Electrospun Polyvinyl Alcohol/Nanodiamond ...

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Journal of Biomaterials and Tissue Engineering Vol. 4, 1–8, 2014

Electrospun Polyvinyl Alcohol/Nanodiamond Composite Scaffolds: Morphological, Structural, and Biological Analysis Amanee D. Salaam1 ∗ , Manoj Mishra3 , Elijah Nyairo4 , and Derrick Dean2 1

Department of Biomedical Engineering, University of Alabama at Birmingham (UAB), Birmingham, AL 35294, USA 2 Department of Materials Science and Engineering, University of Alabama at Birmingham (UAB), Birmingham, AL 35294, USA 3 Department of Biological Science, Alabama State University (ASU), Montgomery, AL, 36101, USA 4 Department of Physical Science, Alabama State University (ASU), Montgomery, AL 36101, USA

Keywords: Nanodiamond, Polyvinyl Alcohol, Electrospinning, Morphology, Cell Viability, Cell Adhesion.

1. INTRODUCTION Electrospinning has become one of the most widely used methods for fabricating polymeric fibers for biomedical applications such as drug delivery and tissue engineering.1–4 It is a straight forward technique to obtain fibers with repeatedly controlled dimensions— which are excellent for mimicking extracellular matrix (ECM) components.3 4 Electrospinning involves the use of a high voltage supply (typically DC), a syringe filled with polymer solution (or melt), and a grounded collector to create continuous fibers on the micro- or nanoscale. Over the last 10 years, electrospinning has been investigated using several natural and synthetic, biodegradable and permanent materials.5–9 Since electrospun nanofibers have a high surface area that permits for loading large amounts of drug, these nanofibers are being used for delivering numerous types of therapeutics.10–12 ∗

Author to whom correspondence should be addressed.

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Polyvinyl alcohol (PVA) has been used frequently in electrospinning and biocomposites, as it is a biologically compatible water soluble polymer.10 11 13 PVA is attractive for use in drug delivery because it is fully degradable in vivo and dissolves easily in water at around 80 C.14 15 Composite PVA electrospun nanofibers have been investigated previously for drug delivery.16 17 Drugloaded electrospun PVA nanofibers have displayed better release characteristics in comparison to drug-loaded films.11 PVA nanofibers have also been shown to control protein release under physiological conditions.17 In electrospun drug delivery systems, a huge issue involves mediation of therapeutics. A drug delivery vehicle may be required to improve the efficacy of drug treatment.18 19 Nanodiamond (ND), also referred to as ultradispersed diamond, has garnered attention as a potential drug delivery vehicle.20 21 ND is attractive for drug delivery because of its small particle size (5–10 nm), mechanical, chemical, and biological properties.22 23 ND particles have been shown to be uptaken by cells

2157-9083/2014/4/001/008

doi:10.1166/jbt.2014.1152

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Polyvinyl alcohol (PVA)/nanodiamond (ND) composite scaffolds loaded with 0.1 and 0.5 wt%ND were prepared by electrospinning. The effect of ND content on the morphology, structure, and properties of the scaffolds was characterized with scanning electron microscopy, transmission electron microscopy, differential scanning calorimetry, and mechanical testing. The viability of human mesenchymal stem cells hMSC 7043L, mouse pre-osteoblasts MC3T3-1, and osteosarcoma cells SAOS-2 after exposure to ND was investigated, and the attachment of hMSCs to the composite scaffolds was observed. Morphological analysis revealed uniform fibers with nanoscale diameters. Thermal analysis showed that the crystallinity of the scaffolds decreased as ND concentration increased, with tensile modulus and strength corroborating this trend. Cell viability was found to be concentration dependent as ND is significantly more toxic at 100 g · mL−1 than 5 g · mL−1 . Compared to neat PVA scaffolds, scaffolds loaded with 0.1%ND showed no significant difference in cell attachment or cell morphology suggesting their potential usefulness in biological applications.

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Electrospun PVA/ND Composite Scaffolds: Morphological, Structural, and Biological Analysis

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suggesting that ND in both non-conjugated and conjugated forms could serve as solid phase transmembrane carriers for small molecules such as vaccines and drugs.22 24 It has also been shown that the incorporation of ND in thin films sustains and regulates the release of doxorubicin hydrochloride, a chemotherapy drug used to treat many types of cancer.20 The eventual goal is to develop a composite PVAND nanofibrous matrix capable of regenerating bone tissue removed due to primary bone tumor resection while simultaneously delivering chemotherapy drugs to prevent cancer recurrence. In our system, ND is incorporated with intent to mediate drug release in future studies, but first characterization should be performed to understand the effects that ND will have on the overall properties of the system. To the best of our knowledge, only one study has been published on the development of electrospun polymer composites containing ND.25 In that study, polyacrylonitrile (PAN)-ND composite fibers were fused into thin transparent films with high mechanical properties for electrical applications. However, the structure-property relationships and biocompatibility of composite ND electrospun fibers have yet to be investigated. Thus, this study evaluates the effects of ND concentration on the fiber morphology, crystallinity, mechanical properties, cell viability, and cell attachment.

was performed. Three independent studies were conducted in triplicate; the resulting absorbance data were averaged and standard deviations were calculated (N = 9). A student’s t-test with alpha level of 0.05 was used to determine statistical significance.

