Engineering electrospun multicomponent polyurethane scaffolding platform comprising grapeseed oil and honey/propolis for bone tissue regeneration

Cui Yan Chao; Mohan Prasath Mani; Saravana Kumar Jaganathan

doi:10.1371/journal.pone.0205699

. 2018 Oct 29;13(10):e0205699. doi: 10.1371/journal.pone.0205699

Engineering electrospun multicomponent polyurethane scaffolding platform comprising grapeseed oil and honey/propolis for bone tissue regeneration

Cui Yan Chao ¹, Mohan Prasath Mani ², Saravana Kumar Jaganathan ^3,^4,^5,^*

Editor: Yogendra Kumar Mishra⁶

PMCID: PMC6205588 PMID: 30372449

Abstract

Essential oils play an important role in reducing the pain and inflammation caused by bone fracture.In this study, a scaffold was electrospun based on polyurethane (PU), grape seed oil, honey and propolis for bone tissue-engineering applications. The fiber diameter of the electrospun PU/grape seed oil scaffold and PU/grape seed oil/honey/propolis scaffold were observed to be reduced compared to the pristine PU control. FTIR analysis revealed the existence of grape seed oil, honey and propolis in PU identified by CH band peak shift and also hydrogen bond formation. The contact angle of PU/grape seed oil scaffold was found to increase owing to hydrophobic nature and the contact angle for the PU/grape seed/honey oil/propolis scaffold were decreased because of hydrophilic nature. Further, the prepared PU/grape seed oil and PU/grape seed oil/honey/propolis scaffold showed enhanced thermal stability and reduction in surface roughness than the control as revealed in thermogravimetric analysis (TGA) and atomic force microscopy (AFM) analysis. Further, the developed nanocomposite scaffold displayed delayed blood clotting time than the pristine PU in the activated prothrombin time (APTT) and partial thromboplastin time (PT) assay. The hemolytic assay and cytocompatibility studies revealed that the electrospun PU/grape seed oil and PU/grape seed oil/honey/propolis scaffold possess non-toxic behaviour to red blood cells (RBC) and human fibroblast cells (HDF) cells indicating better blood compatibility and cell viability rates. Hence, the newly developed electrospun nanofibrous composite scaffold with desirable characteristics might be used as an alternative candidate for bone tissue engineering applications.

Introduction

In clinical applications, every year the bone tissue transplant surgery was increased in number insisting the demand for bone tissue grafts. The commercial bone tissue grafts used in the reconstruction of the bone defects were autografts and allografts. But their application in the biomedical applications were reduced owing to many limitations such as increased surgery time, donor site pain, and limited quantity of harvestable bone [1–4]. Hence, it forces the researchers to search for the alternate material (especially artificial tissue scaffolds) as a substitute for bone repair. The scaffold for bone tissue engineering must be biocompatible, porous, enough mechanical strength and proper degradation rate. Further, the developed scaffold must mimic the ECM, to support the tissue structure and also cells to adhere and migrate [5, 6].

In bone tissue engineering, a wide range of polymers were used to fabricate the bone scaffolds. Among, wide range of polymers, the polyurethane (PU) is used in this study to develop a scaffold for bone tissue engineering. PU is a copolymer which composed of both soft and hard segments. It was widely used in tissue engineering applications owing to its biodegradability, good barrier properties and better oxidation stability [7, 8]. Further, by tuning soft and hard segments, the properties of the PU were easily changed and been utilized for various biomedical applications. In general, there are different techniques utilized for the fabrication of PU tissue scaffolds such as electrospinning, solvent casting, freeze drying, batch foaming and injection foaming. In this research, the electrospinning technique was utilized for fabricating the PU scaffolds [1].

Electrospinning is a versatile and cost effective technique which utilizes the high voltage to produce the produce nonwoven nanofibers. The nonwoven nanofibers were found to have high-surface area-to-volume ratio and high porosity which can mimic the native structure of the extracellular matrix of bone tissue [9]. In this technique, by applying high voltage to the syringe needle, the fine nanofibers will draw from the polymer solution and collected on the aluminum foil which is placed on the collector drum [10]. Electrospinning involves many parameters such as applied voltage, flow rate, viscosity of polymer solution and collector distance which influence the fiber diameter of the nanofibers [11, 12].

