Materials Science and Engineering C 49 (2015) 632–639

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Fabrication and characterization of poly-(ε)-caprolactone and bioactive glass composites for tissue engineering applications Ali Mohammadkhah a,1, Laura M. Marquardt b,1, Shelly E. Sakiyama-Elbert b, Delbert E. Day a, Amy B. Harkins c,⁎ a b c

Graduate Center for Materials Research and Center for Biomedical Science and Engineering, Missouri University of Science and Technology, Rolla, MO 65409, USA Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO 63130, USA Department of Pharmacological and Physiological Science, Department of Biomedical Engineering, Saint Louis University, St. Louis, MO 63104, USA

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Article history: Received 1 October 2014 Received in revised form 20 December 2014 Accepted 14 January 2015 Available online 16 January 2015 Keywords: Borate glass Neurite extension Mechanical properties Degradation rate Polymer sheet

a b s t r a c t Much work has focused on developing synthetic materials that have tailored degradation profiles and physical properties that may prove useful in developing biomaterials for tissue engineering applications. In the present study, three different composite sheets consisting of biodegradable poly-ε-caprolactone (PCL) and varying types of bioactive glass were investigated. The three composites were composed of 50 wt.% PCL and (1) 50 wt.% 13–93 B3 borate glass particles, (2) 50 wt.% 45S5 silicate glass particles, or (3) a blend of 25 wt.% 13–93 B3 and 25 wt.% 45S5 glass particles. Degradation profiles determined for each composite showed the composite that contained only 13–93 B3 borate glass had a higher degradation rate compared to the composite containing only 45S5 silicate glass. Uniaxial tensile tests were performed on the composites to determine the effect of adding glass to the polymer on mechanical properties. The peak stress of all of the composites was lower than that of PCL alone, but 100% PCL had a higher stiffness when pre-reacted in cell media for 6 weeks, whereas composite sheets did not. Finally, to determine whether the composite sheets would maintain neuronal growth, dorsal root ganglia isolated from embryonic chicks were cultured on composite sheets, and neurite outgrowth was measured. The bioactive glass particles added to the composites showed no negative effects on neurite extension, and neurite extension increased on PCL:45S5 PCL:13–93 B3 when pre-reacted in media for 24 h. This work shows that composite sheets of PCL and bioactive glass particles provide a flexible biomaterial for neural tissue engineering applications. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Ideal nerve repair biomaterials should be biocompatible and noninflammatory, yet flexible with adequate tensile strength to prevent nerve compression [1]. The materials should be biodegradable with a porosity and permeability to supply adequate oxygen and nutrients [1]. Nerve autografts remain the gold standard in nerve repair and regeneration because of their performance. However, autografts require additional surgery, may cause donor site morbidity, and the loss of nerve function; all reasons that alternative materials in nerve repair and regeneration are needed. Bioresorbable synthetic natural polymers such as type I collagen (for example Neuragen®) have received attention over the last decade due to ease of production and controlled degradation. Type I collagen supports glial cell attachment, proliferation, unidirectional neurite extension, and axonal regeneration in vivo [2–6]. The disadvantages to using natural polymers for nerve conduits include poor mechanical ⁎ Corresponding author at: Department of Pharmacological and Physiological Science, 1402 S. Grand Blvd., Saint Louis University, St. Louis, MO 63104, USA. E-mail address: [email protected] (A.B. Harkins). 1 Equal first authorship.

http://dx.doi.org/10.1016/j.msec.2015.01.060 0928-4931/© 2015 Elsevier B.V. All rights reserved.

