Journal of Biomaterials Science, Polymer Edition
ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsp20
Facile fabrication of aloe vera containing PCL nanofibers for barrier membrane application Princeton Carter, Shekh M. Rahman & Narayan Bhattarai To cite this article: Princeton Carter, Shekh M. Rahman & Narayan Bhattarai (2016) Facile fabrication of aloe vera containing PCL nanofibers for barrier membrane application, Journal of Biomaterials Science, Polymer Edition, 27:7, 692-708, DOI: 10.1080/09205063.2016.1152857 To link to this article: http://dx.doi.org/10.1080/09205063.2016.1152857
Accepted author version posted online: 15 Feb 2016. Published online: 10 Mar 2016. Submit your article to this journal
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Date: 08 November 2017, At: 03:07
Journal of Biomaterials Science, Polymer Edition, 2016 VOL. 27, NO. 7, 692–708 http://dx.doi.org/10.1080/09205063.2016.1152857
Facile fabrication of aloe vera containing PCL nanofibers for barrier membrane application Princeton Carter, Shekh M. Rahman and Narayan Bhattarai
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Department of Chemical, Biological and Bioengineering, North Carolina A&T State University, Greensboro, NC USA
ABSTRACT
Guided tissue regeneration (GTR) is a widely used method in dental surgical procedures that utilizes a barrier membrane to exclude migration of epithelium and ensure repopulation of periodontal ligament cells at the sites having insufficient gingiva. Commercial GTR membranes are typically composed of synthetic polymers that have had mild clinical success mostly because of their lack of proper bioactivity and appropriate degradation profile. In this study, a natural polymer, aloe vera was blended with polycaprolactone (PCL) to create nanofibrous GTR membranes by electrospinning. Aloe vera has proven anti-inflammatory properties and enhances the regeneration of periodontium tissues. PCL, a synthetic polymer, is well known to produce miscible polyblends nanofibers with natural polymers. Nanofibrous membranes with varying composition of PCL to aloe vera were fabricated, and several physicochemical and biological properties, such as fiber morphology, wettability, chemical structure, mechanical strength, and cellular compatibility of the membranes were analyzed. PCL/aloe vera membranes with ratios from 100/00 to 70/30 showed good uniformity in fiber morphology and suitable mechanical properties, and retained the integrity of their fibrous structure in aqueous solutions. Experimental results, using cell viability assay and cell attachment observation, showed that the nanofibrous membranes support 3T3 cell viability and could be a potential candidate for GTR therapy.
ARTICLE HISTORY
Received 26 October 2015 Accepted 8 February 2016 KEYWORDS
Guided tissue regeneration; aloe vera; polycaprolectone; nanofiber; electropsinning
1. Introduction The Centers for Disease Control recently revealed that approximately half of all Americans aged 30 and over or 64 million people have some type of periodontitis. Periodontitis is an inflammatory disease that could lead to the destruction of oral tissues. Guided tissue regeneration (GTR) is a widely used method in dental surgical procedures that utilizes a barrier membrane to exclude migration of epithelium and ensure repopulation of periodontal ligament cells at the sites having insufficient gingiva for proper function. Similar to any other tissue engineering applications such as bone, cartilage, muscle, tendon, etc., an ideal scaffold
CONTACT Narayan Bhattarai © 2016 Taylor & Francis
[email protected]
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suitable for GTR membrane should have adequate biocompatibility, proper degradation profile, adequate mechanical and physical properties.[1] On top of these general characteristics, a GTR membrane should enable cell exclusion separating the gingival flap from the fibrin clot and guard space for the new alveolar bone and the periodontal ligament. Infection caused by either pathogen colonization at the wound site or foreign body response of the material is considered a major reason of GTR failure in clinical settings.[2] Antibacterial biomaterials are one of the greatest interests to resist implant-related infections.[3] Clinically used GTR membranes are composed of biodegradable and non-biodegradable polymers.[4] Polytetrafluoroethylene (PTFE), a synthetic polymer, is the most commonly used non-biodegradable material for GTR membranes.[1] Collagen, a protein from animal sources, is the most commonly used biodegradable material in the clinic for GTR membranes.[5] These membranes unfortunately have several drawbacks. The commercially available membranes composed of different forms of PTFE (e.g. high density and expanded PTFE) are non-biodegradable and require a secondary surgery for their removal. The secondary surgery could result in complications and additional costs. PTFE membranes also lack the chemical and physical features to improve regenerative outcomes. Hence, collagenous membranes offer the bioactivity lacking with synthetic materials, but they do not have sufficient mechanical integrity to maintain space throughout the regenerative process.[6] Synthetic biodegradable polymers, including polyglycolic acid, polylactic acid, and polycaprolactone (PCL) have also been used to make commercial GTR membranes. Membranes composed of these materials are generally biocompatible and easier to surgically place compared to PTFE membranes. These membranes, however, are not clinically useful because of the poor cellular response to them.[5] To rectify the issues observed with traditional GTR membranes, nanofibrous GTR membranes have been proposed. Nanofibrous membranes could maintain the defect space, allow nutrients and waste to enter and leave the defective sites, and positively influence cell behavior because they mimic the natural extracellular matrix (ECM). Electrospinning is the most prevalent manufacturing technique for nanofibrous membranes, because it is simple, reliable, and relatively inexpensive.[1] Natural material aloe vera has been shown to have antimicrobial activity toward gram-negative bacteria, which are the primary microbes implicated in the etiology of periodontitis.[7] Aloe vera also has anti-inflammatory properties,[8] which could mitigate the damage associated with the inflammatory disease, periodontitis. Acemannan sugar in aloe vera has been shown to encourage the regeneration of periodontium tissues (i.e. alveolar bone, periodontal ligament, and cementum). [9–11] Aloe vera is also found to be an effective decontaminating agent for gutta percha cones, a traditional dental filling.[12] PCL, a synthetic polymer, is well known for its use as a material for electrospun nanofibers. [13] This polymer is also useful because it has proven miscibility with a variety of other polymers to produce stable polyblends.[14] Finally, PCL can increase sufficient mechanical strength to the membrane and prevent degradation before the 4–6 weeks time period that is important for regeneration of periodontium tissues. Recently, a few attempts have been made to prepare aloe vera-incorporated PCL nanofibers by electrospinning technique with varying degrees of success.[15–17] Although these recent efforts in fabricating aloe vera-containing nanofibrous structures are encouraging, much remains to be explored and improved, particularly, in perspective of applications for GTR.