In bone tissue engineering various types of innovations have been made to support the bone tissue repair. Miculescu et al presented the starch particles incorporated hydroxyapatite scaffolds for medical applications. The reported that the starch incorporated hydroxyapatite composite was considered to be safe in terms of toxicity and might utilized for bone cements, bone waxes, adhesives or scaffolds [13]. Some studies have reported the several metallic nanoparticles such as ZnO, SiO₂ incorporated composites for biomedical and high performance applications. The addition of nanoparticles showed the improved properties, excellent antibacterial activity, improved cell adhesion and osteoconductivity properties [14–16]. In another study, Voicu et al prepared cellulose acetate membrane for biomedical applications added with sericin. The fabricate membranes showed good osteoblast cell response suggested it as a suitable candidate for osseointegration processes [17]. Hence, these type of works motivated us to study the effect of grapeseed oil, honey and propolis in the bone tissue engineering. The pain and inflammation is created at the tissue near the fracture site when the bone get fractured. They are many treatments available to reduce the pain and inflammation occurred at the fracture site. In early days, the essential oil is used to get relief from the pain at the fracture site. Hence, in this research the grapeseed oil was used to fabricate the bone scaffold. Grape seed oil is obtained from the seeds of grapes and it was a by-product of wine making [18]. It was widely used as cooking oil and also in skin care applications as a cosmetics. Grape seed oil contains 0.8 to 1.5% of phenols and steroids and small amounts of vitamin E [19, 20]. The grape seed oil was reported to possess highest antioxidant capacity (42.18 mmol of Trolox equivalent/g) which was due to the existence of high amount of gallic acid, epicatechin, catechin, proanthocyanidins and procyanidins [21, 22]. The polyphenols present in grape seed oil have ability to inhibit the inflammatory response by preventing the release of arachidonic acid (AA) [21, 23]. Further, the phenolic component present in the grape seed oil was found to be toxic on both bacteria’s (Staphylococcus aureus and Escherichia coli) concluding its antimicrobial effect [21]. In addition to grape seed oil, the propolis is added with grape seed oil to improve the bioactivity in the PU polymer. Further, in a recent study, it was that the observed that the incorporation of propolis and honey into the electrospun membrane resulted in the hydrophilicity behavior [24]. Propolis is a resinous substance obtained by bees from their salivary secretions [25]. The propolis were reported to have many bioactive constituents such as flavonoid, phenolic components, amino acids and some inorganic compounds [26]. Further, it possess various medicinal properties like high adhesive, antibacterial, antifungal, antiviral, antioxidant and anti-inflammatory activities [25]. Honey is produced by bees and it contains numerous biological components like glucose, fructose, sucrose, water, amino acids, vitamins, minerals, and enzymes [27]. Honey on a whole or its constituents have been widely reported to possess various biological and pharmacological properties from wound healing to anti-tumour and from anti-inflammatory to antibacterial activities [28–30]. Further, the components such as flavonoids and phenolic acids present in the honey are reported to have synergistic antioxidant effect [31, 32]. In a recently concluded clinical trial, honey was tested on 102 patients with chronic wounds and ulcers which was not able to cure using conventional wound healing treatment. The results of this study showed that honey could heal these wounds dramatically in 4–7 weeks completely insinuating its wound healing potential [33]. Similarly, a recent study also showed the ability of honey to heal the mandibular bone defects of the Wistar rats efficiently compared to untreated control group [34]. In this study, PU nanofiber containing grape seed oil, honey and propolis were electrospun using electrospinning technique. For the first time, the combination effect of hybrid scaffolds based on honey and propolis with the grape seed oil was studied. For the fabricated PU and PU/grape seed oil/honey/propolis scaffold, the physiochemical characteristics, blood compatibility parameters and cytocompatibility studies were investigated to analyze its effect for bone tissue engineering.

Materials and methodology

Materials

The medical grade Tecoflex EG-80A polyurethane was obtained from Lubrizol and dissolved in dimethylformamide (DMF) solvent (Sigma Aldrich, UK). The grape seed oil, honey and propolis were obtained locally. The chemical phosphate buffered saline (PBS) and sodium chloride physiological saline (0.9% w/v) were purchased from Sigma-Aldrich, Kuala Lumpur, Malaysia. The reagents such as rabbit brain activated cephaloplastin, calcium chloride (0.025 M), and thromboplastin (Factor III) used in the blood compatibility studies were purchased from Diagnostic Enterprises, Solan, India.