2. MATERIALS AND METHODS

2.4. Morphological and Structural Characterization of the Scaffolds The morphology of the electrospun PVA and PVA-ND composite scaffolds was observed using a scanning electron microscope (SEM, JEOL-7000). Image J software was

2.1. Materials PVA (95% hydrolyzed) with a number average molecular weight of 95000 (Acros Organics, New Jersey, US) and ND hard gel containing approximately 20% water (NanoCarbon Research Institute Ltd., Osaka, Japan) were used throughout this study. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay (ATCC, Manassas,VA) and the fluorimetric ECM cell adhesion array kit (Chemicon, Temecula, CA) were used for biological analysis. 2.2. Cell Viability After Exposure to ND Human osteosarcoma cells (Saos-2), mouse pre-osteoblasts (MC3T3-E1), and human mesenchymal stem cells 7043L (hMSC) were exposed to various concentrations of ND. Cell viability after ND exposure was evaluated by MTT assay to determine acceptable concentrations of ND for composite scaffold fabrication. The cell lines were maintained in complete media comprised of Dulbecco’s Modified Eagle Medium (DMEM) with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). Briefly, cells were plated in triplicate (approximately 2 × 104 cells/well) in 48-well culture plates and incubated at 37 C and 5% CO2 overnight. On the experiment day, complete media was removed and replaced with media containing 0, 5, or 100 g · mL−1 ND. Cells were then incubated for 1, 2, or 7 days. After each incubation time, MTT assay 2

2.3. Scaffold Fabrication via Electrospinning Neat PVA solutions were prepared for electrospinning by adding 1 g of PVA to 10 g of distilled water (10 wt%). PVA solutions containing 0.1 and 0.5%ND were prepared by first sonicating the appropriate amount of ND in 10 g of distilled water for 30 minutes, and then adding 1 g of PVA. All solutions were magnetically stirred overnight and then heated to 80 C for 30 minutes using a water bath. The solutions were allowed to cool to room temperature before use. Scaffolds were fabricated via electrospinning the PVA and PVA-ND solutions at a high voltage of 15 kV (M826, Gamma High-Voltage Research, Ormond Beach, FL). A syringe pump (Small Parts, Inc., FL) was used to regulate the feeding rate of the polymer solutions to 0.2 mL · hr−1 . The resulting fibers were collected at ambient temperature on a static collector made of aluminum foil (about 10 × 10 cm) set 20 cm away from the tip of the needle and stored in a dessicator overnight to remove residual water.

Fig. 1. Schematic of dimensions for tensile test specimens.

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Fig. 3. Cell growth represented as fold increase in viable cells over 1, 3 and 7 days for hMSC 7048L, Saos-2, and MC3T3-E1 exposed to 0, 5, and 100 g · mL−1 ND.


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Fig. 2. Cell viability was determined with MTT assay after exposure to 0, 5, or 100 g · mL−1 ND for 1, 3, or 7 days. Three cell lines were investigated: (a) human mesenchymal stem cells, hMSC 7043L; (b) mouse pre-osteoblasts, MC3T3-E1; and (c) osteosarcoma, SAOS-2. ∗ Indicates a significant difference in absorbance using student’s t-test (p < 005).


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used to obtain 100 diameter measurements and generate a fiber diameter frequency distribution. The dispersion of the ND within the composite nanofibers was observed with transmission electron microscopy (TEM, FEI Tecnai T12), and ND aggregate sizes were determined with Image J. Differential scanning calorimetry (DSC, TA Instruments Q100) was used to monitor the structural behavior (i.e., melting temperature and enthalpy of fusion) of the composite scaffolds. A temperature ramp from ambient temperature to 300 C was scanned at a rate of 10.0 C · min−1 under nitrogen atmosphere. Crystallinity was determined by the fusion enthalpy method, which calculates the percent crystallinity by relating change of enthalpy for the experimental sample to that of 100% crystalline PVA (H = 1386 J/g).14

