by Ellis et al. using B. subtilis as biological indicator.44 In contrast to previous reports,48 the use of 5% peracetic acid (200 ppm, 10 MPa, 1 h, 60 °C) did not lead to complete sterilization of the spore strips (not reported). This analysis also confirms previous results reported by the group of Matthews on the fundamental importance of the additives to accomplish terminal sterilization in a reasonable treatment time.42,43 Terminal Sterilization on lTS Thermoset and FRC Composite. Three conditions out of those reported in Figure 3, ensuring terminal sterilization, were selected to explore further their effect on light polymerized and postcured thermoset samples. In particular, the following three conditions were considered at p = 27 MPa and 40 °C: (A) 1 h, 60 μL (600 ppm) H2O2; (B) 2 h, 20 μL (200 ppm) H2O2; (C) 4 h, 20 μL (200 ppm) H2O2. The flexural modulus (Ef) and the flexural strength at break (σf) of lTS bars were measured prior to and after the terminal sterilization treatment (Figure 4a). It can be seen that the latter mechanical parameter is slightly affected by the scCO2 treatment. In fact, a decrease in σf of about 30% was found regardless of the conditions used. A different trend was found for the flexural modulus, Ef, where basically no differences were detected among the untreated thermosets and the lTS samples treated under the (A) and (B) conditions. However, when condition (C) was used, a reduction of approximately 20% in Ef was noticed for lTS. Considering that the highest variation in Ef and σf was found under (C) conditions, it was resolved to repeat the mechanical measurements in the presence of longitudinally oriented glass fibres inside the thermoset, that is, with lTS-FRC (Figure 4b). In this case, no significant modification in both Ef and σf was noticed, so stressing the fundamental contribution of the glass fibers used for reinforcement to the overall mechanical performance. Surface hardness (Hv) measurements were performed on light polymerized and postcured disks, that is, lTS disks (Figure 5a). It was found that Hv of the thermoset decreased upon increasing treatment time with scCO2, while no dependence on the concentration of the additive was detected. Surface hardness measurements were repeated on lTS disks containing S-glass braid fibres. It must be noted that, at variance with the results on the mechanical properties of lTS-FRC, the treatment with scCO2 under the most harsh conditions, namely, 27 MPa, 40 °C, 200 ppm H2O2 for 4 h, induced a reduction of its surface hardness (Figure 5b). The possibility of using scCO2 under terminal sterilization conditions to remove part of the unreacted monomers (BisGMA and TEGDMA) was explored by means of HPLC analyses (Figure 6a). The peaks pertaining to TEGDMA and BisGMA can be separated very efficiently, to allow for their quantitative determination. The commercial BisGMA gives rise to two distinct peaks in a 1:3 ratio which, by combining 13C

Figure 2. Viability of S. aureus plated on a lTS disk prior to and after the treatment with scCO2 (p = 20 MPa, 40 °C, 4 h). Each bar represents the average of two independent measurements. Inset: lTS thermoset disks with S. aureus (106 colonies) untreated (I) and treated with scCO2 (same conditions; II) immersed overnight in growth medium.

phylus spores were used. Four spore strips were placed in the CO2 reactor at different positions and, after the treatment, they were immersed in the growth medium containing phenol red. According to the manufacturer’s protocol, when the red color persists in the vial after 8 days at 55 °C and no turbidity is detected, it can be stated that the strip has been terminally sterilized. Terminal sterilization conditions were screened at 27 MPa, 40 °C as a function of the treatment time and of the amount of H2O2 in the reactor (Figure 3). It can be seen that,

Figure 3. Terminal sterilization of G. stearothermophylus spore strips as a function of the presence of H2O2 in the reactor vessel and of time exposure. Blue circles represent conditions ensuring sterilization of all the strips. Red circles represent conditions for which no strip was sterilized. Blue/red circles represent conditions in which only some of the strips were sterilized. Inset: Example of assessment of terminal sterilization. Comparison between spore strips untreated (A) and terminally sterilized (B) immersed in the growth medium containing phenol red for 8 days.

in the absence of additives, terminal sterilization cannot be accomplished. On the contrary, the addition of H2O2 at the final concentration of 200 ppm led to terminal sterilization of all the spore strips after a 4 h treatment. The amount of additive used is a fundamental parameter, as reducing its concentration to 50 ppm does not guarantee, in 4 h, the terminal sterilization according to the manufacturer’s protocol. The presence of H2O2 at 200 ppm is sufficient to achieve 1155