PU and PU composite solutions

For fabricating PU membrane, 9 wt% of PU solution was prepared by dissolving calculated amount of PU in DMF and stirred overnight at room temperature to obtain clear homogeneous solution. Similarly, the grape seed oil, honey and propolis homogeneous solution were prepared for 9 wt% obtained by adding calculated amount of grape seed oil, honey and propolis in DMF and stirred 1 hr maximum. For fabricating PU/grape seed oil solution, the prepared 9 wt% of PU solution was doped with 9 wt% of prepared grape seed oil solution in the ratio of 7:2 (v/v%). Similarly, for PU/grape seed oil/honey/propolis solution, the prepared 9 wt% homogeneous PU solution was mixed with 9 wt% of homogeneous grape seed oil, honey and propolis solution at a ratio of 7:1:1 (v/v%) respectively. In composite solution preparation, the solutions were stirred for 2 hrs maximum at room temperature for even dissolution.

Electrospinning technique

The prepared solutions were placed in 10 ml syringes with 18-G stainless steel needle and loaded in the syringe pump. All the prepared PU and composite solutions were electrospun at a voltage of 10.5 kV with flow rate of 0.5 ml/h and collector distance placed at 20 cm. The nanofibers were obtained on the aluminum foil were carefully detached and dried under vacuum at room temperature to remove any residual DMF content.

Physio-chemical characterizations

Scanning electron microscopy (SEM)

SEM analysis was performed to observe the morphological details of electrospun membranes. Prior to capturing photomicrographs, the samples were gold coated. Then, the coated samples were imaged to obtain the SEM images of the electrospun membrane and the average fiber diameter was calculated using Image J software.

Contact angle measurements

The water contact angle measurements were measured using VCA Optima contact angle measurement unit to determine the wetting ability of the electrospun scaffold. To begin, a small piece of electrospun membrane was paced on the measuring surface and water droplets of 0.5 μL were dispensed on the testing membrane and the static image of the water droplet on the testing membrane was captured high-resolution video camera. The contact angle was measured automatically using computer integrated software.

Mechanical testing

The mechanical properties of fabricated scaffolds were tested using uniaxial testing machine. To begin, the rectangular samples with size 40 mm× 15 mm were cut and mounted between the grips. After gripping, the deformation was measured at a cross head speed of 5 mm min⁻¹ with load of 500 N. Finally, the tensile strength and elongation at break were determined from the resulting stress/strain curves.

Fourier transform infrared spectroscopy (FTIR)

The chemical compositions of the prepared composites were inspected using a FTIR analysis. A small piece of electrospun membrane was placed on the sensor surface and the spectra was inspected. The spectra was recorded at wavelength of 600–4000 cm⁻¹ at 4 cm⁻¹ resolution with average of 32 scans per minute. The spectra was baseline corrected and the peaks were identified using Speckwin software.

TGA analysis

The thermal stability of the electrospun membrane was studied using PerkinElmer TGA 4000 unit. A small piece of sample (3 mg) was placed on the measuring unit and the heating rate was performed. The TGA analysis was carried out under dry nitrogen atmosphere at an ascending rate of 10°C/min with temperature range of 30–1000°C. TGA and DTG curve was drawn from the obtained data’s using excel sheet.

AFM analysis

To calculate the surface roughness, AFM analysis was performed using Nanowizard, JPK instruments. A small piece of sample was placed on the scanning surface and the scanning was performed at room temperature in normal atmosphere. The scanning was performed in 20 * 20 μm sizes the 3D image with 256 * 256 pixels was captured using JPKSPM data processing software.

Blood compatibility analysis

APTT and PT assay

APTT assay was used to determine the intrinsic pathway of the blood clot while the PT assay determines the extrinsic pathway. To begin the APTT assay, the developed samples were washed with PBS and incubated at 37°C for 30 min. After incubation, the samples were mixed with 50 μl of PPP for 1 min 37°C followed by adding 50 μL of rabbit brain cephaloplastin reagent and CaCl₂ (0.025 M) solution for 3 min 37°C. The mixture was gently stirred which results the formation of the blood clot and APTT was measured. For the PT assay, the procedures were same as the APTT assay where the electrospun membrane mixed with 50 μl of PPP was further incubated with 50 μl of thromboplastin reagent (Factor III) for 3 min at 37°C. Finally, the blood clot formation was done by stirring and PT was measured [35].