properties and batch-to-batch variability [7–9]. Resorbable synthetic polymers have been used extensively to repair peripheral nerves due to lower cost, simple fabrication, and proven efficacy [1]. Compared to an autograft, these synthetic materials eliminate the shortcomings of the autograft [1]. Because of their biodegradability, poly-ε-caprolactone (PCL) and its copolymers have been used for soft tissue regeneration applications [10] including peripheral nerves [11–14]. PCL slowly degrades in vivo and its degradation can take several years depending upon its molecular weight [15]. Furthermore, the degradation rate of PCL can be altered by polymerization with other polymers such as poly-lactic acid (PLA) [15]. Addition of inorganic materials such as bioactive glass to a biodegradable polymer improves the mechanical strength [10] and enhances the wetting properties of certain polymers [16–18], which can improve cell adhesion [16–19]. Lei et al. have shown that by adding 30 wt.% bioactive glass microspheres to PCL films, the elastic modulus increases by ~ 6 times and the contact angle decreases by ~ 50% compared to 100% PCL films [17,18]. Pre-osteoblast MC3T3-E1 cells were shown to spread more uniformly on a PCL/bioactive glass (BG) composite compared to a pure PCL film [17,18]. BGs are effective in biomedical applications that include bone repair [20] and peripheral nerve repair [21]. For example, Jeans et al. used a rigid glass tube made from a sodium calcium

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phosphate glass as a nerve conduit to repair facial nerves in sheep [21]. Starritt et al. used a controlled release glass wrap that was biocompatible, biodegradable, porous and flexible, to repair a facial nerve [22]. In the Starritt study, the sutureless method was compared to the standard microsurgical suture method to show that the glass did not cause any inflammation or prevent the nerve from regenerating. They concluded that the glass wrap was an acceptable alternative to a conventional suture technique [22]. In this study, we determined the physical and mechanical properties of the composite polymer/BG sheets and their ability to support nerve growth and survival. Here, we show that thin sheets of different BG/ PCL composition have different degradation characteristics. We report on the microstructure, mechanical properties, and biocompatibility of these composites with isolated neurons and whole dorsal root ganglion (DRG) neurons before and after degradation of the composites. 2. Materials and methods 2.1. Materials Neurobasal medium, B27, penicillin/streptomycin, fetal bovine serum, and 0.25% trypsin-EDTA, were purchased from Life Technologies (Carlsbad, CA). Triton X-100, Kaugh's F12 (F12K), HEPES, sodium hydroxide, and sodium bicarbonate were purchased from Sigma-Aldrich (St. Louis, MO). Bovine serum albumin was purchased from Millipore (Billerica, MA). Collagen type I was purchased from BD Biosciences. Nerve growth factor (NGF) was purchased from Peprotech (Rocky Hill, NJ). Fertilized white leghorn Specific Pathogen Free (SPF) eggs were purchased from Sunrise Farms (Catskill, NY). 2.2. Fabrication of bioactive glass/poly-(ε-caprolactone) composites To fabricate the BG composites, 1.14 g of PCL (molecular weight ~ 70,000 GPC Scientific Polymer Products Inc., Ontario, NY) was dissolved in 15 mL of chloroform (Sigma Aldrich, St. Louis, MO). The desired amount of glass particles was then added to the PCL/chloroform solution, stirred for 30 min and ultrasonicated several times at 1 min intervals. The 1393-B3 borate glass powder consisted of minus 20 μm particles with d90, d50 and d10 values of 13.37, 3.96 and 1.61 μm, respectively. The 45S5 silicate glass powder consisted of minus 20 μm particles with d90, d50 and d10 values of 12.40, 3.73, and 1.66 μm, respectively. The glass/PCL mixture was poured onto a polished glass plate and a film, 8 cm wide and 50 cm in length, of the mixture was tape-casted using a Dr. Blade set at a thickness of 600 μm. The composite film was dried at room temperature for 30 min, removed from the glass plate, and stored in a desiccator. The thickness of the dried films, measured with a micrometer at several locations, was 60 ± 10 μm. Hereafter, the surface of the film that contacted the glass plate is referred to as the “glass-side”; whereas, the (top) surface of the film that was in contact with the air is referred to as the “air-side.” The composition of the 13–93 B3 borate glass (in wt.%) is: 53 B2O3, 20 CaO, 12 K2O, 6 Na2O, 5 MgO, and 4 P2O5. The composition of the 45S5 silicate glass (in wt.%) is: 45 SiO2, 24.5 CaO, 24.5 Na2O, and 6 P2O. Irregular particles smaller than 20 μm in diameter of the 13–93 B3 and 45S5 glass were kindly provided by MO-SCI Corp (Rolla, MO) with the same wt.% composition. The following composites were fabricated: 50 wt.% PCL/50 wt.% 13–93 B3 (denoted as “B3”), 50 wt.% PCL/ 50 wt.% 45S5 (denoted as “45S5”), 50 wt.% PCL/25 wt.% B3/25 wt.% 45S5/(denoted as “blend”), and control 100 wt.% PCL (denoted as “PCL”). 2.3. Scanning electron microscopy Scanning electron microscopy (SEM) was performed on dried specimens coated with Au/Pd using a Field Emission Hitachi S-4700