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In this work, a facile method of blending aloe vera with PCL in different ratios was first developed. Electrospinning technique was utilized to transform the blend solution into nanofibrous GTR membranes. Toward the potential use of these GTR membranes, their physicochemical properties such as morphology, mechanical strength, wettability, chemical structure, and integrity in aqueous medium were studied. Cellular compatibility of the nanofibrous membranes was studied by seeding 3T3 fibroblast cells on the fibrous membranes. Cell viability was quantified by alamarBlue assay, and cell attachment was characterized by scanning electron microscope (SEM).
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2. Materials and methods 2.1. Materials Samples of aloe vera were obtained commercially from Terry Laboratories (Melbourne, FL, USA, catalog number TN003). Polycaprolactone (PCL, Mn 80 kDa) was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2, 2, 2-Trifluoroethanol (TFE, 99 + %) was obtained from Alfa Aesar (Ward Hill, MA, USA). 2.2. Solution preparation for electrospinning Aloe vera was dissolved in DI water at a concentration of 10 wt%. The solution was vortexed for 5 min to ensure the aloe vera was completely dissolved. PCL solution was prepared by dissolving PCL pellets using 2-2-2-Trifluoroethanol (TFE) at a concentration of 10 wt%. When both the solutions were dissolved, a plastic syringe was used to remove the appropriate amount of each solution to generate the different ratios of PCL/aloe vera: 100/0, 90/10, 80/20, and 70/30. Each solution mixture was vortexed manually for 20 min. When the solution appeared to be well blended, the glass vial was immediately taken to the fume hood to be electrospun. 2.3. Electrospinning To fabricate the nanofibers with various polymer solutions of PCL/aloe vera (i.e. 100/0, 90/10, 80/20, and 70/30), the parameters of applied voltage and tip-to-collector distance were altered until fiber formation was observed. The Spellman CZE1000R (Hauppauge, NY, USA) high-voltage power supply was adjusted to a voltage of 25–30 kV depending on the solution, and the solution was poured into a static syringe held by a test tube holder. The stationary syringe tip was approximately 16–23 cm away from a plastic, cylindrical grounded collector wrapped with aluminum foil. 2.4. Fiber morphology observation and porosity measurement The fiber morphology after various time points was observed by (SEM) (Hitachi SU8000, Japan). Samples from each polymer ratio used were first gold sputter coated using the Polaron SEM coating System for 1 min and 30 s to prevent over coating of the samples. The samples were then loaded into the SEM chamber and imaged using accelerated voltage of 10 kV and current of 5 μA. Additional parameters were adjusted to improve the quality
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of the image at 500 and 10 K magnifications. All images were captured using a 40 or 80-s scanning process. The apparent density and porosity of the membranes were estimated according to the previously reported methods.[18] Briefly, the apparent density and porosity were estimated using Equations (1) and (2).
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( ) g Mass of membrane (g) Apparent density = (1) 3 cm Membrane thickness (cm) × Membrane area (cm2 )
( ) g Apparent density cm3 Porosity (%) = 1 − ( ) × 100 (%) g Bulk density cm3
(2)
2.5. Contact angle measurement
The wettability of electrospun fiber membranes was determined by contact angle (CA) measurement. The CA measurement was carried out using a specially arranged microscope equipped with a camera (Dynamic Contact Angle Tester, Billerica, MA). The DI water droplet (0.25 μL in volume) was used in the CA measurement. The CA experiments were carried out at room temperature and were repeated five times. All CAs were measured within 20 s of placement of the water droplet on the electrospun fiber mat. The wettability of nanofiber samples was analyzed by comparing the CAs of PCL/aloe vera nanofibers with PCL nanofibers (act as internal reference). Five samples of each ratio of nanofibers were used to measure CA. 2.6. Chemical structure analysis To characterize the chemical bonding between PCL and aloe vera, Fourier Transform Infrared Spectroscopy (FTIR) spectra were obtained using a Varian 670 spectrophotometer (Santa Clara, CA, USA). The data were collected between 4000 cm−1 and 400 cm−1. Four scans were used to collect the spectra. ATR or attenuate total reflectance FTIR was used, so that the fiber samples could be directly analyzed without further processing (i.e. making a KBr pellet). The powdered aloe vera, as provided from the manufacturer, was tested along with fiber samples from each time point of the degradation study. The IR spectra were used to confirm the absence or presence of aloe vera in the nanofibrous membranes. 2.7. Tensile mechanical testing Measurements were carried out according to previously published methods.[19,20] Fiber samples were cut to have a 5 mm width and L = 16 mm length. The thickness for all of the fiber meshes was measured using a Digimatic micrometer. Fiber samples were center aligned and mounted within a Lg (gauge length) = 6 mm window cut from a paper template and affixed using two-sided tape as shown in Figure 1. The top and bottom dimensions of the paper holder are also listed below. The additional paper allowed the sample to be held by the clamps of the Shimadzu machine (North America Analytical and Measuring Instruments AGS-X series obtained from Columbia, MD).
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Figure 1. Dimensions of paper holder used for tensile testing.
Trapezium Lite X (Shimadzu Corporation, Columbia, MD) software was used to collect the tensile data with the parameters of displacement rate set to V1 = 10 mm/min and data acquisition time to 100 ms for a 50 N load cell. The load (P) is the force applied to the sample. Additionally, the software was set to display data in time (seconds), force (Newtons), and stroke (millimeters). Before clamping the sample in the machine, the force was calibrated and zeroed. The sample holder was then centered and tightened between the bottom and top clamps. Both sides of the sample holder were cut to release the tension of the paper prior to initiating testing. The stroke was zeroed and then testing was started. After each test was completed, the exported raw data, which consisted of the load (P) on the sample and the displacement (d) of the sample, was manipulated in Excel. The strain was calculated by: strain = d∕Lg . and the stress was calculated by: Stress = P∕(cross sectional area). The load was given in Newtons (N) from the computer software. The cross sectional area was computed by multiplying the 5 mm width by the thickness of the samples as measured with the micrometer. These calculated data were then used to make stress-strain curves and to determine Young’s Moduli and ultimate tensile strengths for each of the fiber samples. (n = 5 for each treatment (70/30, 80/20, 90/10, and 100/0)). 2.8. In vitro degradation test Fiber samples from each ratio were cut into rectangular pieces placed in conical tubes and then submerged in phosphate-buffered saline (PBS) solution. The conical tubes were then placed in a water bath held at 37.5 °C. After time points of 0 day, 3 days, 1 week, and 3 weeks, the samples were removed from the PBS solutions, rinsed with DI water, patted dried, and then allowed to completely dry in the open air. After completely drying, each sample was probed using SEM.
