Effect of HA Nanoparticles on Adsorption of Vitamin D3 on Super-Hydrophobic PA6 Nanofibrous Scaffold

ABSTRACT

Vitamin D₃ has significant roles in bone growth and the prevention of osteoporotic fractures. The present study investigated the effect of hydroxyapatite nanoparticles decoration onto polyamide-6 nanofibrous scaffold on adsorption behaviour of Vitamin D₃. To synthesize the nanofibrous scaffold, an electrospinning device was used, and the surface of the scaffold was characterized by scanning electron microscopy, energy dispersive spectrometry, Fourier transform infrared spectroscopy, and measurement of the water contact angle. The antibacterial activity test against E. coli and S. aureus bacteria indicated no such activity of pristine and hydroxyapatite decorated scaffold. The results demonstrated that a hydrophobic and high porous scaffold was formed, and hydroxyapatite nanoparticles were distributed inside the polyamide-6 nanofibers homogenously. Results showed that the hydroxyapatite nanoparticles improved the adsorption efficiency of polyamide-6 scaffold. It was found that the amount of adsorbed Vitamin D₃ molecules onto the polyamide-6/HA scaffold was rapid during the first hour of immersion (24.4 ng.cm^-3), then declined over the next 3 h, and eventually reached a stable percentage of about 10.3 ng.cm^-3. This phenomenon appears to be related to the high adsorption potential of porosities and the hydrophobic nanofibers during the first stage of immersion and non-occupied hydroxyapatite ceramic sites during the final stage of immersion.

Keywords
Hydroxyapatite; Nanofibrous scaffold; Surface adsorption; Vitamin D₃

1. INTRODUCTION

Vitamin D₃ (VD₃) is an essential organic compound that has a significant role in bone growth and is required in the production of collagen, absorption of calcium and phosphorus, and the regulation of the immune system [¹-⁵]. On the other hand, the lack of VD₃ is a major health problem [⁶,⁷]. According to clinical interpretations, the adequate blood level of VD₃ is 30-36 ng.ml^-1, and above 100-150 ng.ml^-1 is considered as toxic [⁸].

Plenty of studies have focused on the biological properties of VD₃ and there are very few reports about colloidal and particle systems for the delivery of VD₃ [⁹,¹⁰]. However, there have been no reports about the physiochemical behaviour of VD₃ in relation to nanoporous media such as electrospun nanofibrous scaffolds. Nanofibrous scaffolds, which are fabricated by electrospinning, have numerous applications in tissue engineering due to having a polymer-ceramic composite scaffold and a nanoporous structure [¹¹-¹³]. The scientific importance of the study of VD₃ adsorption by a PA6/HA nanofibrous scaffold is explained by the critical role of VD₃ in bone growth. A balanced amount of VD₃ in the surrounding environment has a significant role in bone regeneration during long term scaffold implantation.

Hydroxyapatite (HA) is well known as a bioactive ceramic added to many artificial bone tissues and implants for orthopedic surgeries [¹⁴-¹⁶]. Not only polymer/HA composites exhibit the chemical and mechanical stability of natural bones [¹⁷,¹⁸], but also they can be designed in the form of porous materials that accelerate the circulation of essential biochemical agents such as VD₃ [¹⁹]. Given the significant chemical (e.g. stability in human body) and mechanical (e.g. tensile strength and toughness) properties of polyamides, polyamide-6 (PA6) is utilized as the polymeric base of many biocomposite scaffolds [²⁰,²¹].

Adsorption can occur through different routes by physical, chemical or physiochemical reactions between adsorbent and adsorbed species [²²,²³]. The hydrophobicity of the surface of the adsorbent has a significant role in the adsorption of vitamins and proteins [²⁴-²⁶]. Hydrophilic surfaces are wetted by the surrounding environment and do not allow more amino acid molecules (e.g VD₃) to adsorb onto the scaffold [²⁷]. HA NPs are bound to the super hydrophobic PA6 fibers, helping achieve greater VD₃ adsorption by these nanoparticles during implantation.

