Time-dependent evaluation of mechanical properties and in vitro cytocompatibility of experimental composite-based nerve guidance conduits

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

4 (2011) 1266–1274

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/jmbbm

Research paper

Time-dependent evaluation of mechanical properties and in vitro cytocompatibility of experimental composite-based nerve guidance conduits X.F. Zhang a , H. O’Shea a , S. Kehoe b,c,∗ , D. Boyd b,c,∗∗ a Cork Institute of Technology, Ireland b Department of Applied Oral Sciences, Dalhousie University, 5981 University Ave., Halifax, NS, B3H 4R2, Canada c School of Biomedical Engineering, Dalhousie University, Halifax, NS, B3H 4R2, Canada

A R T I C L E

I N F O

A B S T R A C T

Article history:

The use of nerve guidance conduits to repair peripheral nerve discontinuities has

Received 11 February 2011

attracted much attention from the biomaterials community, with many resorbable and

Received in revised form

non-resorbable materials in clinical use. However, a material with ideal biocompatibility,

11 April 2011

sufficient mechanical properties (to match that of the regenerating nerve) coupled with a

Accepted 15 April 2011

suitable degradation rate, has yet to be realized. Recently, potential solutions (composite

Published online 22 April 2011

nerve guidance conduits) which support the emerging philosophy of allowing synthetic materials to establish key interactions with cells in ways that encourage self-repair

Keywords:

(i.e. ionic mediators of repair such as those observed in hard tissue regeneration) have

Peripheral nerve regeneration

been proposed in the literature; such composites comprise specially designed bioactive

Nerve discontinuities

phosphate-free glasses embedded in degradable polymeric matrices. Whilst much research

Composite based nerve guidance

has focussed on the optimization of such composites, there is no published literature

conduits

on the performance of these experimental compositions under simulated physiological

Young’s modulus

conditions. To address this key limitation, this paper explores the time-dependent

Ultimate tensile stress (UTS)

variations in wet-state mechanical properties (tensile modulus and ultimate tensile

Cytocompatibility

strength) for NGC composites containing various compositions of PLGA (at 12.5, and 20 wt%), F127 (at 0, 2.5 and 5 wt%) and various loadings of Si–Na–Ca–Zn–Ce glass (at 0 and 20 wt%). It was observed that Young’s modulus and ultimate tensile strength of these composites were in the range 5–203 MPa and 1–7 MPa respectively, indicating comparable mechanical performance to clinical materials. Furthermore, an analysis of the cytocompatibility of experimental compositions showed comparable (in some instances R superior), compatibility when compared with the commercial product Neurolac⃝ . Based

on current synthetic devices and the demands of the indication, the CNGCs examined in this work offer appropriate mechanical properties and compatibility to warrant enhanced development. c 2011 Elsevier Ltd. All rights reserved. ⃝

∗ Corresponding author. Tel.: +1 902 494 1255. ∗∗ Corresponding author at: Department of Applied Oral Sciences, Dalhousie University, 5981 University Ave., Halifax, NS, B3H 4R2, Canada. Tel.: +1 902 494 6347. E-mail addresses: [email protected] (S. Kehoe), [email protected] (D. Boyd). c 2011 Elsevier Ltd. All rights reserved. 1751-6161/$ - see front matter ⃝ doi:10.1016/j.jmbbm.2011.04.013

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

1.

Introduction

Peripheral nerve injury (PNI) remains a challenging clinical problem that affects 2.8% of trauma patients annually (Belkas et al., 2004; Wang and Cai, 2010). Annually, over 360,000 cases of upper extremity paralytic syndromes are reported in the US with an additional 300,000 peripheral nerve injuries in Europe (Noble et al., 1998). Currently, this is estimated to result in over 8.5 million restricted activity days and almost 5 million bed/disability days (Belkas et al., 2004; Intiso et al., 2010). Over the past 20 years, much research has focussed on developing bioengineered nerve guidance conduits (NGCs) that have tailored properties and dimensions to meet the requirements of peripheral nerve regeneration (using either natural or synthetic materials). Research is ongoing to enhance clinical outcomes post-PNI and simplify the surgical procedure associated with large defects. Design specifications for an ideal NGC require that it be: noncytotoxic, highly permeable (yet prevents fibrous tissue infiltration and interactions between the myofibroblasts and axon growth), sufficiently flexible to allow functionality, and possess suitable degradation rates to provide guidance for regenerative axons. The microenvironment within such devices must favor peripheral nerve regeneration whilst concurrently minimizing swelling and inflammatory responses (Kehoe et al., in press-a). To date, 11 commercially available NGCs and nerve protectant wraps have been approved by the US Food and Drug Administration (FDA). A comprehensive review of the materials and efficacy for each of these devices has recently been published by the authors (Kehoe et al., in press-a). In brief, present state-ofthe-art materials have yielded excellent progress, however much research and development is still required to generate new approaches to combine all the design requirements into an ideal NGC construct. Specifically, a material with ideal biocompatibility, sufficient mechanical properties (to match that of the regenerating nerve) coupled with a suitable degradation rate, has yet to be realized. Composite materials combining bioresorbable polymers and bioactive glass phases have attracted recent interest in soft tissue engineering, as they offer exceptional potential in tailoring physical, biological and mechanical properties (Boccaccini and Maquet, 2003). Bioresorbable poly ∑ ( -caprolactone) (PCL), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) PLGA copolymers are attractive as matrix materials for scaffolds in tissue engineering (Maquet et al., 2004; Murphy et al., 2010). These polymers have demonstrated efficacy in clinical use as resorbable sutures, meshes, and in drug delivery systems (Chen et al., 2002), and are of interest in nerve repair (Bini et al., 2004; Keeley et al., 1991; Kiyotani et al., 1996; Luis et al., 2007). Of additional interest, Hench (2009) has R recently demonstrated that Bioglass⃝ bonds successfully to both hard and soft tissues. In addition, Bunting et al. (2005) have reported the growth of cultured rat Schwann cells R and fibroblasts onto Bioglass⃝ in vitro. Moreover, this group provided in vivo evidence of axonal regeneration through R a silastic NGC filled with Bioglass⃝ fibers. Consequently, the investigation of new bioactive glass compositions designed to mediate specific host responses in nerve regrowth applications is valuable in terms of promoting peripheral nerve regeneration and self-repair. The authors’