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2.9. Culture of 3T3 fibroblast cells
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NIH/3T3 cells (a mouse fibroblast cell line) were purchased from the American Tissue Type Culture Collection (Manassas, VA). The growth medium was Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (10,000 units/mL of penicillin and 10,000 μg/mL of streptomycin). The cells were cultured in a 75 cm2 culture flask and maintained in a tissue culture incubator at 37 °C and 5% CO2 atmosphere. The culture medium was replaced every 2 days. After reaching about 80% confluence, the cells were detached by 0.025% trypsin and 0.01% EDTA in PBS solution and transferred to centrifuge tube containing culture medium. After centrifugation, the cells were resuspended in fresh culture medium and counted using a hemocytometer before seeding to nanofiber samples. 2.10. Cell viability study Nanofiber samples were attached to 12-mm square-diameter coverslips using biocompatible, silicone-based elastomeric glue (i.e. Kwik-Sil). Each fiber sample was first wrapped on a coverslip and glued at the back of the glass, so that front side with the porous structure was available for cell attachment and infiltration. The nanofiber samples were sterilized in 24-well plates by incubating in 70% ethanol for 24 h and rinsed 3 times with sterile DI water and then basal medium prior to cell seeding. A 1-mL aliquot of medium containing 20,000 cells was seeded on each nanofiber sample. The cells were grown in a tissue culture incubator at 37 °C and 5% CO2 atmosphere. Triplicates of each sample were plated with cells. The cells were also seeded on glass coverslips without nanofiber samples to be used as control group. The cells were cultured for 5 days, and the culture medium was replaced every 2 days. The alamarBlue assay ((Life Technologies, Grand Island, NY)) was used to analyze the cell viability of 3T3 fibroblast cells grown on nanofiber samples. After 1, 3, and 5 days of cell seeding, the coverslips were transferred to new plates, washed twice with PBS and incubated with 10 vol% of alamarBlue reagent in DMEM with 10% FBS for 2 h. Aliquots of 400 μL of assay solution were removed from the wells with the mats and transferred to a 96-well culture plate for fluorescent measurements on a Spectra max Gemini XPS microplate reader (Molecular Devices, Sunnyvale, CA) at λex 530 nm and λem 590 nm. The relative fluorescent units were converted to a percent of the average values for cells in control wells. 2.11. Cell attachment study After the determination of cell viability using the alamarBlue assay, the cells growing on the mats were fixed and cell attachment was observed by SEM. The cells were washed 3 times with PBS and fixed with 4% glutaraldehyde (pH 7.4) for 20 min. After fixing, the samples were briefly rinsed with DI water and dehydrated by sequential incubations in 50, 70, and 100% ethanol at room temperature. The samples were air dried inside a fume hood for 24 h and then sputter coated with Au for 1 min 30 s at 15 mA. The samples were imaged with SEM at an accelerating voltage of 10 kV and current of 5 μA.
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2.12. Statistical analysis All quantitative data, except cell viability data were analyzed for significance with Minitab 16 statistical software. Cell viability data were analyzed with OriginPro software. Data were analyzed using one-way analysis of variance (ANOVA). Pairwise comparisons were conducted using the Post hoc Tukey’s test. The α-value was set to 0.05 and p-values less than 0.05 were considered statistically significant.
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3. Results and discussion In this research, GTR membranes were fabricated using electrospun PCL/aloe vera blends at several different concentrations. PCL is a nonionic polymer that is soluble in range of solvents, mainly halogenated organic solvents. We chose TFE, a water-miscible fluorinated alcohol to dissolve PCL. TFE has been found to be a good solvent to create nanofibers by electropsinning [23]. Due to the electronegativity of the trifluoromethyl group, this alcohol exhibits a strong acidic nature. Thus, TFE helps to form heterocyclic complexes between PCL and aloe vera through hydrogen bonding. PCL/aloe vera solutions were prepared by mixing PCL and aloe vera in TFE and DI water, respectively, both at a fixed concentration of 10 wt%. Solutions with PCL/aloe vera ratios in the range 70:30–90:10 were investigated. Jet elongation occurs during electrospinning, and rapid solvent evaporation and phase separation occur simultaneously due to jet elongation.[21] Volatility and vapor pressure are two important parameters for rapid solvent evaporation and phase separation. We selected TFE because of its volatility and high vapor pressure (70 mm Hg) at room temperature so that electrospinning could evaporate and separate TFE completely. 3.1. Fiber morphology and porosity In tissue engineering, fiber morphology and porosity are two important parameters for cell growth and proliferation. Figure 2 shows SEM images of nanofibers spun from solutions with three different ratios PCL/aloe vera. These experimental results have shown that solutions with higher PCL/aloe vera ratios had better electrospinnability. At lower PCL/aloe vera ratios, a mixture of beads and fibers were obtained. As solution viscosity decreases at reduced concentration, the charged jet fragments into discrete droplets before reaching the collector. This happens due to the effect of the applied voltage and surface tension of the polymeric solution.[22] As the viscosity increases at an increased polymeric concentration, the chain entanglement between polymeric chains improves and nanofibers are formed. In this study, stable and completely ‘bead-free’ solid nanofibers were obtained at a PCL/ aloe vera ratio of 70/30 or higher. Solutions with PCL/aloe vera ratios in the range 70/30– 90/10 yielded cylindrical nanofibers with a mean diameter in the range of 300–600 nm. Solutions of PCL/aloe vera with 60/40 ratio or lower were also tested. These results are not included in this study because of their poor morphology, i.e. ‘beads on fiber’ and weak handleability. Fibers with nanoscale diameters are important because they have been shown to positively influence cellular behavior. This property could lead to improved regenerative outcomes for a GTR membrane comprised of nanofibers.
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Figure 2. SEM images of PCL/aloe vera nanofibers with ratios of (a) 100/0, (b) 90/10, (c) 80/20 and (d) 70/30, respectively. Notes: The insets show higher magnification images of each corresponding SEM image. The scale bar in the inset represents 5 μm and scale units for the lower power are on the figures, i.e. 10 tick marks represent 100 μm.