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Figure 4. (a) Flexural modulus (Ef, striped bars, right-y scale) and flexural strength at break (σf, open bars, left-y scale) of lTS bars prior to (untreated) and after scCO2 treatment for different times and amounts of H2O2 (conditions: p = 27 MPa, 40 °C). Values are reported as mean ± s.d. (n = 10). (b) Flexural modulus (Ef, striped bars, right-y scale) and flexural strength at break (σf, open bars, left-y scale) of lTS-FRC bars prior (untreated) and after scCO2 treatment (conditions: p = 27 MPa, 4 h, 40 °C, 20 μL H2O2). Values are reported as mean ± s.d. (n = 10). *p < 0.01 with respect to the untreated sample.

Figure 5. (a) Vickers hardness (Hv) measured on the surface for lTS prior to (untreated) and after scCO2 treatment for different times and amounts of additive (H2O2; conditions: p = 27 MPa, 40 °C). (b) Surface hardness expressed in Vickers for untreated and scCO2 treated lTS-FRC disks (p = 27 MPa, 4 h, 40 °C, 20 μL H2O2). Values are reported as mean ± s.d. (n = 10). *p < 0.01 with respect to the untreated sample.

thermoset did not increase in dimension after the terminal sterilization. Terminal Sterilization of lTS Disks Coated with a Bioactive System. Light-polymerized and postcured thermoset disks were coated with chitlac-bearing nanosilver particles (chitlac-nAg) according to a procedure previously reported5 and the antimicrobial properties of the coating were tested prior to and after the terminal sterilization (Figure 8). Noncoated lTS disk was used as control for which no antibacterial activity was detected. In agreement with previous results,5 the coating with chitlac-nAg led to a net reduction of approximately 5.5 log in the number of CFU. The chitlac-nAg coated lTS terminally sterilized with scCO2 (p = 27 MPa, 40 °C, 4 h, 200 ppm H2O2), still showed a very marked antibacterial activity toward S. aureus despite a slight decrease in the log reduction of CFU (approximately 4.5) with respect to the noncoated resin.

NMR and 135 DEPT data (not reported), can be ascribed to the presence of two different isomers (Figure 6a). The comparison was made between untreated and scCO2 treated thermoset disks, which were (i) light polymerized and (ii) light polymerized and postcured (Figure 6b). It can be seen that light-induced postcuring has a relevant effect on the amount of both unreacted monomers. In fact, the unreacted BisGMA monomer is reduced from 28.6 mg/gresin in the case of light polymerization to 8.9 mg/gresin after postcuring. Similarly, unreacted TEGDMA is reduced from 10.7 to 2.3 mg/gresin when going from light polymerization to postcuring. Focusing on Figure 6b, it can be seen that when only light polymerization is performed, the treatment with scCO2 under C) conditions leads to a reduction as high as 60.4 ± 15.5% for unreacted BisGMA and 53.9 ± 14.5% for unreacted TEGDMA, respectively. A decrease in unreacted monomer content for the thermoset was also detected for the postcured thermoset; in this case BisGMA and TEGDMA were reduced by approximately 41.6 + 17.2% and 57.1 + 16.9%, respectively, with respect to the untreated sample. The integrity of the lTS thermoset prior to and after terminal sterilization under C) conditions was assessed by means of μCT (Figure 7). No detectable dimensional variation of the resin bar was noticed nor was CO2 inducing bubble formation found. Moreover, the small bubbles present within the

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DISCUSSION Sterilization is a fundamental step of biomaterials development which often turns into a bottleneck. For the ability to engage specific receptors on cell surface, several bioactive biomaterials have been proposed in the recent years as innovative implants for tissue restoration/replacement. However, the drawbacks of the material connected with terminal sterilization always emerge and question the possibility that these very advanced 1156


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Figure 6. (a) HPLC chromatogram of coinjection of BisGMA (two isomers) and TEGDMA. (b) Amount of BisGMA (open bars, right-y scale) and TEGDMA (striped bars, left-y scale) in thermoset disks light polymerized and after postcuring. Values are reported as mean ± s.d. (n = 4). *p < 0.01 and **p < 0.05 with respect to the untreated sample.