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(Hitachi-Japan). All of the SEM was done with 5 kV accelerating voltage and 10 mA current. 2.4. Degradation of composites in simulated body fluid (SBF) Two samples, 3 cm × 3 cm, were cut from each composite film and placed in 50 mL of SBF composed of the following (in mM) 137.5 NaCl, 3 KCl, 2 MgCl2, 2.6 CaCl2, 4.2 NaHCO3, 1 K2HPO4, 0.5 Na2SO4, and 50.6 Tris–Cl, pH 7.4 at 37 °C [23]. After desired time intervals, the two samples were removed from the SBF, rinsed with distilled water and dried at 50 °C for 12 h. The weight loss of each sample was measured and the residual SBF solution was analyzed by inductive coupled plasma spectroscopy (ICP) for boron, silicon, calcium, and phosphorus. 2.5. Mechanical testing of composites Uniaxial tensile properties of each composite were measured with a MTS Criterion C42 machine (MTS Systems, USA) with a 100 N load cell and 10 mm/min extension rate. Dogbone shape samples were cut using a cutting die (2.75 mm at the narrowest point and 7.5 mm gage length; custom specimen die, ODC Tooling & Molds, Waterloo, Ontario). Elastic modulus, peak stress, peak load, and strain at break were calculated for each sample using TestWorks TW Elite software. Composites were tested unreacted (control), and reacted in neurobasal media (37 °C) for 3 and 6 weeks prior to experimentation. 2.6. Biocompatibility and neurite extension on BG/PCL composites Six millimeter diameter punches of composite sheets were used to evaluate neurite outgrowth on the BG/PCL composites that were either unreacted or pre-reacted for 24 h in cell culture media. Whole DRG were extracted from E11–12 leghorn chicks and placed on the air-side of the composite punches that were coated with Type VII (100 μg/mL) collagen. The whole DRG was incubated in neuronal growth media (neurobasal media supplemented with B27 and 50 ng/mL NGF [24]) at 37 °C in a 5% CO2 incubator. After 72 h, Calcein-AM, a “Live” stain (Life Technologies, Grand Island, NY) was added to assess overall health and visualize neurite extensions on the composite sheets. Fluorescent images were acquired of the neurite outgrowth and whole DRG with an inverted Olympus IX70 microscope at 2× objective with a CCD camera (Magnifire, Olympus). Average neurite extension was determined by subtracting the radius of the DRG body from the radius of the outermost DRG neurite extension measured by ImageJ software. 2.7. Statistical analysis Statistical analysis was performed using Statistica software (StatSoft, Tulsa, OK) with comparative ANOVA analysis and a Scheffé post hoc test with α b 0.05. Sample sizes were at least n = 6 for uniaxial tensile testing per condition. At least 18 whole DRGs were analyzed per condition in neurite extension studies. All graphs and values are displayed as mean ± standard error of the mean. 3. Results 3.1. Microstructure of BG/PCL composites The fabrication method of the glass/polymer composite sheet resulted in differences in the two surfaces, air-side and glass-side, of the glass/ polymer sheets. The air-side, or what was the original top surface of the composite sheet, was rougher to the touch than the glass-side or the original bottom surface of the composite sheet. While the cast film was still liquid, a portion of the suspended glass particles agglomerated, moved upward to the air-side of the film and formed islands of particles (Fig. 1A). The roughness of the air-side of the composite sheet is attributed to these islands of glass particles. Representative light microscope