Porosity of these nanofiber membranes was in the range of 85–74%, i.e. PCL/aloe vera: 100/0 (85 ± 3%), 90/10 (83 ± 4%), 80/20 (79 ± 3%), and 70/30 (74 ± 2%). Generally, 60–90% porosity is preferred for GTR membrane to ensure sufficient gas and nutrient exchanges.[18] 3.2. Integrity of nanofibrous structure in aqueous medium The degradation of the nanofibrous membranes was assessed qualitatively by visual inspection with SEM. Representative images from the degradation study are displayed from the first time point (0 day) and the final time point (3 weeks). For GTR application, PCL/aloe vera nanofibers are expected to maintain their structural integrity in an aqueous environment. This is important, because GTR membranes should maintain their structure and space to allow the oral tissues to properly regenerate. The structural integrity of PCL/aloe nanofibers in an aqueous environment was examined by immersing nanofibers in PBS for up to 3 weeks. Aloe is a water soluble protein, so it was expected that aloe-based electrospun fibrous membranes would dissolve quickly in water at 37 °C. Thus, it was of practical interest to study the effect of the amount of aloe in PCL/aloe nanofibers on the integrity of the nanofibrous structure in buffer solution. As shown in Figure 3, the membranes made of nanofibers containing 10–30 wt % aloe retained fibrous structure after immersion in PBS up to 3 weeks. No significant change in morphology was observed. Thus, these fibers
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Figure 3. SEM images in the top panel (a–d) and bottom panel (e–h) display the corresponding morphology of nanofibers after first time point (0 day) and final time point (3 weeks) of degradation tests, respectively. Notes: Images (a, e), (b, f), (c, g) and (d, h) represent the PCL/aloe nanofibers with ratios of 100/0, 90/0, 80/20, and 70/30, respectively. The insets show higher magnification images of each corresponding SEM image. Table 1. Water contact angles (average numbers of five samples) of PCL and PCL/aloe vera nanofiber membranes. Ratios 100/0 90/10 80/20 70/30
Contact angle (CA) 82 78 72 65
were further studied for their wettability, chemical structure, mechanical properties, and cellular compatibility. 3.3. Contact angle CAs, which depend on chemical composition and topographic pattern, reflect the hydrophilicity of membrane, relating to protein absorption and cell attachment characteristics of scaffolds.[23] Table 1 shows the results of contact angle measurement of nanofibrous membranes. Compared with the hydrophobicity of PCL nanofiber membranes, PCL/aloe vera membranes showed hydrophilic characteristics. These results revealed that the surface hydrophilicity of nanofiber membranes increased with the incorporation of aloe vera as the CAs decreased. A high water CA means the material is hydrophobic and thus exhibits poor cell–material interactions. For nanofibers, we observed significant differences in the CAs between PCL and PCL/aloe vera nanofibers. Such an improvement in hydrophilicity will increase tissue regeneration and the biodegradation rate of the membranes. 3.4. Structural characterization by FTIR The FTIR spectra were measured to confirm the blend of aloe vera with PCL and examine the relative strengths of characteristic peaks changing with increasing aloe vera concentration. Figure 4 shows representative FTIR spectra of electrospun PCL, aloe vera, and PCL/
Journal of Biomaterials Science, Polymer Edition PCL/Aloe (90/10)
PCL/Aloe (70/30)
Aloe
PCL/Aloe (80/20)
PCL/Aloe (70/30) 3 Week
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Absorbance (A.U.)
1590
PCL
701
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1000
1500
2000
2500
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3500
Wave number (cm-1)
Figure 4. FTIR spectra of PCL, aloe vera, and PCL/aloe vera nanofibers.
aloe nanofibers. The characteristic features of PCL and aloe in the PCL/aloe nanofibers were observed in the spectra. PCL spectrum shows characteristic carbonyl (C=O) peak at 1720 cm−1, CH2 stretching at 2950 cm−1 (asymmetric) and 2865 cm−1 (symmetric), C–O stretching at 1050 cm−1, C–O–C stretching at 1240 cm−1 (asymmetric).[24,25] The main constituent of aloe extract is mannose-6-phosphate. It contain phosphate group, hydroxyl group, and ring oxygen [26] Phosphate stretching frequency appeared at 1060 cm−1. The absorption band around 3314 cm−1 represent the presence of hydrogen bonded N–H stretching,[27] characteristic of amino acids. The absorption band at 2920 cm−1 is due to the symmetrical and asymmetrical C–H stretching of the CH2 groups which is also characteristic of the presence of aliphatic (C–H) groups in the aloe vera. The absorption band at 1719 cm−1 is characteristic of CO stretching and indicates the presence of carbonyl groups. The strong absorption band at 1590 cm−1 is due to CC stretching, which indicates the presence of vinyl ether and aloin compound.[28,29] PCL/aloe nanofibers showed the characteristic bands of aloe in addition to the characteristic peaks of PCL, but the relative strengths of these peaks changed with increasing aloe vera concentration of the nanofibers. Unfortunately, majority of the strong absorption bands of aloe falls in a region overlapped by the PCL absorptions except absorption band at 1590 cm−1. Therefore, in this study the characteristic absorption band at 1590 cm−1 was used as a reference peak to confirm the presence of aloe in the nanofibers. The favorable molecular interaction between the PCL and aloe is required to maintain their structural and mechanical integrity for GTR application, because aloe vera’s antimicrobial and other bioactive functions are needed throughout the 3–6-week regenerative period. As the body’s aqueous environment is considered to be the primary source of polymer degradation, PCL/aloe fibrous matrices were tested in vitro at 37°C in a PBS solution for up to 3 weeks. No discernable changes to the structural integrity and chemistry of the nanofiber were identified by SEM images (Figure 3) and FTIR analysis (Figure 4). This was also confirmed by comparing the characteristic absorption band at 1590 cm−1 in FTIR
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Figure 5. Representative stress/strain curve from tested nanofibrous PCL/aloe membrane with ratio of 80/20.
Figure 6. Ultimate strength and Young’s modulus of each fiber sample. Note: Statistical significance of p < 0.05 is indicated by *.
spectra of PCL/aloe (70/30) nanofibers in the degradation study at the 3 weeks time point. The above demonstration of good miscibility between PCL and aloe in the electrospun nanofibers may be attributed to intermolecular hydrogen bonding between PCL and aloe. This is consistent with the extended degradation rate of PCL observed in the literature. The hydrophobic nature of the polymer gives scaffolds degradation times of up to 1 year. 3.5. Mechanical properties Tensile tests were performed on electrospun membranes from each of the following PCL/aloe vera solution ratios: 100/0, 90/10, 80/20, and 70/30. Figure 5 is a representative curve of the tensile data collection. The Young’s modulus was found by first fitting a line to the elastic (initial linear) region of the curve in Figure 5. The equation of this best fit line was then calculated and the resulting slope was the modulus. The ultimate tensile strength was calculated by determining the maximum stress that a material can withstand before failing or breaking.