4 (2011) 1266–1274

1267

have reported evidence of controlled degradation rates in a novel phosphate-free Si–Na–Ca–Zn–Ce glass system (Zhang et al., 2011). The component ions have each been reported in the literature to have potential as suitable candidates for this indication. Calcium transients in growth cones in vivo are regarded as a major determinant of axonal extension rates and provide for growth cone guidance (Gallo and Letourneau, 1999; Gomez and Spitzer, 2000; Letourneau et al., 1994). There is also a growing awareness that zinc plays a role as a signaling substance through the body (Frederickson et al., 2005), in particular zinc ions are liberated to the synaptic space during normal neuronal activity. Evidence has also demonstrated the oxide of the rare earth element (REE) cerium as a biocompatible anti-oxidant, capable of protecting nerves from oxidative injury during regeneration (Das et al., 2007). In order to deploy these glasses within a NGC an appropriate matrix based on the ideal requirements of such devices is necessary. Recently, Oh et al. (2006) have developed PLGA/F127 composites for peripheral nerve repair, with the view point of increased F127 content reducing the hydrophobic nature of PLGA; thus enhancing its biocompatibility in vivo. Pluronic F127 (F127) is a poly(ethylene oxide)-poly(propylene oxide)poly(ethylene oxide) (PEO–PPO–PEO) triblock copolymer that can undergo sol–gel transition, depending on its concentration and ambient temperature. It has water-solubility and carries a low toxicity. Approved by the FDA for use in the human body, F127 has been found to significantly enhance the rate of wound and burn healing with several Pluronic-based formulations shown to effectively prevent postoperative adhesions or reduce adhesion area after surgery (Vlahos et al., 2001). The present work investigates the potential for further exploitation of the glass system developed by the authors to favorably alter PLGA (75:25)/F127-based NGC mechanical properties and in vitro cytocompatibility, the later being examined with respect to the commercially available R PCL-based NGC Neurolac⃝ (Polyganics B.V, Netherlands). Numerous data has been reported on the mechanical properties (static and dynamic, compressive and tensile) for various experimental NGC constructs derived from a wide array of polymeric biomaterials (Wang and Cai, 2010). This recent review reports tensile moduli of: 206–345 MPa (PCL); 70 ±31.1 MPa (PCL acrylate); 138 ± 17 MPa (PCL fumarate); ∼8 MPa (PLGA); 1.0 ± 0.4 MPa (PLLA) and 1.9–46 MPa (polyurethane (PU)). However, most reports in the literature only investigate the mechanical measurements of PLGAbased NGCs in a dry state at room temperature (Bender et al., 2004; Oh and Lee, 2007; She et al., 2008). To address this key limitation, the paper explores the time-dependent variations in wet-state mechanical properties (tensile modulus and ultimate tensile strength) for NGC composites containing various compositions of PLGA (at. 12.5, and 20 wt%), F127 (at. 0, 2.5 and 5 wt%) and various loadings of Si–Na–Ca–Zn–Ce glass (at. 0 and 20 wt%).

2.

Materials and methods

2.1.

Glass synthesis and characterization

A glass with composition (mol. fraction) 0.5SiO2 –0.2CaO–0.13 ZnO–0.14Na2 O–0.03CeO2 was synthesized. Analytical grade

1268

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

Table 1 – Compositions of composite nerve guidance conduits (CNGC). Deucalex composites (CNGC) CNGC-M CNGC-D CNGC-B CNGC-J CNGC-I CNGC-F

PLGA (wt%)

12.5 20 20 12.5 12.5 20

Pluronic F127 (wt%)

Bioactive glass (wt%)

2.5 5 0 5 0 2.5

20 20 20 0 0 0

reagents: silicon dioxide, calcium carbonate, zinc oxide, sodium carbonate and cerium oxide (Sigma Aldrich, Wicklow, Ireland) were weighed as appropriate and thoroughly mixed by shaking (30 min) in a plastic container. Each batch of powder was placed in platinum crucibles (50 mL), then fired (1520 ◦ C, 1 h) using Laboratory Chamber Furnaces (BRF 16/5, Elite Thermal Systems Ltd. UK) and shock quenched into water. The resulting glass frit was dried in an oven (120 ◦ C, 1 day), ground and sieved (<45 µm aperture) to retrieve glass powder and stored in dry desiccators. X-ray Diffraction (XRD) analysis of the glass showed that the material was fully amorphous as previously reported (Zhang et al., 2011).

2.2.

Polymer solution preparation

PLGA with a lactic to glycolic acid mole ratio, 75:25 (Mw, 113 kDa; IV, 0.74 dL/g; Lot #: LP-443, Lakeshore Biomaterials, Birmingham, AL, USA) was dissolved in tetraglycol T3396, (Sigma Aldrich, Wicklow, Ireland) using an airtight container with a volume of 100 mL at 60 ◦ C (at. 5, 12.5 and 20% w/w) in a waterbath to ensure full dissolution occurred. Dissolution occurred overnight. Pluronic F127 (P2443) was used as obtained from Sigma Aldrich (Wicklow, Ireland).