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Fig. 1. Scanning electron and light micrographs of fabricated composite sheets. Optical images are displayed of air-side (A) and glass-side (B) of the B3 composite. After immersion in SBF for 14 days, the images show the changes in the air-side of 100% PCL (C), B3 (D), blend (E), and 45S5 (F) composites. Insets show regions at higher magnification to show the agglomerated glass islands.

images of the air-side and smoother glass-side of the B3 composite are shown in Fig. 1A and B, respectively. All BG/PCL composites had similar surfaces as shown for the B3 glass composite. Noticeable changes in the microstructure and morphology of the composites occurred after immersion in SBF media for 14 days. Both surfaces of all the bioactive glass-containing composites became rougher, and were covered with regions of submicron crystals of hydroxyapatite (HA). Using scanning electron microscopy, the 100% PCL sheet is shown in Fig. 1C, and is compared to that of B3 (Fig. 1D), the blend of

B3 and 45S5 (Fig. 1E), and 45S5 alone (Fig. 1F). The micrographs show that the glass particles agglomerate during the drying process (after the tape casting) on the air-side (top surface) to form islands of glass particles which are likely responsible for the rougher surfaces. After immersion in SBF for 14 days, the agglomerate islands at the air-side of the B3, blend, and 45S5 composites were nearly covered with regions composed of hydroxyapatite crystals (inset higher magnifications, Fig. 1D, E and F) that were identified by X-ray diffraction. The average size of the HA “microspheres” is smaller (3.5 ± 0.8 μm in

Fig. 2. Ultrastructure of composite sheets' cross-section. Scanning electron micrograph of the blend (25% B3, 25% 45S5, 50% PCL) composite is shown. The surface and cross-sectional view were imaged after immersion in SBF for 14 days (left). An expanded image of the section in the red box (right) shows the HA microspheres. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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diameter) for the 45S5 composite compared to the B3 composite (7 ± 1.8 μm). Cross-section scanning electron microscopy images show that roughly spherical shapes composed of HA crystals (Fig. 2A and B) were also visible within the cross section (interior) of the composite film. This image indicates that the BG/PCL composite sheets are permeable to the SBF solution since the bioactive B3 and 45S5 glass particles embedded in the PCL matrix had reacted to form biocompatible HA.

longer times. The P concentration in the composites decreased with time in the first 24 h, and gradually decreased to nearly zero for the 45S5 composite (Fig. 4D). This reduction in P concentration is consistent with the formation of HA which continues until either the Ca or P is consumed. Expanded time scales for the first 24 h for both Ca and P are shown in Fig. 4E and F, respectively.

3.2. Degradation and release profiles in SBF media

Uniaxial tensile testing was performed to assess the mechanical properties of the unreacted composites and compared to those reacted with media for 3 and 6 weeks. Peak stress, strain at break, and elastic modulus were calculated for all composite samples (Fig. 5). Compared to unreacted composites, peak stress was only significantly affected after 6 weeks in cell culture media for 100% PCL polymer sheets (Fig. 5A). PCL alone resulted in higher peak stresses than any of the BG composite sheets at any time point (Fig. 5A). The blend and 45S5 composites did not show any significant change in peak stress (Fig. 5A) or strain at break (Fig. 5B), perhaps due to the slower degradation profile of 45S5 bioactive glass (Fig. 5B). The elastic moduli, shown in Fig. 5C, also indicate stiffer properties of PCL and 45S5 composite sheets compared to the B3 composite sheets. As expected, the blend of 45S5 and B3 glasses yielded moduli between the B3 and 45S5 composite sheets. In addition, PCL and 45S5 composite trended toward stiffer moduli with increased time in media, though these results were not significant. Composites containing B3 did not vary in stiffness with incubation time.