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The mechanical properties of the scaffolds were determined in accordance with ASTM International standard, ASTM D882, for tensile testing of thin film plastics. Specimens were cut from the scaffolds to tensile test dimensions as shown in Figure 1. A Minimat® mechanical testing system equipped with a 20 N load cell was used to strain specimens at a rate of 5 mm/min. The tensile moduli and tensile strengths were averaged for each set of specimens (N = 4), and standard deviations were calculated. 2.5. Analysis of Cell Attachment on Composite Scaffolds The attachment of hMSCs to the neat PVA, PVA + 0.1%ND, and PVA + 0.5%ND composite scaffolds was evaluated using a ECM cell adhesion array kit, which monitors expression of several ECM component proteins including collagen I, collagen II, collagen IV, fibronectin, vitronectin, tenascin, and laminin. hMSCs were seeded onto the scaffolds at approximately 1×106 cells·mL−1 and incubated for 2 hours at 37 C. The unbound cells were washed away, and bound cells were lysed and detected using fluorescence. Two independent studies were performed with each in duplicate (N = 4). The data were averaged and standard deviations were calculated. A student’s t-test was performed using an alpha level of 0.05. To observe cell morphology, light microscope images were acquired for cells on tissue culture plastic, neat PVA scaffolds, and electrospun composite scaffolds after incubation for 24 hours.

3. RESULTS AND DISCUSSION Cell viability was evaluated by MTT assay for hMSC, MC3T3-E1, and Saos-2 after exposure to 0, 5, and 100 g· mL−1 ND. Cell viability was found to be both time and dose dependent, as shown in Figure 2. Though there was no significant decrease in cell viability observed for any of the cell lines after one day of ND exposure, a significant decrease in cell viability was observed for all three

Fig. 4. Scanning electron microscope images of electrospun fibrous mats. (A) Neat PVA, (B) PVA + 0.1%ND, and (C) PVA + 0.5%ND. Images were taken at 1000× magnification using an accelerating voltage of 20 kV. Scale bar denotes 1 micron.

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Fig. 5. Frequency distribution of fiber diameter and average diameter of PVA nanofibers containing 0, 0.1, and 0.5% nanodiamond.


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Fig. 6. Transmission electron microscope images of PVA nanofibers containing 0.1%ND (A) and 0.5%ND (B). Red arrows indicate nanodiamond aggregates within electrospun nanofibers. Images were taken at 26000× magnification using an accelerating voltage of 80 kV.


Table I.

Summary of detonation nanodiamond aggregate sizes.

0.1%ND 0.5%ND

Average aggregate diameter (nm)

Standard deviation (nm)

Diameter range (nm)

39.0 93.4

16.5 82.6

14.8–101.2 21.6–358.9

was achieved. For the nanofibers containing 0.1%ND, the fiber diameter ranged from 128 to 588 nm; the average fiber diameter was 338 nm. With the 0.5%ND nanofibers, the diameter ranged from 128 to 390 nm, and the average fiber diameter was 253 nm. There was a trend observed of decreased fiber diameter with increased ND concentration. This was largely due to the chemical structure of ND which possesses a graphitic shell; this graphitic shell possibly increased the solution’s electrical conductivity, which directly led to decreased fiber diameter. A similar observation was reported for PAN/ND composite electrospun nanofibers.25 TEM revealed that the ND particles aggregated within the electrospun fibers despite the use of a hydrophilic polymer and aqueous solvent (Fig. 6). The aggregates were 0.8 As received PVA Neat PVA Nanofibers PVA+0.1%DND Nanofibers PVA+0.5%DND Nanofibers

0.6

Heat Flow (W/g)

0.4 0.2 0.0 –0.2 –0.4 –0.6 –0.8 –1.0 –1.2 50 Exo Up

100

150

Temperature (°C)

200

250 Universal V4.5A TA Instruments

Fig. 7. Differential scanning calorimetry thermograms for as received PVA, neat, and composite PVA-ND electrospun scaffolds.