Figure 7. μCT slice of the same lTS bar prior to (a) and after (b) the treatment with scCO2 (p = 27 MPa, 40 °C, 4 h, 200 ppm H2O2).

biomaterials will reach the market. As FDA regulations are very

recently emerged as a promising alternative to harsh treatments

strict on the requirements for terminal sterilization, very few

such as drying heating, autoclaving, and gamma ray. Indeed,

treatments are presently accepted. Supercritical CO2 has

supercritical fluid technology has been used on a commercial 1157


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onto paper strips. The relationship between treatment time and amount of additive used was explored. Figure 3 shows that the increase in the pressure of the vessel to 27 MPa is not sufficient for terminal sterilization to occur unless it is accompanied by the addition of H2O2. While the 4 h treatment in the presence of 200 ppm of hydrogen peroxide performed in the present work confirms previous reports by Matthews and co-workers,42 Figure 3 extends the time-additive amount relationship guaranteeing terminal sterilization. Indeed, the present results show that the exposure time to scCO2 for terminal sterilization can be cut to only 30 min with a 3-fold increase in the amount of the additive in the reaction vessel. This result is of particular importance as the net reduction of operating times brings about a notable reduction in the costs associated with the sterilization procedure. It should be mentioned that by increasing the hydrogen peroxide concentration in the vessel to 600 ppm, terminal sterilization can be obtained with an overall pressure as low as 16 MPa, which brings about a net reduction in operating costs. Hydrogen peroxide is expected to impact the overall performance of the thermoset to be used as biomaterial. This was assessed by examining the main mechanical properties and surface hardness of the thermoset materials. Figure 4 shows that the presence of H2O2 did in fact induce a slight decrease in the strength at break on the lTS which does not seem to be influenced by the amount of additive. On the other hand, the flexural modulus seems to be affected by H2O2 only when a concentration of 200 ppm is used for 4 h. These results might be explained by invoking some oxidative reaction on the resin induced by the additive. This is well sustained by the results on surface hardness decrease for lTS disks upon terminal sterilization in different conditions (Figure 5). In this case, there seemed to be a linear relationship between the hardness decrement and the treatment time (R2 = 0.986), while the amount of additive seemed to have a minor importance. However, additional data are needed for these analyses to be conclusive. It is interesting to note that the introduction of the glass fibers into the thermoset, besides the obvious increase in mechanical properties of the bars (Figure 4), reduced the adverse effects of terminal sterilization. In fact, Ef and σf are basically unmodified after the terminal sterilization even in the most harsh conditions (p = 27 MPa, 40 °C, 4 h, 200 ppm). However, this was not the case when the surface hardness was considered (Figure 5). This could be a consequence of the embedding of glass fibers within the composite thermoset disk (lTS-FRC disk); they do not protrude from the surface, the composition of which is then basically represented by pure thermoset. Therefore, in the composite the decrease of the surface hardness, which was induced by the oxidative degradation due to H2O2, is highly comparable to the one displayed by the light polymerized and postcured thermoset (Figure 5). One of the major drawbacks in the use of light cured BisGMA/TEGDMA materials for dental restoration is connected to the release of unreacted monomers, which have been reported to be cytotoxic, genotoxic, and mutagenic.55−58 This drawback still exists when these thermoset resins are proposed for orthopedic implants where contact with body fluids and tissues is unavoidable. In view of these considerations, scCO2 can play a dual role when used on the present thermoset materials. In fact, not only can an effective terminal sterilization, under proper conditions, be reached, but the

Figure 8. Viability of S. aureus seed onto unmodified lTS thermoset disks (control), chitlac-nAg coated lTS thermoset disks (nAg-NT), chitlac-nAg coated lTS thermoset disks treated with scCO2 (p = 27 MPa, 40 °C, 4 h, 200 ppm H2O2) (nAg-scCO2).