Degradation of the 100% PCL and the three composites of BG/PCL in SBF was determined in two ways: weight loss measurements and analysis of the SBF solution with ICP for B, Si, Ca, and P, as a function of time. The weight loss degradation profile for each composite is shown in Fig. 3 for 14 days of SBF reaction. Weight loss for all three BG composites rapidly increased for the first 3 days and reached a maximum for the duration of the time period. The B3 composite had the largest weight loss, and weight loss for the 100% PCL film was negligible over the 14 day period. An ideal maximum weight loss for each composite was calculated by assuming that the measured weight loss is due to (i) all of the bioactive glass in the composite reacting to form stoichiometric HA and (ii) no weight loss is attributable to the degradation of PCL. An ideal weight loss calculated in this way is 32.5% for B3, 30% for the blend, and 28% for 45S5. The weight loss profile for the B3 composite was closest to the calculated percent weight loss compared to the calculated ideal loss. The concentration of B, Si, Ca, and P present in the SBF solution in which the composites or the 100% PCL (control) were immersed was measured (Fig. 4). The average concentration of B released from the B3 and blend composites reached its highest value in ~72 h (Fig. 4A). As expected, the maximum concentration of B for the blend composite is about half that of the B3 composite, as the blend contains 25 wt.% of the B3 glass composite. The 45S5 composite does not contain B. The concentration of Si ions (Fig. 4B) released from the 45S5 and blend composites plateaued at approximately 145 h. Most of the change in the Ca concentration occurred in the first 24 h, although the change (increase) is small compared to the nominal 100 ppm Ca concentration in the starting SBF media (Fig. 4C). The increase in Ca concentration for the B3 composite is attributed to the Ca released by the fast-reacting B3 glass, which temporarily increases the overall super-saturation of the SBF. Eventually, this leads to the precipitation of the insoluble HA material and accounts for the small decrease in Ca concentration in the SBF at

3.3. Mechanical property testing

3.4. Neurite extension Response of DRG neurons to the PCL and composite BG polymer sheets was determined by the length of neurite outgrowth from whole DRG cultured on each polymer material (Fig. 6). A representative image of a whole DRG is shown in Fig. 6A and demonstrates that the DRG survive and their neurites extend after 72 h in culture on the PCL or the composites. Addition of BG particles to PCL showed no inhibition in neurite extension, and in fact, significantly increased neurite extension when cultured on the 45S5 composites compared to PCL control sheets (Fig. 6B). Pre-reacted composite sheets cultured in media for 24 h prior to addition of whole DRG were also tested to determine whether reaction with cell culture media altered the glass properties to affect neurite outgrowth. In the pre-reacted case, only the B3 composites showed a significant increase in neurite outgrowth, reaching similar neurite lengths as unreacted 45S5 composite sheets. Although there was a trend for the 45S5 to decrease with pre-reacted sheets compared to unreacted sheets, there was no significant difference. Finally, in order to ensure that neurite outgrowth on the different composite sheets was not a result of change in pH due to the glass degradation, we examined the change in media pH caused by the glass composition degradation over the 72 h culture period. Despite the faster degradation of B3 composite sheets, little change in pH was observed when B3 was in culture media for 72 h compared to PCL alone (7.5 ± 0.05 compared 7.4, respectively). Composite sheets of 45S5 microparticles that were tested were ~1.5× larger than the PCL alone (or the B3 above). The 45S5 composites resulted in a somewhat larger pH change compared to the PCL (7.9 ± 0.06 vs. 7.4, respectively). Neuronal media was mixed to pH of 7.5 and 8.0, and used to grow whole DRG on tissue culture plastic without glass composites. There was no significant difference in neurite outgrowth between the pH media conditions and that of control pH of 7.4 (data not shown). 4. Discussion

Fig. 3. Degradation profiles for composite sheets. Weight loss for PCL-based bioactive glasses is shown over 14 days of immersion of the composites in SBF at 37 °C. The percent weight loss for B3 (■), blend (●), and 45S5 (♦) is shown compared to the 100% PCL (▲). The splines are for guidance only.