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Figure 7. Cell viability results of 3T3 cells grown on PCL/aloe fibers. Notes: Cells were grown on glass coverslips without fiber samples (control) or on glass coverslips holding mats of PCL/aloe fibers with ratios of 100/0, 90/10, 80/20, and 70/30, respectively. Statistical significance of p
ISSN: 0920-5063 (Print) 1568-5624 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsp20
Facile fabrication of aloe vera containing PCL nanofibers for barrier membrane application Princeton Carter, Shekh M. Rahman & Narayan Bhattarai To cite this article: Princeton Carter, Shekh M. Rahman & Narayan Bhattarai (2016) Facile fabrication of aloe vera containing PCL nanofibers for barrier membrane application, Journal of Biomaterials Science, Polymer Edition, 27:7, 692-708, DOI: 10.1080/09205063.2016.1152857 To link to this article: http://dx.doi.org/10.1080/09205063.2016.1152857
Accepted author version posted online: 15 Feb 2016. Published online: 10 Mar 2016. Submit your article to this journal
Article views: 202
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Citing articles: 5 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbsp20 Download by: [Purdue University Libraries]
Date: 08 November 2017, At: 03:07
Journal of Biomaterials Science, Polymer Edition, 2016 VOL. 27, NO. 7, 692–708 http://dx.doi.org/10.1080/09205063.2016.1152857
Facile fabrication of aloe vera containing PCL nanofibers for barrier membrane application Princeton Carter, Shekh M. Rahman and Narayan Bhattarai
Downloaded by [Purdue University Libraries] at 03:07 08 November 2017
Department of Chemical, Biological and Bioengineering, North Carolina A&T State University, Greensboro, NC USA
ABSTRACT
Guided tissue regeneration (GTR) is a widely used method in dental surgical procedures that utilizes a barrier membrane to exclude migration of epithelium and ensure repopulation of periodontal ligament cells at the sites having insufficient gingiva. Commercial GTR membranes are typically composed of synthetic polymers that have had mild clinical success mostly because of their lack of proper bioactivity and appropriate degradation profile. In this study, a natural polymer, aloe vera was blended with polycaprolactone (PCL) to create nanofibrous GTR membranes by electrospinning. Aloe vera has proven anti-inflammatory properties and enhances the regeneration of periodontium tissues. PCL, a synthetic polymer, is well known to produce miscible polyblends nanofibers with natural polymers. Nanofibrous membranes with varying composition of PCL to aloe vera were fabricated, and several physicochemical and biological properties, such as fiber morphology, wettability, chemical structure, mechanical strength, and cellular compatibility of the membranes were analyzed. PCL/aloe vera membranes with ratios from 100/00 to 70/30 showed good uniformity in fiber morphology and suitable mechanical properties, and retained the integrity of their fibrous structure in aqueous solutions. Experimental results, using cell viability assay and cell attachment observation, showed that the nanofibrous membranes support 3T3 cell viability and could be a potential candidate for GTR therapy.
ARTICLE HISTORY
Received 26 October 2015 Accepted 8 February 2016 KEYWORDS
Guided tissue regeneration; aloe vera; polycaprolectone; nanofiber; electropsinning
1. Introduction The Centers for Disease Control recently revealed that approximately half of all Americans aged 30 and over or 64 million people have some type of periodontitis. Periodontitis is an inflammatory disease that could lead to the destruction of oral tissues. Guided tissue regeneration (GTR) is a widely used method in dental surgical procedures that utilizes a barrier membrane to exclude migration of epithelium and ensure repopulation of periodontal ligament cells at the sites having insufficient gingiva for proper function. Similar to any other tissue engineering applications such as bone, cartilage, muscle, tendon, etc., an ideal scaffold
CONTACT Narayan Bhattarai © 2016 Taylor & Francis
[email protected]
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suitable for GTR membrane should have adequate biocompatibility, proper degradation profile, adequate mechanical and physical properties.[1] On top of these general characteristics, a GTR membrane should enable cell exclusion separating the gingival flap from the fibrin clot and guard space for the new alveolar bone and the periodontal ligament. Infection caused by either pathogen colonization at the wound site or foreign body response of the material is considered a major reason of GTR failure in clinical settings.[2] Antibacterial biomaterials are one of the greatest interests to resist implant-related infections.[3] Clinically used GTR membranes are composed of biodegradable and non-biodegradable polymers.[4] Polytetrafluoroethylene (PTFE), a synthetic polymer, is the most commonly used non-biodegradable material for GTR membranes.[1] Collagen, a protein from animal sources, is the most commonly used biodegradable material in the clinic for GTR membranes.[5] These membranes unfortunately have several drawbacks. The commercially available membranes composed of different forms of PTFE (e.g. high density and expanded PTFE) are non-biodegradable and require a secondary surgery for their removal. The secondary surgery could result in complications and additional costs. PTFE membranes also lack the chemical and physical features to improve regenerative outcomes. Hence, collagenous membranes offer the bioactivity lacking with synthetic materials, but they do not have sufficient mechanical integrity to maintain space throughout the regenerative process.[6] Synthetic biodegradable polymers, including polyglycolic acid, polylactic acid, and polycaprolactone (PCL) have also been used to make commercial GTR membranes. Membranes composed of these materials are generally biocompatible and easier to surgically place compared to PTFE membranes. These membranes, however, are not clinically useful because of the poor cellular response to them.[5] To rectify the issues observed with traditional GTR membranes, nanofibrous GTR membranes have been proposed. Nanofibrous membranes could maintain the defect space, allow nutrients and waste to enter and leave the defective sites, and positively influence cell behavior because they mimic the natural extracellular matrix (ECM). Electrospinning is the most prevalent manufacturing technique for nanofibrous membranes, because it is simple, reliable, and relatively inexpensive.[1] Natural material aloe vera has been shown to have antimicrobial activity toward gram-negative bacteria, which are the primary microbes implicated in the etiology of periodontitis.[7] Aloe vera also has anti-inflammatory properties,[8] which could mitigate the damage associated with the inflammatory disease, periodontitis. Acemannan sugar in aloe vera has been shown to encourage the regeneration of periodontium tissues (i.e. alveolar bone, periodontal ligament, and cementum). [9–11] Aloe vera is also found to be an effective decontaminating agent for gutta percha cones, a traditional dental filling.[12] PCL, a synthetic polymer, is well known for its use as a material for electrospun nanofibers. [13] This polymer is also useful because it has proven miscibility with a variety of other polymers to produce stable polyblends.[14] Finally, PCL can increase sufficient mechanical strength to the membrane and prevent degradation before the 4–6 weeks time period that is important for regeneration of periodontium tissues. Recently, a few attempts have been made to prepare aloe vera-incorporated PCL nanofibers by electrospinning technique with varying degrees of success.[15–17] Although these recent efforts in fabricating aloe vera-containing nanofibrous structures are encouraging, much remains to be explored and improved, particularly, in perspective of applications for GTR.