The present study has evaluated a nanobiomaterial, fabricated by the electrospinning procedure, in terms of its adsorption of VD₃, which is essential for bone growth. For this purpose, PA6 and PA6/HA nanofibrous scaffolds was firstly synthesized by electrospinning and then characterized by SEM, EDS, FTIR, and WCA measurements. Finally the adsorption behaviour of VD₃ was fundamentally investigated to determine the responses of the electrospun scaffolds to VD₃ molecules.

2. MATERIALS AND METHODS

2.1 Scaffold preparation

The HA NPs were synthesized in accordance to the wet chemical precipitation method commonly described in literature [²⁸,²⁹]. The polymer solution was prepared by dissolving 2 and 20 wt% of HA NPs and PA6 granules (1015b UBE, M_w; 15,000 g.mol^-1) in formic acid (Sigma Aldrich, 64-18-6), respectively. The components were dispersed in solution by the application of stirring and ultrasonication for 10 min at room temperature. The homogenous solution was spun by means of electrospinning which was setup with the following conditions: 15 kV apply voltage, 0.5 ml h^-1 feed rate and 10 cm distance. The collected fibers were dried on a flat aluminium foil and then cut to appropriate size for further investigations.

2.2 Characterization of scaffolds

The thickness of the scaffolds was measured using a micrometer and the average of 10 measurements was reported. In order to study the morphology of the electrospun scaffold, the sample was sputter coated with gold and then scanning electron microscopy (JEOL- JSM 840A SEM) was applied at an accelerating voltage of 5 kV. Further investigations were conducted with ImageJ software (ImageJ 1.38, NIH, USA) to determine the diameter of the fibers and surface porosity. The morphology and composition of HA NPs and also the elemental distribution within the PA6/HA scaffold were analysed using SEM equipped with EDS setup (model Vega\TScan-20 kV). Functional molecular groups formed in the composite scaffold were evaluated using the Fourier transform infrared (FTIR) technique (Nicolet Avatar 360 FT-IR spectrometer, 4000 cm^-1 to 400 cm^-1). The hydrophobicity of the scaffolds during the first minute was studied regarding water contact angle measurement (WCA) in three different locations of the samples by using a contact angle meter (VCA Optima-AST products, Billerica, MA).

2.3 Antibacterial activity test

The antibacterial activity test of PA6 and PA6/HA scaffolds were performed in a solution contacting two kinds of bacteria in accordance to the ASTM G21-1996. The S. aureus (ATCC 25923) and E. coli (ATCC 25922) were the gram-positive and gram-negative bacteria in the solution, respectively. The samples were incubated for 24 h at 37 ˚C to evaluate their antibacterial activity.

2.4 VDs adsorption procedure

A 1 ml VD₃ ampoule (Cholecalciferol) containing 6.16 mg of was dissolved in 37.5 ml of deionized water by stirring at room temperature. Both sides of the PA6 and PA6/HA scaffolds (1cm×1cm) were vertically exposed to 8 ml of VD₃ solution (160 ng.ml^-1) at room temperature as shown schematically in Fig. 1. Similarly, 7 scaffolds were immersed in distinct cells in order to study the effect of soaking time on adsorption of VD₃ onto mentioned scaffolds during a period of 7 h. The quantity of adsorbed VD₃ (Q_t) on the scaffolds at any time was calculated according to the following equation [²²];

Figure 1
Set-up of VD₃ adsorption test.

$$Q_t=\frac{\left(C_i-C_f\right)·V}{A·T}$$ (1)

where C_i and C_f (mg.ml^-1) are the initial and remaining concentration of VD₃ in solution, respectively. V (ml) is volume of the VD₃ solution, A (cm²) and T (cm) stand for area and thickness of scaffolds respectively. The remaining concentration of VD₃ in solution was calculated using Beer's law [³⁰]. For this purpose, the adsorption intensity of the surrounding solution was measured by a UV-VIS instrument (model: Cary, 100 Cone) and C_f was calculated according to the calibration curve.