Development of tunable materials that can be used to provide ideal properties for tissue regeneration has been sought after for decades. From natural to synthetic materials, studies have focused on generating materials that provide both physical and biochemical support for tissue

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Fig. 4. Ion release profiles for composite sheets. Released concentration of B (A), Si (B), Ca (C) and P (D) is shown as a function of hours of immersion in SBF. The first 24 h of Ca (E) and P (F) is shown on an expanded time scale.

remodeling and regeneration [25]. Bioactive glasses have been shown to be a promising avenue for promoting tissue growth into bone defects [26], yet may have the potential for use in soft tissue applications such as nerve regeneration and angiogenesis [24,27]. Here we have investigated the development of composite bioactive glass polymer materials with varying degradation and ion release profiles. In addition, the physical and mechanical properties of the polymer composite sheets were examined along with the ability of the polymer sheets to support neurite growth in vitro. In order to develop a material that may be used to promote axon regeneration as a nerve guidance conduit, three bioactive glass polymer sheet composites were generated. The composite materials

were composed of 50:50 wt.% poly-(ε-caprolactone) (PCL) and either (1) 45S5 bioactive glass microparticles, (2) 13–93 B3 microparticles, or (3) a 25:25 blend of 45S5 and 13–93 B3 microparticles. PCL was chosen as the base polymer to form these composite sheets as it has been used extensively in nerve regeneration studies as a conduit material. Accordingly, a 100% PCL polymer sheet was used as a control material for all experiments reported here. While fabricating composite sheets, the glass particles were initially dispersed in the PCL/chloroform liquid and as it formed the solid composite, the glass microparticles agglomerated and formed islands on the air-side of the composite film/sheet. This agglomeration of glass particles could be due to an interaction between the hydrophobic PCL

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Fig. 5. Mechanical properties from tensile testing. Peak stress (A), strain at break (B) and elastic modulus (C) are shown for the composites unreacted (black bars), after pre-reaction for 3 weeks (gray bars) or 6 weeks in media (white bars). The peak stress and strain at break values for B3, blend, and 45S5 are significantly lower than the 100% PCL for each condition (*p b 0.05). The 6 week pre-reacted PCL is significantly higher in peak stress compared to the unreacted or the 3 week pre-reacted PCL (#p b 0.05). No other values of either peak stress or strain at break were significantly different within the composite types for the unreacted or pre-reacted conditions. In contrast, the elastic modulus for the B3 was significantly lower for each of the unreacted and pre-reacted conditions compared to PCL (*p b 0.05); and 45S5 was significantly higher than the B3 for each of the unreacted and pre-reacted conditions (#p b 0.05).