2.3. Fabrication of composite nerve guidance conduits (CNGC) The CNGCs examined in this research paper were previously identified as candidate compositions for peripheral nerve regeneration based on a preliminary DOE analysis, which evaluated (in a dry state) a Box–Behnken design matrix of 13 compositions; identification of suitable candidates was based on mechanical strength (UTS), ease of processability, and retention of structural stability (Kehoe et al., 2011). For clarity, the alphabetical identification of each compositional variation used in previous literature has been retained for this research paper. Six CNGC compositions (Table 1) were prepared for this study based on a modified immersion precipitation technique described previously (Kehoe et al., 2011). The wt% of the F127 phase is relative to the PLGA content and not the overall composite. The wt% of the glass phase is relative to the PLGA/F127 composite content. Of the thirteen compositional variants investigated in this study, 6 were found to have structural integrity to match natural peripheral nerves (ref); whereas compositions containing 5 wt% PLGA and 40 wt% glass did not. In brief, variable concentrations of F127 (0%–5% w/w, PLGA base) gel were incorporated into prepared (12.5%–20% w/w)

4 (2011) 1266–1274

PLGA solutions at 60 ◦ C and allowed to dissolve. F127 was used as a hydrophilic additive to PLGA, with tetraglycol (glycofurol) used as a nontoxic co-solvent for PLGA and F127. A given amount of glass (0%–20% w/w) powder was subsequently dispersed into the polymer solution using ultrasonification (Model UCB-30, Spectrolab Instrument Pvt. Ltd) for 60 min. Calcium alginate hydrogels (rod-shaped) were fabricated by the injection of 4% (w/w) sodium alginate, NaC6 H7 O6 , (medium viscosity: W201502: Sigma Aldrich, Wicklow, Ireland) into 2% (w/w) calcium chloride, CaCl2 , solution (C4901: Sigma Aldrich, Wicklow, Ireland) using a stainless steel 316 syringe needle, pipetting blunt 90◦ tipTM (Sigma Aldrich, Wicklow, Ireland) of gauge size, 14. The diameter obtained for the prepared calcium alginate hydrogel rods was controlled via the injection process: using different needle gauge sizes in order to obtain the desired inner diameter of the resulting NGC. The prepared calcium alginate hydrogel rod (diameter, ∼1.5 mm) with water saturation was immersed into the PLGA/F127/glass mixture solution (10 mL) for a period of 3 min at room temperature. The coating thickness (that is, the CNGC wall thickness) can be increased with a subsequent increase in the calcium alginate hydrogel immersion time in the PLGA/F127/glass solution. PLGA/F127/glass solution was precipitated onto the surface of the calcium alginate hydrogel rod by the diffusion of water molecules from the hydrogel rod into the PLGA/F127/glass solution (in tetraglycol co-solvent). After washing the PLGA/F127/glass coated calcium alginate hydrogel rod in excess water to remove any residual tetraglycol, the calcium alginate hydrogel was removed (slid out) from the outer PLGA/F127/glass thin walled tube to form the final PLGA/F127/glass CNGC. Following fabrication, the CNGC was submersed in deionized water and 25% (w/v) glycerine for 24 h in order to protect the tube walls during the post-drying process as per methodology outlined by Wen and Tresco (2006). The CNGCs were subsequently suspended in separate test tubes in the laminar flow hood to dry (48 h) for evaporation of any residual solvent under atmospheric pressure. The CNGCs were cut into segments (30 mm length × 1.5 mm inner diameter) using a surgical blade to avoid any compression of the thin walled membrane and stored under moisture-free conditions in desiccators (at. <10 ◦ C) for subsequent testing.

2.4.

Commercial control

R The commercial nerve guidance conduit Neurolac⃝ , Product No. NER-500-015T, Lot No. NGA2008090311 (Polyganics BV, Netherlands) was used as a control for cytocompatibility analysis.

2.5. Mechanical property study over time in wet-state conditions CNGC specimens (25 mm(±5 mm) length × 1.8(±0.2 mm) diameter) and Neurolac (30 mm × 1.5 mm) were incubated in 10 mL of phosphate buffered saline (PBS, Sigma Aldrich, Ireland) at 37 ◦ C for 1, 3, 7, 28 days (n = 3 for each material) under dynamic condition (frequency = 2 Hz) representative of the physiological condition (ISO 10993-14, 2009). After

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

incubation at each time point the uni-axial tensile strength and Young’s Modulus for each material was measured using an Instron 3345 test machine (10 mm min−1 ), test data was recorded using Bluehill Ver.2 software. The tensile test was performed on wet specimens at room temperature (Kehoe et al., in press-b). All sample dimensions were accurately measured using vernier calipers and the measured average dimensions recorded.

2.6.

Preparation of CNGC extracts

The CNGCs segments (30 mm length×1.5 mm inner diameter) prepared in Section 2.3 were submersed in 10 mL of sterile tissue culture water (Sigma Aldrich, Ireland) for 3, 7 and 28 day (n = 3) time periods. Each specimen was stored in polypropylene tubes maintained at 37 ◦ C in a shaking waterbath (Stuart Sb40, Reagecon, Shannon, Ireland), agitated at 2 Hz (longitudinal movement). After each time period, the CNGC specimens were removed and its filtrate (filtered through Grade 5 Whatman filter paper) retained and stored at 4 ◦ C; prior to in vitro evaluation.