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cell lines after 3 days of incubation with 100 g · mL−1 ND. For hMSC and MC3T3-E1 cell lines, there was no significant difference in cell viability after exposure to 5 g · mL−1 ND, but cell viability decreased significantly with exposure to 100 g · mL−1 ND (Figs. 2(a) and (b)). Dose dependent toxicity has been reported by a number of research groups when investigating ND and other carbonbased nanomaterials such as carbon nanotubes (CNT).26–28 When a toxic response is elicited due to carbon nanomaterials, the mechanism of cell death is typically related to oxidative stress.29 30 ND has shown no evidence of inducing cell membrane injury or intracellular oxidative stress.31 Furthermore, ND does not interfere with any known cellular processes,24 but the decrease in cell viability has been attributed to apoptosis.31 There was a preferential toxicity of SAOS-2 cells to ND, as both 5 and 100 g · mL−1 ND concentrations exhibited significantly lower cell viability after 3 days of exposure (Fig. 2(c)). Overall proliferation of hMSC and MC3T3-E1 cells was not inhibited by exposure to ND (Figs. 3(a) and (b)), but slight growth inhibition was observed in SAOS-2 cells between 3 and 7 days after incubation with 5 g · mL−1 and 100 g · mL−1 ND (Fig. 3(c)). Electrospun composite PVA-ND scaffolds were fabricated with 0.1 and 0.5% ND as these loadings were within the 5 g · mL−1 deemed acceptable in the cell viability studies. The SEM images displayed in Figure 4 show the morphology of the electrospun fibers. Neat PVA fibers were found to be uniform with a bead-free morphology (Fig. 4(a)). The fiber diameter was continuous in the nanoscale range, and scaffold porosity was interconnected. The frequency distribution of the fiber diameter was somewhat broad, ranging from 162 to 762 nm with the average being 361 nm (Fig. 5). There was no surface roughness observed on the composite polymer fibers indicating that most of the ND had been encapsulated within the fibers (Figs. 4(b) and (c)). The morphology of the composite scaffolds was also bead-free (Figs. 4(b) and (c)), but a narrower distribution of fiber diameters

Electrospun PVA/ND Composite Scaffolds: Morphological, Structural, and Biological Analysis Table II. Summary of DSC data for as received PVA, neat nanofibers, and composite nanofibers.

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As Received PVA PVA nanofibers PVA + 0.1%ND nanofibers PVA + 0.5%ND nanofibers

Melting temperature

Heat of fusion

Percent crystallinity (%)

201 210 205 207

61.3 35.1 30.0 16.3

44.2 25.3 21.6 11.7

encapsulated within the PVA nanofibers, but the aggregate size increased with ND concentration. As summarized in Table I, the aggregate size for 0.1%ND fibers, ranged from about 15 to 101 nm. The aggregate size in the 0.5%ND fibers ranged more broadly from about 22 to 359 nm. The increase in the average aggregate diameter was possibly due to the rich surface chemistry of the ND particles which led to particle-particle attraction. Though ND already possess carboxylic groups on their surface which help to deaggregate the particles when dispersed in various solvents, further oxidation by treating with hydrochloric acid could greatly improve dispersion. Differential scanning calorimetry was used to determine the thermal-structural behavior of the scaffolds. In the literature, the melting temperature (Tm ) and crystallinity determined by the fusion enthalpy for PVA were 230 C and ∼ 45%, respectively.14 32–34 The DSC thermograms for as received PVA, electrospun PVA nanofibers, and PVAND composite nanofibers are shown in Figure 7, and the thermal analysis findings are summarized in Table II. The thermographs for all electrospun nanofibers displayed two endotherms—the first around 150 C possibly due to the less ordered crystallitesand the second at approximately 210 C representing the true melting behavior. The disruption in the chain packing observed in the 150 C peak was presumably due to the rapid solvent evaporation and crystallization experienced during electrospinning. The Tm values observed were all somewhat lower than published values for electrospun PVA nanofibers, but similar to those reported by Yamamoto et al.35 The lower Tm and degrees of crystallinity could be attributed to using a high molecular weight polymer that is only 95% hydrolyzed. The percent crystallinity for the as received

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PVA powder was around 44%. However for the neat PVA nanofibers, the degree of crystallinity decreased to about 25%, which was acceptable for a partially hydrolyzed polymer.32 Crystallinity for the PVA + 0.1%ND fibers was comparable to that of the neat PVA fibers at ∼ 22%; yet, for PVA + 0.5%ND fibers the crystallinity decreased to ∼ 12%. In comparison to the as received PVA granules, the percent crystallinity first decreased due to the processing technique (i.e., electrospinning) and then further decreased as ND concentration increased. The decrease in crystallinity in relation to ND concentration was mainly due to the bulky particles disrupting the packing ability of the polymer chains into the crystal lattice. The average tensile modulus and strength for the neat PVA nanofibrous mats were found to be 84.6 MPa and 7 MPa, respectively, which are well within the published range for electrospun PVA.3436 The average tensile moduli (Fig. 8(a)) for PVA nanofibrous mats with 0.1% and 0.5%ND loadings were 51.2 and 32.4 MPa, respectively. The modulus of the composite scaffolds decreased in comparison to the neat scaffolds; a similar trend was observed for the crystallinity as a function of ND loading. It is well established that the crystallinity of a polymer directly relates to the mechanical properties exhibited by that polymer.37 In general, semi-crystalline and crystalline materials behave more stiffly, or have a higher modulus of elasticity; amorphous materials are usually tougher or stronger. Hence, the average tensile strengths (Fig. 8(b)) for the PVA nanofibrous mats with 0.1%ND and 0.5%ND were 8.6 and 9.7 MPa, respectively. Cell attachment to the ECM is needed to build a multicellular organism. Cell adhesion molecules (CAMs) bind cells to the surface providing critical signals to the cell about its surroundings. Expression of CAMs can be used to predict if cells will have proper adhesion and migration. hMSCs were seeded on the neat PVA and PVA-ND composite scaffolds, and their CAMs expression was quantified using a fluorimetric ECM cell adhesion array kit. The resulting expression of CAMs by hMSCs is displayed in Figure 9. There was a significant increase in expression of vitronectin and tenascin for the ND loaded scaffolds. This is interesting because vitronectin expression has been