surface and hydrophilic glass particle surfaces [28] or to surface tension effects that carry the particles to the air/liquid interface during the drying process. In addition, the agglomeration of glass particles on the air-side of the composite sheets causes the surface to feel rougher compared to the glass-side. Even with prolonged reaction in SBF, up to 2 weeks, the islands were easily distinguished by touch on the air-side. The formation of the HA particles may be due to a nucleation and crystallization-like process, as opposed to a simple dissolution/ precipitation process seen with other calcium containing bioactive glasses immersed in phosphorus containing media, such as SBF or body fluids [29]. When the glass particles on the surface or inside the PCL polymer react in SBF, the glass dissolves and releases calcium, which reacts with the phosphorus in the SBF solution. This leads to the formation of small hydroxyapatite (HA) particles, which deposit on the surface of the PCL as well as on any undissolved/unreacted glass particles. Eventually, these clusters could grow large enough to combine with one another to form larger assemblies of HA assuming an adequate supply of Ca and P in the media. It is important to consider desirable degradation profiles that match the rate of healing and tissue regeneration when generating materials for specific applications. Unlike bone tissue engineering, soft tissue applications often require a material that provides an initial physical support structure, but one that will degrade in weeks or months to allow proper growth or healing of new tissues. For example, one of the reasons silicone or similar material conduits fail as nerve guidance conduits is their lack of degradation. As new tissues form, the continued presence of silicone prevents tissue growth and impedes regeneration due to compression [30,31,9,32]. Here, we studied the degradation properties

of PCL alone, as a commonly used material control, and PCL with bioactive borate glass particles, bioactive silicate glass particles, or a blend of both. The B3 composite, unlike the 45S5 and the blend composites, reached its ideal weight loss in ~3 days, thus all of the 13–93 B3 borate glass in the B3 composite had fully reacted to form HA in that time. On the other hand, the measured weight loss for the slower reacting 45S5 composite only reached about one half of its calculated ideal weight loss which indicates that only about one half of the 45S5 glass had reacted to form HA. The reaction rate of the 45S5 silicate glass in SBF is known to be much slower than the reaction rate of the 1393-B3 borate glass [33,34]. The measured weight loss for the blend composite, which contained 25% each of the B3 borate and 45S5 silicate glass, was closer, but still less than its ideal weight loss. This slowed degradation rate of the blend is attributed to the slower reacting 45S5 glass particles that had not fully reacted after 14 days in SBF. Mechanical testing of the PCL and composites indicated that addition of glass, and the type of glass, can significantly alter the tensile properties. The borate bioactive glass, when added to the PCL, significantly decreases the elastic modulus. However, addition of the silicate based (45S5) glass did not significantly alter the modulus. As expected, the blend of the two glasses resulted in mechanical properties that lay between the two types of the glass in the blend. The elastic modulus of the PCL sheets matched similar reported values, and while the addition of B3 glass altered the modulus, it still fell within the range reported for similar materials used in conduit applications (0.1–400 MPa) [9,35]. Previously reported composite sheets made with PCL and different silicate glass nanoparticles indicated a decrease in tensile strength and elongation at break from 100% PCL sheets similar to our findings [16].

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regeneration in an in vivo sciatic nerve injury model [36]. Thus as we have seen in vitro, perhaps the incorporation of glass particles into PCL sheets may improve the effectiveness of the material as a nerve guide conduit. Interestingly, however, when composite sheets were pre-reacted for 24 h prior to DRG seeding, the positive effect seen with 45S5 particles was decreased to normal levels while neurite extension increased when cultured on B3. Perhaps the conversion to HA leads to a more growth permissive substrate, either through improved adhesion or by some unknown mechanism. 5. Conclusions Overall, this study shows that the degradation rates, reaction with body-like fluids, mechanical properties, and permissive structure for neuronal outgrowth hold promise for potential use of bioactive B3 glass composites in nerve regeneration. Conflict of interest No author has a conflict of interest. Acknowledgments This work was funded in part by NSF DGE-1143954 (LMM) and a Saint Louis University Presidential Research Fund (ABH) and a grant from the Center for Biomedical Science and Engineering at the Missouri University of Science and Technology. The authors acknowledge the assistance of Dr. Scott Sell for use of his MTS Machine and critical reading of this manuscript. We thank Mr. Jason Papke for his research assistance.