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In this work, a facile method of blending aloe vera with PCL in different ratios was first developed. Electrospinning technique was utilized to transform the blend solution into nanofibrous GTR membranes. Toward the potential use of these GTR membranes, their physicochemical properties such as morphology, mechanical strength, wettability, chemical structure, and integrity in aqueous medium were studied. Cellular compatibility of the nanofibrous membranes was studied by seeding 3T3 fibroblast cells on the fibrous membranes. Cell viability was quantified by alamarBlue assay, and cell attachment was characterized by scanning electron microscope (SEM).
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2. Materials and methods 2.1. Materials Samples of aloe vera were obtained commercially from Terry Laboratories (Melbourne, FL, USA, catalog number TN003). Polycaprolactone (PCL, Mn 80 kDa) was obtained from Sigma-Aldrich (St. Louis, MO, USA). 2, 2, 2-Trifluoroethanol (TFE, 99 + %) was obtained from Alfa Aesar (Ward Hill, MA, USA). 2.2. Solution preparation for electrospinning Aloe vera was dissolved in DI water at a concentration of 10 wt%. The solution was vortexed for 5 min to ensure the aloe vera was completely dissolved. PCL solution was prepared by dissolving PCL pellets using 2-2-2-Trifluoroethanol (TFE) at a concentration of 10 wt%. When both the solutions were dissolved, a plastic syringe was used to remove the appropriate amount of each solution to generate the different ratios of PCL/aloe vera: 100/0, 90/10, 80/20, and 70/30. Each solution mixture was vortexed manually for 20 min. When the solution appeared to be well blended, the glass vial was immediately taken to the fume hood to be electrospun. 2.3. Electrospinning To fabricate the nanofibers with various polymer solutions of PCL/aloe vera (i.e. 100/0, 90/10, 80/20, and 70/30), the parameters of applied voltage and tip-to-collector distance were altered until fiber formation was observed. The Spellman CZE1000R (Hauppauge, NY, USA) high-voltage power supply was adjusted to a voltage of 25–30 kV depending on the solution, and the solution was poured into a static syringe held by a test tube holder. The stationary syringe tip was approximately 16–23 cm away from a plastic, cylindrical grounded collector wrapped with aluminum foil. 2.4. Fiber morphology observation and porosity measurement The fiber morphology after various time points was observed by (SEM) (Hitachi SU8000, Japan). Samples from each polymer ratio used were first gold sputter coated using the Polaron SEM coating System for 1 min and 30 s to prevent over coating of the samples. The samples were then loaded into the SEM chamber and imaged using accelerated voltage of 10 kV and current of 5 μA. Additional parameters were adjusted to improve the quality
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of the image at 500 and 10 K magnifications. All images were captured using a 40 or 80-s scanning process. The apparent density and porosity of the membranes were estimated according to the previously reported methods.[18] Briefly, the apparent density and porosity were estimated using Equations (1) and (2).
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( ) g Mass of membrane (g) Apparent density = (1) 3 cm Membrane thickness (cm) × Membrane area (cm2 )
( ) g Apparent density cm3 Porosity (%) = 1 − ( ) × 100 (%) g Bulk density cm3
(2)
2.5. Contact angle measurement
The wettability of electrospun fiber membranes was determined by contact angle (CA) measurement. The CA measurement was carried out using a specially arranged microscope equipped with a camera (Dynamic Contact Angle Tester, Billerica, MA). The DI water droplet (0.25 μL in volume) was used in the CA measurement. The CA experiments were carried out at room temperature and were repeated five times. All CAs were measured within 20 s of placement of the water droplet on the electrospun fiber mat. The wettability of nanofiber samples was analyzed by comparing the CAs of PCL/aloe vera nanofibers with PCL nanofibers (act as internal reference). Five samples of each ratio of nanofibers were used to measure CA. 2.6. Chemical structure analysis To characterize the chemical bonding between PCL and aloe vera, Fourier Transform Infrared Spectroscopy (FTIR) spectra were obtained using a Varian 670 spectrophotometer (Santa Clara, CA, USA). The data were collected between 4000 cm−1 and 400 cm−1. Four scans were used to collect the spectra. ATR or attenuate total reflectance FTIR was used, so that the fiber samples could be directly analyzed without further processing (i.e. making a KBr pellet). The powdered aloe vera, as provided from the manufacturer, was tested along with fiber samples from each time point of the degradation study. The IR spectra were used to confirm the absence or presence of aloe vera in the nanofibrous membranes. 2.7. Tensile mechanical testing Measurements were carried out according to previously published methods.[19,20] Fiber samples were cut to have a 5 mm width and L = 16 mm length. The thickness for all of the fiber meshes was measured using a Digimatic micrometer. Fiber samples were center aligned and mounted within a Lg (gauge length) = 6 mm window cut from a paper template and affixed using two-sided tape as shown in Figure 1. The top and bottom dimensions of the paper holder are also listed below. The additional paper allowed the sample to be held by the clamps of the Shimadzu machine (North America Analytical and Measuring Instruments AGS-X series obtained from Columbia, MD).
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Figure 1. Dimensions of paper holder used for tensile testing.
Trapezium Lite X (Shimadzu Corporation, Columbia, MD) software was used to collect the tensile data with the parameters of displacement rate set to V1 = 10 mm/min and data acquisition time to 100 ms for a 50 N load cell. The load (P) is the force applied to the sample. Additionally, the software was set to display data in time (seconds), force (Newtons), and stroke (millimeters). Before clamping the sample in the machine, the force was calibrated and zeroed. The sample holder was then centered and tightened between the bottom and top clamps. Both sides of the sample holder were cut to release the tension of the paper prior to initiating testing. The stroke was zeroed and then testing was started. After each test was completed, the exported raw data, which consisted of the load (P) on the sample and the displacement (d) of the sample, was manipulated in Excel. The strain was calculated by: strain = d∕Lg . and the stress was calculated by: Stress = P∕(cross sectional area). The load was given in Newtons (N) from the computer software. The cross sectional area was computed by multiplying the 5 mm width by the thickness of the samples as measured with the micrometer. These calculated data were then used to make stress-strain curves and to determine Young’s Moduli and ultimate tensile strengths for each of the fiber samples. (n = 5 for each treatment (70/30, 80/20, 90/10, and 100/0)). 2.8. In vitro degradation test Fiber samples from each ratio were cut into rectangular pieces placed in conical tubes and then submerged in phosphate-buffered saline (PBS) solution. The conical tubes were then placed in a water bath held at 37.5 °C. After time points of 0 day, 3 days, 1 week, and 3 weeks, the samples were removed from the PBS solutions, rinsed with DI water, patted dried, and then allowed to completely dry in the open air. After completely drying, each sample was probed using SEM.