3. RESULTS

3.1 Characterization of the scaffold

Prior to the fabrication of scaffolds, the synthesized HA powder was characterized regard to the morphology and composition. Fig.2 a and b present the SEM micrograph and EDS spectrum of HA powder, respectively. As can be seen, the semi-spherical particles with average size of 25.2 ± 3 nm were obtained via wet chemical precipitation method. These specifications are appropriate for blending in electrospun solution. The Ca/P ratio in HA NPs was also calculated based on the EDS spectrum, and it was found that mentioned ratio was 1.62 indicating the well coincidence with theoretical value [¹⁶]. The typical morphology of the prepared PA6 and PA6/HA scaffolds are shown in Fig. 2 c and d. It is obvious that HA NPs have been incorporated in the final microstructure of the scaffold. The HA NPs have been marked by circles, demonstrating the uniform distribution of HA aggregates within the nanofibers (NFs). According to their morphology, some NPs are attached to the stems of the fibers and some NPs are precipitated together at the junction points of the fibers. The physical specifications of nanofibrous scaffolds are given in Table 1. The obtained results declare the formation of extremely high porous, two-dimensional scaffolds, which are promising for the tissue engineering and drug delivery [³¹]. Moreover, the different chemical composition of nanofibrous scaffolds may make the different manner in VD₃ adsorption regard to the surface properties.

Figure 2
(a) SEM micrograph and (b) EDS of HA NPs, (c) PA6 and (d) PA6/HA SEM morphology of prepared scaffolds, the HA-NP is marked by circle.

Table 1
Physical specifications of nanofibrous scaffolds

Scaffold	Thickness of scaffold (µm)	Fibers diameter (nm)	Surface porosity (%)
PA6	120 ±10	265 ±10	3 ±2.3
PA6/HA	130 ±10	382 ±30	1 ±5.1

In addition, EDS analysis was assigned to ensure the incorporation of HA NPs into the PA6 scaffold. The microstructure and EDS analysis of a typical area of the PA6/HA scaffold are presented in Fig. 3. The white spots in the back scattered mode SEM image are attributed to HA aggregates and the grey spots belong to the junction of PA6 NFs (see Fig. 3.a). In EDS analysis, the highest peak in the spectrum shown in Fig.3.b at 1.5 eV is attributed to the carbon (C) element which constitutes the main structure of the PA6 scaffold. The incorporation of HA particles within the nanofibrous structure was also confirmed by EDS due to the presence of Ca and P peaks at 4 and 2 eV, respectively.

Figure 3
(a) BS-SEM image and (b) EDS analysis of typical area of PA6/HA scaffold.

In order to identify the functional molecular groups formed in the polymer/ceramic electrospun scaffold, first FTIR spectra of HA NPs and PA6 scaffold were provided as illustrations in Fig.4, and then those were compared with FTIR spectrum of PA6/HA scaffold. The prominent twin peaks at 1546 and 1641 cm⁻¹ demonstrate the polyamide nature of the scaffold. The N-H stretching vibration bond of PA6 was detected at 3298 cm⁻¹, and CH₂ asymmetric stretching bonds were recognized by 1265, 1368, 1460, and 2931 cm⁻¹ bands. The bands at 2861 cm⁻¹ and 1170 cm⁻¹ are attributed to stretching and wagging vibrations bond of CH₂, respectively [³²]. Furthermore, characteristic bands related to the HA particles were also observed in the FTIR spectrum of PA6/HA. These included a low intensity band at 928 cm^-1 assigned to the stretching vibration of P-O(H) and transmission bands at 582 cm^-1 and 1075 cm^-1 attributed to the bending and stretching vibrations of P-O bonds, respectively. The other bands, such as those transmitted at 711 and 3086 cm^-1, are related to the polyamide nature of the matrix [³³]. Therefore, based on the experimental data from SEM, EDS and FTIR, it can be elucidated that HA NPs were incorporated inside of the PA6 NFs matrix without the creation of any new bonds.

Figure 4
FTIR spectrum of (a) HA NPs, (b) PA6 and (c) PA6/HA scaffold.