Fig. 6. Whole DRG extends neurites on the composites. Images of whole DRG stained with Calcein AM show that the composites support outgrowth of neurites from the body of the DRG (A). Scale bar = 100 μm. The average neurite outgrowth on each of the composites for either unreacted or pre-reacted for 24 h shows that the whole DRG is able to grow and survive cultured on the composites for 72 h. The unreacted 45S5 composite exhibited significantly greater outgrowth than the outgrowth on any of the other unreacted PCL or polymer sheets (*p b 0.05). After pre-reaction for 24 h, the B3 polymer sheet exhibited significantly greater outgrowth than the blend composite (#p b 0.05), but not significant differences with the 100% PCL or the 45S5 composite.

However, this material also saw increase in elastic modulus with the addition of silicate glass particles indicating a more rigid material. Our silicate composite sheets saw little change in elastic modulus in 100% PCL and our B3 composite sheets indicated a decrease in elastic modulus. This decrease in elastic modulus and rigidity may be useful in designing nerve guidance conduits. For other tensile properties such as peak stress and strain at break, only the addition of glass caused a significant decrease, but not the type of glass. As described above, glass composites exposed to SBF led to conversion of the glass to HA, thus we were interested to determine how the pre-reaction of the glass in media could affect the mechanical properties. After 6 weeks pre-reacted in media, PCL and 45S5 sheets appeared to have a slight increase in elastic modulus, while B3 actually decreased, and the blend of the glass types did not change. Thus, it is likely that the faster degradation of the borate bioactive glass leads to a less elastic material. However, all 4 composites demonstrated a decrease in the strain at break as time in media increased, indicating that they became more brittle as they degraded. To evaluate the effect that addition of bioactive glass particles to PCL had on neurite extension, whole DRG outgrowth was studied on the composite sheets and compared to commonly used 100% PCL. In this study, none of the added glasses negatively affected neurite extension compared to PCL alone. In fact, the neurites growing on the 45S5 composite showed a significant increase in length. According to previous reports, the addition of 45S5 glass particles significantly improved

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Ali Mohammadkhah is a PhD graduate from Missouri University of Science and Technology. He obtained his Masters and PhD in Materials Science & Engineering. His research interests are the use of bioactive glass and composites in hard and soft tissue repair and regeneration.

Laura M. Marquardt is doctoral graduate from Washington University in St. Louis. She acquired her Bachelor's of Science degree in Biomedical Engineering at Saint Louis University. Her research interests include tissue engineering, regenerative medicine, and biomaterials. She is currently a postdoctoral fellow at Stanford University.

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Shelly E. Sakiyama-Elbert is a professor and Associate Chair of Biomedical Engineering at Washington University. She obtained a PhD at California Institute of Technology in Chemical Engineering. Her research focuses on developing biomaterials for drug delivery and stem cell transplantation to treat nerve injury. She has authored over 65 publications and 8 patents. She is a fellow of the Biomedical Engineering Society and the American Institute of Medical and Biological Engineering.

Delbert E. Day is Curators' Professor Emeritus of Materials Science and Engineering and Senior Investigator (formerly Director) of the Graduate Center for Materials Research at Missouri University of Science and Technology. He obtained his PhD in Glass Technology from the Pennsylvania State University. His research interests include bioactive glasses for hard and soft tissue repair, glass microspheres for drug and radiation therapy, and nuclear waste vitrification. He is a member of the National Academy of Engineering, a Distinguished Life Member and Fellow of the American Ceramic Society, has authored over 390 technical publications and holds 62 US and foreign patents.

Amy B. Harkins is an Associate Professor at Saint Louis University, with a primary appointment in the School of Medicine in the Department of Pharmacological and Physiological Science and a secondary appointment in Biomedical Engineering. She obtained her PhD at the University of Pennsylvania in Neuroscience, and continued her research as a postdoctoral fellow at the University of Chicago. Her interests include improving nerve regeneration using novel biomaterials as 3D scaffolds.

Fabrication and characterization of poly-(ε)-caprolactone and bioactive glass composites for tissue engineering applications.

Much work has focused on developing synthetic materials that have tailored degradation profiles and physical properties that may prove useful in devel...
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