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2.9. Culture of 3T3 fibroblast cells
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NIH/3T3 cells (a mouse fibroblast cell line) were purchased from the American Tissue Type Culture Collection (Manassas, VA). The growth medium was Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (10,000 units/mL of penicillin and 10,000 μg/mL of streptomycin). The cells were cultured in a 75 cm2 culture flask and maintained in a tissue culture incubator at 37 °C and 5% CO2 atmosphere. The culture medium was replaced every 2 days. After reaching about 80% confluence, the cells were detached by 0.025% trypsin and 0.01% EDTA in PBS solution and transferred to centrifuge tube containing culture medium. After centrifugation, the cells were resuspended in fresh culture medium and counted using a hemocytometer before seeding to nanofiber samples. 2.10. Cell viability study Nanofiber samples were attached to 12-mm square-diameter coverslips using biocompatible, silicone-based elastomeric glue (i.e. Kwik-Sil). Each fiber sample was first wrapped on a coverslip and glued at the back of the glass, so that front side with the porous structure was available for cell attachment and infiltration. The nanofiber samples were sterilized in 24-well plates by incubating in 70% ethanol for 24 h and rinsed 3 times with sterile DI water and then basal medium prior to cell seeding. A 1-mL aliquot of medium containing 20,000 cells was seeded on each nanofiber sample. The cells were grown in a tissue culture incubator at 37 °C and 5% CO2 atmosphere. Triplicates of each sample were plated with cells. The cells were also seeded on glass coverslips without nanofiber samples to be used as control group. The cells were cultured for 5 days, and the culture medium was replaced every 2 days. The alamarBlue assay ((Life Technologies, Grand Island, NY)) was used to analyze the cell viability of 3T3 fibroblast cells grown on nanofiber samples. After 1, 3, and 5 days of cell seeding, the coverslips were transferred to new plates, washed twice with PBS and incubated with 10 vol% of alamarBlue reagent in DMEM with 10% FBS for 2 h. Aliquots of 400 μL of assay solution were removed from the wells with the mats and transferred to a 96-well culture plate for fluorescent measurements on a Spectra max Gemini XPS microplate reader (Molecular Devices, Sunnyvale, CA) at λex 530 nm and λem 590 nm. The relative fluorescent units were converted to a percent of the average values for cells in control wells. 2.11. Cell attachment study After the determination of cell viability using the alamarBlue assay, the cells growing on the mats were fixed and cell attachment was observed by SEM. The cells were washed 3 times with PBS and fixed with 4% glutaraldehyde (pH 7.4) for 20 min. After fixing, the samples were briefly rinsed with DI water and dehydrated by sequential incubations in 50, 70, and 100% ethanol at room temperature. The samples were air dried inside a fume hood for 24 h and then sputter coated with Au for 1 min 30 s at 15 mA. The samples were imaged with SEM at an accelerating voltage of 10 kV and current of 5 μA.
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2.12. Statistical analysis All quantitative data, except cell viability data were analyzed for significance with Minitab 16 statistical software. Cell viability data were analyzed with OriginPro software. Data were analyzed using one-way analysis of variance (ANOVA). Pairwise comparisons were conducted using the Post hoc Tukey’s test. The α-value was set to 0.05 and p-values less than 0.05 were considered statistically significant.
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3. Results and discussion In this research, GTR membranes were fabricated using electrospun PCL/aloe vera blends at several different concentrations. PCL is a nonionic polymer that is soluble in range of solvents, mainly halogenated organic solvents. We chose TFE, a water-miscible fluorinated alcohol to dissolve PCL. TFE has been found to be a good solvent to create nanofibers by electropsinning [23]. Due to the electronegativity of the trifluoromethyl group, this alcohol exhibits a strong acidic nature. Thus, TFE helps to form heterocyclic complexes between PCL and aloe vera through hydrogen bonding. PCL/aloe vera solutions were prepared by mixing PCL and aloe vera in TFE and DI water, respectively, both at a fixed concentration of 10 wt%. Solutions with PCL/aloe vera ratios in the range 70:30–90:10 were investigated. Jet elongation occurs during electrospinning, and rapid solvent evaporation and phase separation occur simultaneously due to jet elongation.[21] Volatility and vapor pressure are two important parameters for rapid solvent evaporation and phase separation. We selected TFE because of its volatility and high vapor pressure (70 mm Hg) at room temperature so that electrospinning could evaporate and separate TFE completely. 3.1. Fiber morphology and porosity In tissue engineering, fiber morphology and porosity are two important parameters for cell growth and proliferation. Figure 2 shows SEM images of nanofibers spun from solutions with three different ratios PCL/aloe vera. These experimental results have shown that solutions with higher PCL/aloe vera ratios had better electrospinnability. At lower PCL/aloe vera ratios, a mixture of beads and fibers were obtained. As solution viscosity decreases at reduced concentration, the charged jet fragments into discrete droplets before reaching the collector. This happens due to the effect of the applied voltage and surface tension of the polymeric solution.[22] As the viscosity increases at an increased polymeric concentration, the chain entanglement between polymeric chains improves and nanofibers are formed. In this study, stable and completely ‘bead-free’ solid nanofibers were obtained at a PCL/ aloe vera ratio of 70/30 or higher. Solutions with PCL/aloe vera ratios in the range 70/30– 90/10 yielded cylindrical nanofibers with a mean diameter in the range of 300–600 nm. Solutions of PCL/aloe vera with 60/40 ratio or lower were also tested. These results are not included in this study because of their poor morphology, i.e. ‘beads on fiber’ and weak handleability. Fibers with nanoscale diameters are important because they have been shown to positively influence cellular behavior. This property could lead to improved regenerative outcomes for a GTR membrane comprised of nanofibers.
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Figure 2. SEM images of PCL/aloe vera nanofibers with ratios of (a) 100/0, (b) 90/10, (c) 80/20 and (d) 70/30, respectively. Notes: The insets show higher magnification images of each corresponding SEM image. The scale bar in the inset represents 5 μm and scale units for the lower power are on the figures, i.e. 10 tick marks represent 100 μm.