scale for extraction, fractionation, cleaning, and pasteurization purposes and, recently, for sterilization of allografts.48 Very recently, scCO2 has been proposed for the terminal sterilization of UHMWPE, proving that a 6-log reduction in the number of viable spores can be achieved without altering the physicalchemical properties of the material. In the present case, scCO2 was used for terminal sterilization of thermoset materials and composites obtained from thermal- and light-polymerized BisGMA and TEGDMA. One of the main concerns regarding the use of scCO2 is associated with its plasticizing effect, as already reported for different polymer-based materials.49−52 This might present some serious drawbacks when Tg is lowered below body temperature because of the rubber-like behavior the biomaterial might display upon implantation. Figure 1 shows that in the present case the Tg of the thermoset is not affected by the scCO2 treatment; this is probably a consequence of the high reticulation among the reactive residues BisGMA and TEGDMA within the sample51,53,54 which, in the case of the thermal-induced polymerization, is approximately 90%, as determined by FT-IR (not reported). The depressurization of the vessel, with the transition from supercritical fluid to gas phase, is another critical step for the thermoset sample, as cracks or bubble formation and propagation might alter its mechanical properties and limit its use for hard tissue replacement. However, this risk for thermoset composite is ruled out by Figure 1, which shows that, after a treatment with scCO2 at 20 MPa, 40 °C for 4 h, there is basically no variation in the flexural modulus and strength at break. This conclusion is sustained by the μCT slices recorded on the same thermoset bar prior to and after the scCO2 treatment, which did not show detectable variations on the thermoset (Figure 7). The 4 h treatment with scCO2 at 20 MPa and 40 °C led to a marked reduction in the number of viable cells of S. aureus seeded on the surface of the thermoset disks (Figure 2) and it can therefore be defined as an efficient sanification method. These results are in contrast with those of Dehghani and coworkers who have recently reported only a partial deactivation in the absence of H2O2.44 It is very likely that this discrepancy can be traced back to the higher pressure, exposure time, and temperature used in the present work. To achieve a terminal sterilization, the scCO2 treatment has to completely inactivate spores of G. stearothermophilus loaded 1158


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peculiar solvent properties of CO2 in supercritical conditions allow for an efficient removal of both unreacted BisGMA and TEGDMA from light polymerized and postcured materials (Figure 6). The reduction of about 60% for the two unreacted monomers present in the thermoset material, with the reduction in potential cytotoxicity, represents another main advantage of the terminal sterilization based on scCO2 over other sterilization techniques. Besides the overall mechanical performance of the lTS thermoset, it is important to focus on the biological interactions between the biomaterial surface and the biological environment. This is the very first event to take place and it is fundamental for biological tissue-biomaterial integration. In this sense, the coating of biomaterials with bioactive signals is constantly sought in order to improve its integration within surrounding tissues. However, for the limited stability of bioactive molecules normally used, the sterilization method could dramatically hamper the use of advanced biological techniques. We had previously developed a coating4 based on a lactose-modified chitosan (chitlac) with nanosilver, which endowed the thermoset with very good antibacterial properties.5 However, the terminal sterilization of these bioactive coatings raises a serious concern. Figure 8 shows that the terminal sterilization performed with scCO2 in the most harsh condition (p = 27 MPa, 40 °C, 4 h, 200 ppm H2O2) led to a limited decrease in antibacterial efficacy of the coating, but still endows the material with a very good activity against S. aureus. Additional work is needed to shed light on the rationale of the reduction of the antibacterial activity. It is probable that the presence of H2O2 induces some oxidation of the nanoparticles thus hampering, to some extent, their activity. In addition, the presence of scCO2 at high pressure might remove some of the more exposed layers of the coatings, thus, reducing the amount of available and exposed antimicrobial nanoparticles.

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel.: +39 040 558 3682. Fax: +39 040 558 3691. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the EU-FP6 Project “NEWBONE” (Contract No. 026279-2) and by the Italian Ministry of Education (PRIN2009 (2009FXT3WL) granted to S.P.). The financial support of the University of Trieste to I.D. (through the InterDepartment Center for Molecular Medicine, CIMM) is acknowledged. The authors thank Dr. Andrea Travan, Ph.D., for helpful discussions and Frederique Amrouni and Suzanne Cann for proofreading the manuscript.

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REFERENCES

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CONCLUSIONS

The present work explores the potential of scCO2 for the terminal sterilization of bioactive biomaterials. Conditions were selected to ensure a SAL of 10−6 for both active bacteria and spores. In the latter case, the need for additional additives was mandatory. Different conditions allowing for terminal sterilization were examined and a relationship between treatment time and amount of additive was preliminarily found. The results show that the use of supercritical CO2 for terminal sterilization induces a small variation in the mechanical properties and surface hardness of the BisGMA/TEGDMA thermoset and composite without affecting the structural integrity of the construct. Moreover, the solvent properties of scCO2 allow for the removal of unreacted cytotoxic monomers with a net gain in the overall biocompatibility of the thermoset material for its use in biomedical applications. It should be mentioned that a BisGMA/TEGDMA resin modified with a coating containing silver nanoparticles showed a very limited loss of antimicrobial properties upon terminal sterilization with scCO 2 proving the compatibility of these sterilization conditions with the use of bioactive materials. The present work should be regarded as the onset of an extensive screening of scCO2 in the sterilization of bioactive biomaterials. Specifically, further research is needed into the effect of the present terminal sterilization on growth factors and adhesion peptides and proteins. 1159


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