4 (2011) 1266–1274

1269

MTT solubilization solution equal to original culture medium volume (1 mL) was added in each well of the plates to dissolve the resulting formazan crystal. Each well was mixed using a pipette in order to enhance dissolution of the crystals. The spectrophotometric absorption was measured (TriStar LB 941, Berthold Technologies, US) at a wavelength of 570 nm and the background absorption was recorded at a wavelength of 650 nm (Boyd et al., 2009; Murphy et al., 2010), then subtracted from the 570 nm measurement to get the accurate spectrophotometric absorption of the testing samples (Boyd et al., 2009; Murphy et al., 2010). Cell viability was calculated by comparison with the positive control (100%) using the following equation (ISO 10993-5, 2009): Cell Viability = 100∗ OD570e /OD570c

(1)

where: OD570e is the mean value of the measured optical density of the extracts of the experiment sample. OD570c is the mean value of the measured optical density of the positive control.

2.7.

In vitro cytotoxicity test

2.8.

2.7.1.

Cell culture of mouse fibroblast cell line L929

Each analysis was performed in triplicate and all data were expressed as means ± standard deviation (SD). All the measurements were analyzed using Prism Ver.5.03 software (GraphPad software Inc.) and carried out using Student’s t-test, with a significance level of P < 0.05. The mechanical property data were subdivided into four groups by their incubation periods of 1, 3, 7, and 28 days. The cell viability results were divided into two groups relative to their PLGA (12.5 wt%, 20 wt%) content.

The established mouse fibroblast cell line L929 (European Collection of Cell Cultures (ECACC), NCTC clone 929) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma Aldrich, Ireland) supplemented with 10% fetal calf serum (FCS, Sigma Aldrich, Ireland), 1% (2 mM) L-glutamine (Sigma Aldrich, Ireland), and 1% Tryptose Phosphate Broth TPB (Sigma Aldrich, Ireland). Cells were incubated in T-75 flasks (Sarstedt, Ireland) at 37 ◦ C in a 5% CO2 incubator, cell culture media was changed every 2–3 days. When the cells reached confluency (∼70%), they were detached using 0.25% trypsin EDTA Solution (Sigma Aldrich, Ireland), centrifuged and re-suspended in fresh culture media with an appropriate cell concentration (0.1 mL) of cells to new 75 mL flasks to create a new single cell suspension until desired passage was reached.

2.7.2.

Cell viability assay

L929 cells were used for 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cells (1 mL) were seeded at a density of 1 × 104 /mL in 24 well plates (Sarstedt, Ireland). Culture media was used as a negative control in row 1 and culture media plus cells used as a positive control in row 2. Plates were then incubated for 24 h in a cell culture incubator at 37 ◦ C (5% CO2 , 37 ◦ C, > 90%humidity) (Boyd et al., 2009). 24 h later, 100 µL of sterile tissue culture water was added to negative and positive control wells. 100 µL of experimental samples (Section 2.5) were added to appropriate wells for testing. Analysis of each extract (prepared as per Section 2.4) was performed in triplicate (n = 3 extracts per condition), with 3 cell viability analyses performed on each extract). The plate was then incubated for another 24 h in a cell culture incubator at 37 ◦ C (5% CO2 , 37 ◦ C, > 90% humidity) (Boyd et al., 2009; Murphy et al., 2010). Following a 24 h incubation period, an amount equal to 10% of culture medium (100 µL) of MTT was added in the each well of 24 plates. Plates were then returned to the incubator for 4 h. Subsequently, an amount of

Statistical analysis

3.

Results

3.1.

Ultimate tensile strength (UTS)

Fig. 1 compares the UTS for each CNGC at each discrete incubation period (1, 3, 7, and 28 days). The UTS for each CNGC composition after 1day incubation (Fig. 1(a)) is in the range of 1–4 MPa. No significant difference in UTS was observed between CNGC-M, D, B, and F. The observed UTS for CNGC-J and I was significantly less when compared with that of CNG-M and B. CNG-I exhibited the lowest mean UTS at 1MPa, significantly less than that of CNG-F (3 MPa). The UTS for each CNGC after the 3 days incubation (Fig. 1(b)) is in the range 3–7 MPa. CNGC-D exhibited the highest mean UTS at 7 MPa. The following significant differences are noted after 3 days incubation; The UTS of CNGC-M was significantly higher than the UTS of CNGC-B, J, and F. The UTS of CNGCD was significantly higher than CNGC-B, J, I, and F. The UTS of CNGC-I (4 MPa) was significantly higher than the UTS of CNGC-B (2 MPa) and J (3 MPa). The UTS for each CNGC after 7 days incubation (Fig. 1(c)) is in the range 5–7 MPa. No significant difference was noted between the UTS of the strongest CNGCs (c. 7 MPa): CNGCM, D, B, and F. The UTS of CNGC-I (5 MPa) was significantly lower than CNGC-M (7 MPa), and F (7 MPa). The UTS of CNGCJ (5 MPa) was significantly lower than UTS of CNGC-B (7 MPa).

1270

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

5 4 3 2 1 0

d 10 8 6 4 2 0

Ultimate Tensile Strength (MPa)

Ultimate Tensile Strength (MPa)

c

Ultimate Tensile Strength (MPa)

b Ultimate Tensile Strength (MPa)

a

4 (2011) 1266–1274

10 8 6 4 2 0

8 6 4 2 0

Fig. 1 – Bar graph comparison of UTS for each CNGC at each incubation period; (a) 1 day, (b) 3 days, (c) 7 days and (d) 28 days. (∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ p < 0.0005).

With the exception of CNGC-D, which showed peak UTS after 3 days incubation, each CNGC exhibited peak UTS after 7 days incubation. It was found that UTS of CNGC-M remained stable over all incubation periods; while the UTS of CNGC-D increased between 7 and 28 day incubation periods. The UTS of CNGC-I and J significantly increased from 1 to 3 days, and from 3 to 7 days. The UTS of CNGC-B declined between 1 and 3 days. The UTS of CNGC- F had increased significantly between 3 and 7 day incubation periods, subsequently decreasing between 7 and 28 days.