Porosity of these nanofiber membranes was in the range of 85–74%, i.e. PCL/aloe vera: 100/0 (85 ± 3%), 90/10 (83 ± 4%), 80/20 (79 ± 3%), and 70/30 (74 ± 2%). Generally, 60–90% porosity is preferred for GTR membrane to ensure sufficient gas and nutrient exchanges.[18] 3.2. Integrity of nanofibrous structure in aqueous medium The degradation of the nanofibrous membranes was assessed qualitatively by visual inspection with SEM. Representative images from the degradation study are displayed from the first time point (0 day) and the final time point (3 weeks). For GTR application, PCL/aloe vera nanofibers are expected to maintain their structural integrity in an aqueous environment. This is important, because GTR membranes should maintain their structure and space to allow the oral tissues to properly regenerate. The structural integrity of PCL/aloe nanofibers in an aqueous environment was examined by immersing nanofibers in PBS for up to 3 weeks. Aloe is a water soluble protein, so it was expected that aloe-based electrospun fibrous membranes would dissolve quickly in water at 37 °C. Thus, it was of practical interest to study the effect of the amount of aloe in PCL/aloe nanofibers on the integrity of the nanofibrous structure in buffer solution. As shown in Figure 3, the membranes made of nanofibers containing 10–30 wt % aloe retained fibrous structure after immersion in PBS up to 3 weeks. No significant change in morphology was observed. Thus, these fibers
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Figure 3. SEM images in the top panel (a–d) and bottom panel (e–h) display the corresponding morphology of nanofibers after first time point (0 day) and final time point (3 weeks) of degradation tests, respectively. Notes: Images (a, e), (b, f), (c, g) and (d, h) represent the PCL/aloe nanofibers with ratios of 100/0, 90/0, 80/20, and 70/30, respectively. The insets show higher magnification images of each corresponding SEM image. Table 1. Water contact angles (average numbers of five samples) of PCL and PCL/aloe vera nanofiber membranes. Ratios 100/0 90/10 80/20 70/30
Contact angle (CA) 82 78 72 65
were further studied for their wettability, chemical structure, mechanical properties, and cellular compatibility. 3.3. Contact angle CAs, which depend on chemical composition and topographic pattern, reflect the hydrophilicity of membrane, relating to protein absorption and cell attachment characteristics of scaffolds.[23] Table 1 shows the results of contact angle measurement of nanofibrous membranes. Compared with the hydrophobicity of PCL nanofiber membranes, PCL/aloe vera membranes showed hydrophilic characteristics. These results revealed that the surface hydrophilicity of nanofiber membranes increased with the incorporation of aloe vera as the CAs decreased. A high water CA means the material is hydrophobic and thus exhibits poor cell–material interactions. For nanofibers, we observed significant differences in the CAs between PCL and PCL/aloe vera nanofibers. Such an improvement in hydrophilicity will increase tissue regeneration and the biodegradation rate of the membranes. 3.4. Structural characterization by FTIR The FTIR spectra were measured to confirm the blend of aloe vera with PCL and examine the relative strengths of characteristic peaks changing with increasing aloe vera concentration. Figure 4 shows representative FTIR spectra of electrospun PCL, aloe vera, and PCL/
Journal of Biomaterials Science, Polymer Edition PCL/Aloe (90/10)
PCL/Aloe (70/30)
Aloe
PCL/Aloe (80/20)
PCL/Aloe (70/30) 3 Week
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Absorbance (A.U.)
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Wave number (cm-1)
Figure 4. FTIR spectra of PCL, aloe vera, and PCL/aloe vera nanofibers.
aloe nanofibers. The characteristic features of PCL and aloe in the PCL/aloe nanofibers were observed in the spectra. PCL spectrum shows characteristic carbonyl (C=O) peak at 1720 cm−1, CH2 stretching at 2950 cm−1 (asymmetric) and 2865 cm−1 (symmetric), C–O stretching at 1050 cm−1, C–O–C stretching at 1240 cm−1 (asymmetric).[24,25] The main constituent of aloe extract is mannose-6-phosphate. It contain phosphate group, hydroxyl group, and ring oxygen [26] Phosphate stretching frequency appeared at 1060 cm−1. The absorption band around 3314 cm−1 represent the presence of hydrogen bonded N–H stretching,[27] characteristic of amino acids. The absorption band at 2920 cm−1 is due to the symmetrical and asymmetrical C–H stretching of the CH2 groups which is also characteristic of the presence of aliphatic (C–H) groups in the aloe vera. The absorption band at 1719 cm−1 is characteristic of CO stretching and indicates the presence of carbonyl groups. The strong absorption band at 1590 cm−1 is due to CC stretching, which indicates the presence of vinyl ether and aloin compound.[28,29] PCL/aloe nanofibers showed the characteristic bands of aloe in addition to the characteristic peaks of PCL, but the relative strengths of these peaks changed with increasing aloe vera concentration of the nanofibers. Unfortunately, majority of the strong absorption bands of aloe falls in a region overlapped by the PCL absorptions except absorption band at 1590 cm−1. Therefore, in this study the characteristic absorption band at 1590 cm−1 was used as a reference peak to confirm the presence of aloe in the nanofibers. The favorable molecular interaction between the PCL and aloe is required to maintain their structural and mechanical integrity for GTR application, because aloe vera’s antimicrobial and other bioactive functions are needed throughout the 3–6-week regenerative period. As the body’s aqueous environment is considered to be the primary source of polymer degradation, PCL/aloe fibrous matrices were tested in vitro at 37°C in a PBS solution for up to 3 weeks. No discernable changes to the structural integrity and chemistry of the nanofiber were identified by SEM images (Figure 3) and FTIR analysis (Figure 4). This was also confirmed by comparing the characteristic absorption band at 1590 cm−1 in FTIR
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Figure 5. Representative stress/strain curve from tested nanofibrous PCL/aloe membrane with ratio of 80/20.
Figure 6. Ultimate strength and Young’s modulus of each fiber sample. Note: Statistical significance of p < 0.05 is indicated by *.
spectra of PCL/aloe (70/30) nanofibers in the degradation study at the 3 weeks time point. The above demonstration of good miscibility between PCL and aloe in the electrospun nanofibers may be attributed to intermolecular hydrogen bonding between PCL and aloe. This is consistent with the extended degradation rate of PCL observed in the literature. The hydrophobic nature of the polymer gives scaffolds degradation times of up to 1 year. 3.5. Mechanical properties Tensile tests were performed on electrospun membranes from each of the following PCL/aloe vera solution ratios: 100/0, 90/10, 80/20, and 70/30. Figure 5 is a representative curve of the tensile data collection. The Young’s modulus was found by first fitting a line to the elastic (initial linear) region of the curve in Figure 5. The equation of this best fit line was then calculated and the resulting slope was the modulus. The ultimate tensile strength was calculated by determining the maximum stress that a material can withstand before failing or breaking.
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Figure 7. Cell viability results of 3T3 cells grown on PCL/aloe fibers. Notes: Cells were grown on glass coverslips without fiber samples (control) or on glass coverslips holding mats of PCL/aloe fibers with ratios of 100/0, 90/10, 80/20, and 70/30, respectively. Statistical significance of p