Surface hydrophobicity is one of the significant characteristics which determine the biological response of a surface by affecting the arrangement of biomolecules near to the surface. Hence, the identification of the wettability of the PA6/HA scaffold by means of WCA measurement was very important for understanding its behaviour during immersion in an aqueous solution of VD₃. Fig. 5 shows the variation of WCA on PA6 and PA6/HA scaffold surfaces during the first minute of contact. It is obvious that the PA6 scaffold is super hydrophobic because the average WCA during contact time was higher than 120º. In spite of HA presence, PA6/HA also depicted a hydrophobic surface. Therefore, it is hypothesized that VD₃ molecules can arrange in a stable manner near the hydrophobic surface of the PA6/HA scaffold at preliminary time of adsorption test. This phenomenon allows the molecules to adsorb onto the surface in favourable conditions [³⁴]. However, the WCA on PA6/HA scaffold decreased over one minute due to the unstable surface tension forces between liquid, solid and gas phases (water, scaffold and air respectively) [³⁵].

Figure 5
Variation of WCA on PA6 and PA6/HA scaffold surfaces during the first minute of contact.

3.2 Antibacterial activity

With developing the advanced materials in biomedical application, the antibacterial property of introduced material has been drawn to attention. The assessment results of the antibacterial activity of PA6 and PA6/HA scaffolds against E. coli and S. aureus showed that both scaffolds have no activity against both E. coli and S. aureus bacteria. There are many studies which have demonstrated that PA6 does not exhibit antibacterial activity [³⁶,³⁷]. The HA incorporation inside the PA6 NFs did not influence the antibacterial activity of PA6. The neutralized HA NPs have not interacted with the walls of the gram negative and positive bacteria [³⁸].

3.3 Vitamin D₃ adsorption

Due to the upper limit of VD₃ in the human body and the importance of its values in aqueous solution, the concentration variation of VD₃ in the range of adequate to toxic values was examined according to Beer's law. Fig. 6.a shows the UV spectra of VD₃ solutions with different concentrations within the mentioned range. It shows an absorbance peak at 257 nm, revealing the VD₃ variation in different concentrations from 20 to 160 ng.ml^-1. As shown in Fig. 6.b, the calibration curve was plotted with respect to the aforementioned amounts in order to generate an appropriate linear equation. Experimental data have good correspondence with the line predicted from Beer's law, demonstrating the validity of the data from the examination of VD₃ adsorption. Furthermore, the large correlation coefficient (R²= 0.9836) demonstrated the appropriate dissolution of VD₃ in solvent and the production of a homogenous aqueous solution.

Figure 6
(a) UV spectra of VD₃ solution with different concentration, (b) calibration curve in range between 20 to 160 ng.ml^-1.

The response of the PA6 and PA6/HA scaffolds to VD₃ adsorption over a 7 h period was evaluated by Q_t index given in Eq. 1. Fig. 7 shows the variation of Q_t versus the immersion time. The variation of Q_t display the rather constant amount adsorbed by PA6 scaffold which was about 5 ng.cm^-3. In contrast, the results showed that the PA6/HA scaffold adsorbed VD₃ molecules with high efficiency over the first hour of immersion. The amount of adsorbed VD₃ declined from 24.4 to 8.3 ng.cm^-3 during the period of 1 to 4 h. Finally, adsorption efficiency improved partially and then remained constant during the period of 4 to 7 h. The final recorded amount for adsorbed VD₃ onto PA6/HA scaffold was 10.3 ng.cm^-3. Therefore, it was found that the HA NPs improve the adsorption efficiency, and three distinct stages can be assumed for adsorption: stage I: adsorption, stage II: desorption, and stage III: stability.

Figure 7
Amount of adsorbed VD₃ on PA6 and PA6/HA scaffolds, in period of 7 h.