Fig. 8. Summary of average tensile moduli (a) and tensile strengths (b) for mechanical testing performed on neat PVA, PVA + 0.1%ND, and PVA + 0.5%ND nanofibrous mats (N = 4).

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Fig. 9. Cell adhesion molecule expression after 2 hours of exposure to scaffolds. ∗ Indicates a significant difference when compared to PVA (p < 005).

shown during homeostasis38 and tenascin expression has been linked to bone formation.39 40 This also suggested that ND plays a role in promoting cell attachment, migration, and spreading of cells. Conversely, PVA scaffolds containing 0.5%ND had a significantly lower expression of collagen I, collagen II, and fibronectin. This implied that ND used in higher concentrations decreases adhesion properties. These adhesion properties could be boosted by

incorporating a natural polymer or ECM protein into the system to enhance bioactivity. Though the structure of ND differs from that of CNTs and carbon nanofibers, similar chemistry exists possibly leading to similar biological responses. A previous study from our group with polycaprolactone (PCL)-carbon nanofiber composite scaffolds showed that the inclusion of CNF did not appear to significantly affect cellular morphology of hMSCs.41 Similar to the results from that study, the morphology of hMSCs seeded on PVA-ND composite scaffolds was not significantly different from the morphology of cells seeded on tissue culture plastic (Fig. 10(A)) and neat PVA scaffolds (Fig. 10(B)). As shown in Figures 10(C) and (D), the cells attached to the composite scaffolds and adapted spread morphology. Cell attachment is vital for initiating other cell processes. Consequently, it can also be predicted that the cells will also have good proliferation and migration on the composite scaffolds. We observed that PVA scaffolds formed a fast dissolving hydrogel structure in vitro, which could potentially be an issue for supporting tissue growth. The current system could potentially be improved by controlling the degradation characteristics of the scaffold. One approach would be to impart varying degrees of crosslinking to the PVA. Another approach could be to blend the current system with another polymer that has slower degradation kinetics such as PCL.

RESEARCH ARTICLE Fig. 10. Comparison of cell morphology after seeding on scaffolds for 24 hours. (A) Cells only; (B) cells on PVA; (C) cells on PVA + 0.1%ND; and (D) cells on PVA + 0.5%ND.


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4. CONCLUSION

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This study focused on developing and characterizing PVAND composite scaffolds for use in biological applications. The effect of ND concentration on cell viability, morphological and structural properties, and cell attachment was evaluated. It was determined that 5 g · mL−1 ND does not significantly compromise cell viability of hMSC and MC3T3-E1 cell lines. Consequently, composite scaffolds were fabricated with ND content within the determined limit corresponding to 0.1 and 0.5%. Of the scaffolds tested in this study, PVA + 0.1%ND displayed optimal morphological, structural, and biological properties when characterized using SEM, TEM, DSC, tensile testing, and cell attachment assay. The PVA + 0.1%ND scaffolds consisted of a bead-free nanofibrous interconnected matrix with good dispersion of ND as the average aggregate size was small enough for drug delivery applications (i.e., < 100 nm). PVA + 0.1%ND scaffolds also showed no significant difference in hMSC cell attachment and morphology when compared to neat PVA scaffolds, though more work may be done to improve bioactivity such as incorporating natural polymers or bioactive molecules. Acknowledgments: The authors gratefully acknowledge Melissa Chemento of the UAB Electron Microscopy Core for assistance with the transmission electron microscope imaging and Yuan Yuan Ma for assistance with the cell viability studies. This work was completed with support from the National Science Foundation Graduate Research Fellowship Program.

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Received: xx xxxx xxxx. Accepted: xx xxxx xxxx.

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