3.2. Fig. 2 – The wet-state UTS of each CNGC compared by the incubation periods of 1, 3, 7, 28 days. (∗ P < 0.05, ∗∗ P < 0.005).

The UTS for each CNGC after 28 days incubation (Fig. 1(d)) is in the range 3–6 MPa. The only significant different was between UTS of CNGC-M (6 MPa) and CNGC-I (3 MPa). Even though the mean UTS of CNGC-D (2.9 MPa) is smaller than I (3.3 MPa), there was no significant difference between the UTS of CNGCM and D due to the larger Standard deviation (SD) error bars in the UTS results of CNGC-D (2.9 ± 0.73 MPa) than the error in the UTS of CNGC-I (3.3 ± 0.21 MPa). Fig. 2 compares the UTS for individual CNGCs as it varies with respect to incubation period. Among the 6 CNGC conduits, M and F had the highest UTS results (about 7 MPa) after 7 day incubation period.

Young’s modulus

Fig. 3 compares Young’s Modulus (E) for each CNGC at each discrete incubation period (1, 3, 7, and 28 days). The value of E for each CNGC after 1day incubation (Fig. 3(a)) were in the range 5–103 MPa. Young’s Modulus of CNGC-I was significantly lower than each CNGC with the exception of CNGC-J (17 MPa). Young’s Modulus of CNGC-B (70 MPa) was significantly higher than each CNGC with the exception of CNGC-D. The value of E for each CNGC after 3 days incubation (Fig. 3(b)) were in the range 9–180 MPa. No significant difference was observed between Young’s Modulus of CNGCD (114 ± 12 MPa) and B (180 ± 58 MPa). Young’s Modulus of CNGC-D and B were significantly greater than those of the other CNGCs (M, J, I and F). Also of note is that Young’s Modulus of CNGC-J (9 ± 7.6 MPa) and I (23 ± 12.2 MPa) were significantly different after 3 days incubation. The value of E for each CNGC after 7 days incubation (Fig. 3(c)) were observed to be in the range 12–93 MPa. Young’s

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

b

150 Young's Modulus (MPa)

Young's Modulus (MPa)

a

100

50

0 M

D B J I 6 CNCC Compositions

250

M D B J I F

200 150 100 50 0

F

M

c

D B J I 6 CNCC Compositions

F

d 300

150

Young's Modulus (MPa)

Young's Modulus (MPa)

1271

4 (2011) 1266–1274

100

50

M D B

200

J I F

100

0

0 M

D B J I 6 CNCC Compositions

M

F

D B J I 6 CNCC Compositions

F

Fig. 3 – Bar graph comparison of Young’s modulus for each CNGC at incubation period; (a) 1 day, (b) 3 days, (c) 7 days and (d) 28 days. (∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ p < 0.0005).

Modulus for CNGC-M (70 MPa) was significantly greater than that of CNGC-J, I, and F. In addition, Young’s Modulus of CNGC-D (93 MPa), was also greater than that of CNGC-J, and I. CNGC-B also had a greater Young’s Modulus than CNGC-I. No significant difference between Young’s Modulus of CNGC-B (88 ± 42 MPa) and J (12 ±2 MPa). However, large variability between the SD of CNGC-B and SD of CNGC- J is noted. The value of E for each CNGC after 28 days incubation (Fig. 3(d)) were observed to be in the range 42–204 MPa. It was observed that CNGC-I showed a significantly reduced modulus (41.8 ±7.3 MPa) when compared with CNGC-B (203 ± 58.2 MPa). Fig. 4 compares the E for each CNGCs as it varies with respect to incubation period. Increased modulus was observed for CNGCs with 20% bioglass (M, D, and B) as compared with those containing 12.5% bioglass (J, I, and F). The only significant change in Young’s Modulus associated with incubation period was observed for CNGC-B between 7 and 28 days. Over all time periods, CNGC-I had the lowest Young’s Modulus of 5 MPa (1 day). CNGC-B had the highest Young’s Modulus of 204 MPa after 28 days.

3.3.

Cytotoxicity results

Cytotoxicity results are presented in Fig. 5 including a stateof-the-art commercial NGC Neurolac. Cell viabilities of > 85% were recorded for all NGCs.

4.

Discussion

As previously stated, the CNGCs examined in this research paper were previously identified as candidate compositions for peripheral nerve regeneration based on a preliminary DOE analysis, which evaluated (in a dry state) a Box–Behnken design matrix of 13 compositions; identification of suitable candidates was based on mechanical strength (UTS), ease of processability, and retention of structural stability (Kehoe et al., 2011). The UTS of the CNGCs examined in this study fell within the range of 1–7 MPa, which contrasts well with the limited literature available on the time-dependent properties of PLGA/F127 polymer conduits for nerve regeneration. Oh et al. (2006) reported that 10 wt% of PLGA augmented with 1–5 wt% of F127 the UTS was 0.05–0.08 kgf/mm2 (i.e. 0.5–0.8 MPa). The increased values of UTS associated with the experimental conduits presented in this work arise due to the increased PLGA content of the CNGC (12.5–20 wt%) and glass loading. In particular, CNGC-B, D, and M (20 wt% glass) generally demonstrated higher mean UTS that glass-free conduits. The exception to this observation occurred for CNGC-B after 3 days incubation where its strength was diminished with respect to CNGCJ and I; a result difficult to explain in light of the body of evidence available. It is important to note that while the glass

1272

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

Fig. 4 – The wet-state Young’s modulus of each CNGC conduits compared by the incubation periods of 1, 3, 7, 28 days. (∗ P < 0.05).