The micrographs pertaining to the adsorption test of the VD₃ on PA6/HA scaffold is presented in Fig. 8. A high porous scaffold with interconnected and open porosities was observed in the sample at the starting point of the test (Fig. 8.a). This appearance dramatically changed only 0.5 h after immersion in VD₃ solution. As it can be seen in Fig. 8.b, not only the surface porosities were blocked by VD₃ molecules but also the fibers were covered by a thin layer of VD₃. Extreme adsorption also occurred for the sample which was immersed for 2 h (see Fig. 8.c). These observations are in strong agreement with results obtained by Q_t calculations. The maximum adsorption was recorded after 1 h immersion, relating to the end of stage I. More micrographs showed that blocked porosities were reopened over time, indicating the release of VD₃ into the surrounding solution. The increase in open porosities and the removal of VD₃ from NFs are demonstrated from the micrographs of the sample immersed from 2 to 4 h. This behaviour coincides with stage II and can be recognized in the area shown in Fig. 7. A similar behaviour was also observed for the immersed sample at 5 h. It is worth mentioning that the amount of adsorbed VD₃ by the last two samples was rather similar. Eventually, a rather blocked porous scaffold was observed throughout the final stage. The related micrograph may predict the long-term appearance of a PA6/HA scaffold implantation.

Figure 8
SEM micrographs of PA6/HA scaffold regarding to different times of immersion in VD₃ solution.

It seems that during stage I, VD₃ suddenly made a concentration gradient to adsorb on an extremely large surface area of the nanofibrous scaffolds. This rapid adsorption occurred due to the high amount of open pores at surface of scaffolds, as well as the super hydrophobic nature of the adsorbent. However, incorporation of HA NPs could intensify the adsorption efficiency of PA6 scaffold three of times. Afterwards, the main reason for the release of VD₃ from the scaffolds during stage II was the wetting of the surface after 1 h of immersion. This phenomenon resulted in repulsing VD₃, and thus the amount of adsorbed VD₃ decreased rapidly. This is in strong agreement with results reported in other research which describe that the wettability of polymeric substances is increased by the addition of amino acid species such as proteins and vitamins [5,31]. It is worth mentioning that hydrophilic HA sites could promote the wettability of scaffold too. During stage III, HA sites of scaffolds had significant roles in holding VD₃. This is proven by the rather uniform distribution of adsorbed VD₃ on the scaffold surface as observed in the micrograph shown in Fig. 8 related to the sample at 7 h. Moreover, the amount of adsorbed VD₃ did not change perceptibly because the HA sites were occupied by the previously adsorbed VD₃. Finally, an equilibrium gradient was established between the solution and scaffold resulting in a scaffold with an area occupied by VD₃ for osteoblast progress, and open porous media for necessary biological interactions [³⁹].

4. CONCLUSIONS

The goal of this study was to explore the adsorption behaviour of VD₃ onto electrospun nanofibrous PA6 scaffolds decorated by HA NPs. The SEM micrographs of the scaffold showed a relatively homogenous distribution of HA particles within the PA6 NFs. The average diameter size of fibers and surface porosity of PA6/HA scaffold were 213±30 nm 72.1 ± 5.1 %, respectively. EDS analysis of the PA6/HA scaffold confirmed the presence of HA particles within the fibers, and FTIR spectrum also depicted the incorporation of HA particles within the PA6 matrix, while new bonds were not formed between polymer fibers and ceramic particles. The variation of WCA during one minute signified the hydrophobic nature of the PA6/HA scaffold, helping the scaffold to adsorb more vitamin molecules. The variation of Q_t showed that the adsorption of VD₃ occurred in three stages: (I) adsorption, (II) desorption, and (III) stability. Experimental data showed that amount of adsorbedVD₃ molecules onto the PA6 and PA6/HA scaffolds was about 8.1 and 24.4 ng.cm^-3 over the first hour of immersion, and eventually reaching a stable amount of about 5 and 10.3 ng.cm^-3 respectively, which depicts the significant role of HA NPs in VD₃ adsorption. The morphology study of the PA6/HA scaffold at different times of immersion indicated dramatic changes in fibre thickness and blocked porosities after 1 h of immersion. However, an open porous media with fibers occupied by VD₃ persisted after 7 h of immersion. With the results obtained from the evaluation of VD₃ adsorption by a PA6/HA scaffold, the opportunity of creating totally controlled conditions for bone regeneration within implantations arises.

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Publication Dates

• Publication in this collection
  17 Apr 2020
• Date of issue
  2020

History

• Received
  10 July 2018
• Accepted
  30 Sept 2019