Hemolysis assay

To start the assay, the electrospun membrane (1 cm ×1 cm) was soaked in 0.9% w/v of saline at 37°C for 30 min. After soaking, the samples added with the mixture of citrated blood and diluted saline (4:5 v/v%) for 1 h at 37° C. Then, the samples centrifuged at 3000 rpm for 15 min and OD was measured. The absorbance was measured at 542 nm with pipetted supernatant which indicates the release of hemoglobin. The percentage of hemolysis or hemolytic index was calculated as described earlier [35].

Cell culture and MTS assay

The cell viability of the electrospun scaffolds were determined using HDF cells. Initially, HDF were cultured using DMEM medium with 10% fetal bovine serum and incubated at 37°C and 5% carbon dioxide (CO₂). The medium was replaced for every 3 days. To begin the cell seeding onto the electrospun scaffold, the samples were cut and placed in the 24 well plates and sterilized with 75% of alcohol solution. After sterilizing, the scaffolds were washed with PBS solution. Then, HDF cells with density of 1.0×10⁵ cells were seeded per scaffold in each well and incubated at 37°C with 5% CO₂. The MTS assay was used to determine the cell viability rates in the electrospun membranes after 72 hr incubation. After 3 days culture, the cell seeded scaffolds were washed with PBS and added with 20% of MTS solution (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt) and further incubated for 4 hr. After 4 hr, the culture plates were retrieved and absorbance was measured at 490 nm using spectrophotometer to measure the cell counts in the fabricated membranes.

Statistical analysis

All experiments were performed thrice independently and the Unpaired t-test was carried out to calculate the statistical significance. All experiment results are expressed as mean ± SD and for qualitative experiments, a representative of three images is shown.

Results

SEM investigation

The morphology and EDS of the electrospun PU, PU/grapeseed oil and PU/grape seed oil/honey/propolis scaffold were shown in Figs 1 and 2. Further, Tables 1–3 depicts the elemental analysis for the electrospun PU, PU/grapeseed oil and PU/grape seed oil/honey/propolis scaffold. From the SEM image, it was observed that the prepared PU, PU/grape seed oil and PU/grape seed oil/honey/propolis scaffolds showed uniform fibers without any beads. The fiber diameters of electrospun PU, PU/grape seed oil and PU/grape seed oil/honey/propolis scaffold were found to be 890 ± 116.911 nm, 817 ± 155.45 nm and 601 ± 151.76 nm respectively and the distribution curve was shown in Fig 3.

Fig 1 — SEM images of a) Polyurethane b) Polyurethane/ grape seed oil composites c) Polyurethane/grape seed oil/propolis/honey composites.

Fig 2 — EDS spectrum of a) Polyurethane b) Polyurethane/ Grape seed oil composites c) Polyurethane/Grape seed oil/propolis/honey.

Table 1. Elemental analysis of PU membrane.

Element	Weight (%)	Weight (% σ)	Atomic (%)
Carbon	70.717	1.290	79.645
Oxygen	23.614	1.237	19.966

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Table 3. Elemental analysis of PU/Grapeseed oil/Propolis/Honey composite.

Element	Weight (%)	Weight (% σ)	Atomic (%)
Carbon	73.942	0.771	81.057
Oxygen	22.749	0.754	18.722

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Fig 3 — Fiber diameter distribution of a) Polyurethane b) Polyurethane/ grape seed oil composites c) Polyurethane/grape seed oil/propolis/honey composites.

Table 2. Elemental analysis of PU/Grapeseed oil composite.

Element	Weight (%)	Weight (% σ)	Atomic (%)
Carbon	77.212	0.745	83.349
Oxygen	20.348	0.728	16.490

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FTIR analysis

Fig 4 indicates the absorption bands present in the electrospun PU, PU/grape seed oil and PU/grape seed oil/honey/propolis scaffold. The spectrum of PU showed a wide band at 3323 cm⁻¹ indicating presence of N-H stretching and the peaks at 1597 cm^-1 and 1531 cm^-1 denotes the vibration of NH. The peaks seen at 2939 cm^-1 and 2854 cm^-1 corresponds to the CH₂ stretching and the peak at 1413 cm^-1 was attributed to the vibrations of CH₂. The C = O stretching corresponding to carboxylic groups was shown by a twin peak at 1730 cm^-1 and 1703 cm^-1 and the C-O stretching attributed to alcohol groups were seen at peaks 1221 cm^-1,1104 cm^-1 and 1078 cm^-1 respectively [35]. There were no additional peaks formed in PU/grape seed oil and PU/grape seed oil/honey/propolis scaffold, but there was a change in peak intensity with the incorporation of additives.