Fig. 5 – Cell viability of each CNGC compared with Neurolac. (∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ p < 0.0005).

loadings in this study were fixed, previous examinations of the composition–property relationships in these materials (in their dry state) has quantified the impact of glass content (0–40 wt%) on UTS using a surface response methodology and is available elsewhere in the literature (Kehoe et al., 2011). The data acquired illustrates that under simulated physiological conditions, control of the UTS of the CNGCs can be modulated in the range 1–7 MPa by modification of the concentrations of PLGA and glass component as outlined. It is also clear that the CNGCs are extremely sensitive to the physiological environment. Previous work by the authors has shown that post-synthesis (fully dried), and prior to exposure to aqueous media the CNGCs examined in this work exhibit values of UTS ten times greater than those incubated under physiological conditions; observations which are consistent with the literature. Wu et al. (2006), found that the mechanical properties of biomedical polyesters only retain c. 10 wt% of their ‘dry-state’ properties after exposure to aqueous media and whilst they are evaluated in a wet state. Fig. 3 illustrates Young’s Modulus of all the CNGCs, as expected glass filled composites demonstrated increased E as compared with unfilled materials (Oh et al., 2006). The overall range of E was between 5.5 and 203 MPa. With respect to the data pertaining to Modulus, for glass filled CNGC, it is observed that where significant difference arises (1 and 3 days); such changes are attributable to PLGA content (that is, CNGC- B, CNGC-D, Vs. CNGC-M). However this compositional difference appears to no longer provide for significant changes in strength at 7 and 28 days. With respect

4 (2011) 1266–1274

to the impact of F127 on the E of the CNGCs it is observed that in general F127 does not significantly impact on E for filled or for unfilled materials, with the exception of data collected at 3 days. Critically, and from a clinical standpoint and based on current synthetic devices which have demonstrable safety and efficacy in the indication considered, it is clear from the literature (based on predicate device properties and ex vivo data presented in Table 2) that the CNGCs examined in this work offer comparable mechanical properties to an approved NGC device over the time periods examined. The cell viability of all the CNGCs > 85% for each incubation period and compares well with previous literature which reports viabilities of PLGA scaffolds in the range of 86%–91% (Asti et al., 2010) over 1, 3, 14 day periods (cultured with stem cell lines) and 83%–101% for L929 mouse fibroblasts (Fernandez-Carballido et al., 2008). As is preferable for initial cytotoxicity evaluation, the established cell line L929 was selected from the recognized repositories (ISO 10993-5, 2009). The extraction assay was preferred; (a) to eliminate mechanical trauma of cells (associated with direct cell culture assays), (b) to avoid adverse affects of agar on diffusion (associated with agar diffusion assay) and evaluation of cytocompatibility. The cell viability of CNGC with 12.5 wt% PLGA and 20 wt% bioglass had an inhibitory effect at the start of incubation (1 day and 3 day incubation). This was possibly due to the acid degradation products of PLGA, which lead to the pH decrease in the test extract (Nyilas et al., 1983), and then the acidic product was balanced by the alkali product of bioglass (Zhang et al., 2011; Wu et al., 2009), thus the cell viability increased after 7 day incubation in the naturalized solution (Chang et al., 2007). This inhibitory phenomena also occurred for the cell viability of CNGC-I (12.5 wt% PLGA only), since there was no alkali of bioglass to balance the acidic solution, the inhibitory effect was still observed after 7 days incubation, and the cell viability recovered after that. A similar tendency have been observed by Wu et al. (2009), through proliferation of osteoblast-like cell culture over 1, 3, and 7 day incubation period. In addition, the investigation carried out by Lu et al. (2005) on the response of osteoblasts-like cell on polymer–bioactive glass composites over 1, 5, 7, 14 day have indicated the increase of ALP activity after 7 days for polymer/bioglass composites (10 wt% bioglass), and PLGA had increase ALP activity in 14 days incubation. The cell viability of CNGC-J (12.5 wt% PLGA, 5 wt% F127) was above 100% over all incubation periods, and there was no significant difference between each time periods. The addition F 127 in the CNGC-J could increase the hydrophilicity of the composition; the increased hydrophilicity could increase the interaction between cells and material for direct cell viability test (Oh et al., 2006; Zhang et al., 2004). Nevertheless, the MTT Assay in the research was carried out through extract, and the all cell viabilities recorded represent reasonable levels of cytocompatibility. The cell viability of group (b) (Fig. 5(b)) was comR pared with the cell viability of Neurolac⃝ at each incubation period, CNGC-D, B, and F had significantly higher cell viabilR ity than the cell viability of Neurolac⃝ after 3, 7, and 28 day incubation period respectively (Section 3.3), and there was no inhibitory effect for the CNGCs with 20 wt% PLGA. The potential reason might be the wt% of PLGA, which is a marked factor that it inhibit the cell viability at lower level (12.5 wt%) and

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

4 (2011) 1266–1274

1273

Table 2 – Mechanical property of existing copolymer for NCG application and predicted compositions material property of PLGA in vitro. Material

Incubation time (days)

Maximum strength (MPa)

Young’s Modulus, E (MPa)

Reference

PCL

7 14 28

3.4 (±0.5) 2.6 (±0.2) 2.2 (±0.3)

13.0 (±5.5) 9.9 (±2.8) 3.0 (±1.8)

Meek et al. (2004) Meek et al. (2004) Meek et al. (2004)

PLGA (80:20)

7 14 28

5 3.6 3.4

– – –

Zhao et al. (2010) Zhao et al. (2010) Zhao et al. (2010)

PLGA (70:30)

7 14 28

4.5 3.5 3.8

– – –

Zhao et al. (2010) Zhao et al. (2010) Zhao et al. (2010)

enhance cell viability at higher level (20 wt%). The cell viability of most of the CNGCs was above 100%; a result which must be taken with caution (Murphy et al., 2010) since this could arise from cell DNA damage leading to uncontrolled proliferation of mutated cell. However, the indirect MTT assay is an extrapolation to the in vivo situation, further test are required to monitor the activity undertaken in vivo. Conclusions. This paper has successfully characterized the wet-state, time-dependent mechanical properties (up to 28 days) of experimental CNGC based on (i) a unique, phosphate-free ion leachable glass comprising elements associated with enhanced peripheral nerve regeneration and protection, alongside (ii) a PLGA/F127 polymeric matrix, synthesized as tubular constructs. The data presented in this paper illustrates that the experimental CNGCs offer comparable mechanical properties, and cytocompatibility to conventional NGCs and as such offer significant potential for peripheral nerve repair. Following on from this study, it is now necessary to fully characterize the ion release profiles arising from each composition of CNGC, and correlate this data with enhanced biological evaluations. REFERENCES

Asti, A., Gastaldi, G., Dorati, R., Saino, E., Conti, B., Visai, L., Benazzo, F., 2010. Stem cells grown in osteogenic medium on PLGA, PLGA/HA, and titanium scaffolds for surgical applications. Bioinorganic Chemistry and Applications 831031. Belkas, J.S., Shoichett, M.S., Midha, R., 2004. Peripheral nerve regeneration through guidance tubes. Neurological Research 26 (2), 151–160. Bender, M.D., Bennett, J.M., Waddell, R.L., Doctor, J.S., Marra, K.G., 2004. Multi-channeled biodegradable polymer/cultispher composite nerve guides. Biomaterials 25 (7), 1269–1278. Bini, T., Gao, S., Xu, X., Wang, S., Ramakrishna, S., Leong, K., 2004. Peripheral nerve regeneration by microbraided poly (L-lactide-co-glycolide) biodegradable polymer fibers. Journal of Biomedical Materials Research Part A 68 (2), 286–295. Boccaccini, A.R., Maquet, V., 2003. Bioresorbable and bioactive R polymer/bioglass⃝ composites with tailored pore structure for tissue engineering applications. Composites Science and Technology 63 (16), 2417–2429. Boyd, D., Carroll, G., Towler, M.R., Freeman, C., Farthing, P., Brook, I.M., 2009. Preliminary investigation of novel bone graft substitutes based on strontium–calcium–zinc–silicate glasses. Journal of Materials Science: Materials in Medicine 20 (1), 413–420.

Bunting, S., Di Silvio, L., Deb, S., Hall, S., 2005. Bioresorbable glass fibres facilitate peripheral nerve regeneration. Journal of Hand Surgery 30 (3), 242–247. Chang, C.J., Hsu, S.H., Yen, H.J., Chang, H., Hsu, S.K., 2007. Effects of unidirectional permeability in asymmetric poly(DL-lactic acidco-glycolic acid) conduits on peripheral nerve regeneration: an in vitro and in vivo study. Journal of Biomedical Materials Research, Part B: Applied Biomaterials 83B (1), 206–215. Chen, G.P., Ushida, T., Tateishi, T., 2002. Scaffold design for tissue engineering. Macromolecular Bioscience 2 (2), 67–77. Das, M., Patil, S., Bhargava, N., Kang, J.F., Riedel, L.M., Seal, S., Hickman, J.J., 2007. Auto-catalytic ceria nano-particles offer neuroprotection to adult rat spinal cord neurons. Biomaterials 28 (10), 1918–1925. Fernandez-Carballido, A., Pastoriza, P., Barcia, E., Montejo, C., Negro, S., 2008. PLGA/PEG-derivative polymeric matrix for drug delivery system applications: characterization and cell viability studies. International Journal of Pharmaceutics 352 (1–2), 50–57. Frederickson, C.J., Koh, J.Y., Bush, A.I., 2005. The neurobiology of zinc in health and disease. Nature Reviews Neuroscience 6 (6), 449–462. Gallo, G., Letourneau, P.C., 1999. Axon guidance: a balance of signals sets axons on the right track. Current Biology 9 (13), R490–R492. Gomez, T.M., Spitzer, N.C., 2000. Regulation of growth cone behaviour by calcium: new dynamics to earlier perspectives. Journal of Neurobiology 44 (2), 174–183. Hench, L.L., 2009. Genetic design of bioactive glass. Journal of the European Ceramic Society 29 (7), 1257–1265. Intiso, D., Grimaldi, G., Russo, M., Maruzz, i G., Basciani, M., Fiore, P., Zarrelli, M., Di Rienzo, F., 2010. Functional outcome and health status of injured patients with peripheral nerve lesions. Injury 41 (5), 540–543. ISO 10993-5 (2009). Biological evaluation of medical devicespart 5: tests for in vitro cytotoxicity. Association for the Advancement of Medical Instrumentation/International Org. for Standardization. ISBN: 1570203555. ISO 10993-14 (2009). Biological evaluation of medical devices-part 14: identifiction and quantification of degradation products from ceramics. Association for the Advancement of Medical Instrumentation/International Org. for Standardization. ISBN: 9780580658259. Keeley, R., Nguyen, K., Stephanides, M., Padilla, J., Rosen, J., 1991. The artificial nerve graft: a comparison of blended elastomer-hydrogel with polyglycolic acid conduits. Journal of Reconstructive Microsurgery 7 (2), 93–100. Kehoe, S., Zhang, X.F., Boyd, D., 2011. FDA approved guidance conduits and wraps for peripheral nerve injury: a review of materials and efficacy. Injury (in press-a) doi:10.1016/j.injury.2010.12.030.

1274

J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S

Kehoe, S., Zhang, X.F., Lewis, L., Boyd, D., 2011. Time-dependent effects of physiological environment on the modulus of various PLGA/F127 nerve guidance conduits. Materials Letters, Article (in press-b). Kehoe, S., Zhang, X.F., Boyd, D., 2011. Composition-property relationships for an experimental composite nerve guidance conduit: evaluating cytotoxicity and initial tensile strength. Journal of Materials Science: Materials in Medicine 22 (4), 945. Kiyotani, T., Teramachi, M., Takimoto, Y., Nakamura, T., Shimizu, Y., Endo, K., 1996. Nerve regeneration across a 25-mm gap bridged by a polyglycolic acid-collagen tube: a histological and electrophysiological evaluation of regenerated nerves. Brain Research 740 (1–2), 66–74. Letourneau, P.C., Snow, D.M., Gomez, T.M., 1994. Growth cone motility: substratum-bound molecules, cytoplasmic [Ca2+ ] and Ca(2+ )-regulated proteins. Progress in Brain Research 102, 35–48. Lu, H.H., Tang, A., Oh, S.C., Spalazzi, J.P., Dionisio, K., 2005. Compositional effects on the formation of a calcium phosphate layer and the response of osteoblast-like cells on polymer-bioactive glass composites. Biomaterials 26 (32), 6323–6334. Luis, A.L., Rodrigues, J.M., Amado, S., Veloso, A.P., Armada-DaSilva, P.A.S., Raimondo, S., Fregnan, F., Ferreira, A.J., Lopes, M.A., Santos, J.D., Geuna, S., Varejão, A.S.P., Maurício, A.C., 2007. PLGA 90/10 and caprolactone biodegradable nerve guides for the reconstruction of the rat sciatic nerve. Microsurgery 27 (2), 125–137. Maquet, V., Boccaccini, A.R., Pravata, L., Notingher, I., Jérôme, R., R 2004. Porous poly ([alpha]-hydroxyacid)/bioglass⃝ composite scaffolds for bone tissue engineering. I: preparation and in vitro characterisation. Biomaterials 25 (18), 4185–4194. Meek, M.F., Jansen, K., Steendam, R., van Oeveren, W., van Wachem, P.B., van Luyn, M.J.A., 2004. In vitro degradation and biocompatibility of poly(DL-lactide- epsilon_caprolactone) nerve guides. Journal of Biomedical Materials Research Part A 68A (1), 43–51. Murphy, S., Wren, A.W., Towler, M.R., Boyd, D., 2010. The effect of ionic dissolution products of Ca–Sr–Na–Zn–Si bioactive glass on in vitro cytocompatibility. Journal of Materials Science: Materials in Medicine 21 (1), 2827–2834. Noble, J., Munro, C.A., Prasad, V.S.S.V., Midha, R., 1998. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. Journal of Trauma-Injury Infection and Critical Care 45 (1), 116–122. Nyilas, E., Chiu, T.H., Sidman, R.L., Henry, E.W., Brushart, T.M., Dikkes, P., Madison, R., 1983. Peripheral nerve repair with

4 (2011) 1266–1274

bioresorbable prosthesis. Transactions American Society for Artificial Internal Organs 29, 307–313. Oh, S.H., Kim, J.H., Kim, J.M., Lee, J.H., 2006. Asymmetrically porous PLGA/F127 membrane for effective guided bone regeneration. Journal of Biomaterials Science, Polymer Edition 17 (12), 1375–1387. Oh, S.H., Lee, J.H., 2007. Fabrication and characterization of hydrophyllized porous PLGA nerve guide conduits by a modified immersion precipitation method. Journal of Biomedical Materials Research Part A 80 (3), 530–538. She, Z., Zhang, B., Jin, C., Feng, Q., Xu, Y., 2008. Preparation andin vitro degradation of porous three-dimensional silk fibroin/chitosan scaffold. Polymer Degradation and Stability 93 (7), 1316–1322. Vlahos, A., Yu, P., Lucas, C.E., Ledgerwood, A.M., 2001. Effect of a composite membrane of chitosan and poloxamer gel on postoperative adhesive interactions. American Journal of Surgery 67 (1), 15–21. Wang, S., Cai, L., 2010. Polymers for fabricating nerve conduits. International Journal of Polymer Science doi:10.1155/2010/138686. Wen, X., Tresco, P.A., 2006. Fabrication and characterization of permeable degradable poly(dl-lactide-co-glycolide) (PLGA) hollow fiber phase inversion membranes for use as nerve tract guidance channels. Biomaterials 27 (20), 3800–3809. Wu, C., Ramaswamy, Y., Zhu, Y., Zheng, R., Appleyard, R., Howard, A., Zreiqat, H., 2009. The effect of mesoporous bioactive glass on the physiochemical, biological and drug- release properties of poly(DL-lactide-co-glycolide) films. Biomaterials 30 (12), 2199–2208. Wu, L., Zhang, J., Jing, D., Ding, J., 2006. “Wet-state” mechanical properties of three- dimensional polyester porous scaffolds. Journal of Biomedical Materials Research Part A 76A (2), 264–271. Zhang, X.F., Kehoe, S., Adhi, S.K., Ajithkumar, T.G., Moane, S., O’Shea, H., Boyd, D., 2011. Composition–structure–property (Zn2+ and Ca2+ ion release) evaluation of Si–Na–Ca–Zn–Ce glasses: potential components for nerve guidance conduits. Materials Science and Engineering: C 31 (3), 669–676. Zhang, R., Weng, W., Du, P., Zhao, G., Shen, G., Han, G., 2004. Effect of pluronic F127 on the pore structure of macrocellular biodegradable polylactide foams. Polymers for Advanced Technologies 15 (7), 425–430. Zhao, L., He, C., Cui, L., 2010. The influence of copolymer compositions on the physiochemical and biological properties of poly (lactic-co-glycolic acid) porous scaffolds. Journal of Biomimetics, Biomaterials, and Tissue Engineering 6, 35–44.