Microwave-induced rapid formation of biomimetic hydroxyapatite coating on gelatin-siloxane hybrid microspheres in 10X-SBF solution

Farnaz Ghorbani; Ali Zamanian; Aliasghar Behnamghader; Morteza Daliri Joupari

doi:10.1515/epoly-2017-0196

Article Open Access

Microwave-induced rapid formation of biomimetic hydroxyapatite coating on gelatin-siloxane hybrid microspheres in 10X-SBF solution

Farnaz Ghorbani , Ali Zamanian , Aliasghar Behnamghader and Morteza Daliri Joupari

Published/Copyright: January 25, 2018

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From the journal e-Polymers Volume 18 Issue 3

Abstract

Bioactive materials can attract calcium and phosphate ions in simulated body fluid (SBF) solution to mimic the composition of extracellular matrix (ECM). Rapid biodegradation rate of natural polymers in contact with water-based solutions and time-consuming process of mineralization in SBF led to using concentrated simulated media. Herein, gelatin-siloxane microspheres were fabricated via single emulsion method. Then hybrid spheres were immersed in the modified 10X-SBF solution, and microwave energy (600 W) was expanded for the rapid formation of hydroxyapatite (HA) on the spheres. Results indicated homogeneous coating of microspheres and high similarity of synthesized HA to the bone composition. Increasing intensity of HA-related peaks in Fourier transform infrared spectrum, X-ray diffraction and surface roughness after utilizing microwave-assisted method confirmed high efficiency of this technique in biomimetic mineralization of structures. Cell culture studies with human osteosarcoma cell lines (MG-63) demonstrated that mineralized HA in 10X-SBF solution under microwave treatment could be able to mimic bone ECM for tissue regeneration applications in the shortest time and highest similarity to the natural tissue.

Keywords: 10X-SBF; gelatin microsphere; microwave; mineralization; siloxane

1 Introduction

A large number of investigations have focused on hybrid organic-inorganic structures, which include polymers and ceramics, to achieve a high degree of similarity to natural bone structure and character (1). Chao et al. (2) fabricated gelatin-hydroxyapatite (HA) porous microspheres, and they found that constructs can support new bone mineralization and indicated desirable osteoconductivity. Natural polymers such as collagen and its derivatives, carboxymethyl cellulose (3), silk, chitosan (4), etc., have found versatile applications in biomedical areas due to the resemblance of their features with the human body composition, support cellular proliferation and processability (5). However, lack of osteoconductive and mechanical features in the introduced polymers have been led to the fabrication of blend systems of organic and inorganic components (6). Thus, the presence of a bioactive inorganic component in polymeric matrixes supports the bioactivity behavior while providing required mechanical properties (7).

HA is the primary component of natural bone that has been developed for biomedical applications (8). Various techniques have been introduced for fabrication of HA-contained platforms. Nonetheless, biomimetic mineralization in simulated body fluid (SBF) solution has gained popularity over the past decades due to abundant production, better simulation of the bone extracellular matrix (ECM), the lack of transmission viruses or bacteria agents, compliance with the immune system, etc. (9). Most of the polymeric materials cannot provide proper sites for significant absorption of calcium and phosphate ions in order to nucleation of HA, so they need to modify with functional groups to show bioactive behavior. Silane coupling agents are one of the bioactive modifiers that interact with epoxy and amino groups of organic materials like gelatin. When modified structures immersed in SBF solution, hydrolysis of alkoxy, acetoxy, etc., functional groups by water result to create silanol for bonding with inorganic compounds and fabrication of HA layer (10).

The time-consuming process of HA coating and differences in ion concentration of SBF with the human body led to the introduction of the new formulation by Tas et al. (11). In modified SBF, NaH₂PO₄·H₂O was utilized instead of K₂HPO₄·3H₂O; therefore, chlorine ions became more concentrated, and the concentration of hydrogen carbonate ions reached to the body values (Table 1). Moreover, lack of necessity to the addition of pH adjustment agents terminated more similarity of Tas SBF to the human plasma. Studies have been suggested that 10 times concentrated SBF accelerated the formation of mineralized layers by providing a higher amount of required ions (12), (13), but it cannot provide the homogeneous coating layer led to lack of achieving to optimal physiological response.

Table 1:

Ion concentration of modified SBF compared with the human plasma (11).

Ion	Kokubo SBF (mm)	Tas SBF (mm)	Human plasma (mm)
Na⁺	142.0	142.0	142.0
K⁺	5.0	5.0	5.0
Mg²⁺	1.5	1.5	1.5
Ca²⁺	2.5	2.5	2.5
Cl⁻	147.0	125.0	103.0
HCO₃⁻	4.2	27.0	27.0
HPO₄²⁻	1.0	1.0	1.0
SO₄²⁻	0.5	0.5	0.5

Recently, microwave energy has been expanded because of energy and time-saving and environmentally friendly procedure (14). This technique can support rapid, nanoscale and homogeneous mineralization of three-dimensional structure via mechanisms of dipole interaction and ionic conduction (15) without any adverse effects on the chemical structure of polymers. High degree of uniformity and resemblance of the coating layer with biological HA can be the strength of this technique. The other similar study evaluated the synergistic effect of heating and 10X-SBF for coating the Ti and observed mentioned phenomena (16).

In this study, gelatin microspheres were fabricated by single emulsion technique and were directly cross-linked by GLYMO. Silane-modified spheres were homogeneously coated with HA through immersion in 10X-SBF solution and synergistic effects of concentrated modified SBF and microwave irradiation as a facile and controllable coating method was studied. Microwave-induced HA was compared with conventional coating systems in terms of morphological, chemical and biological analyses. Biological evaluation was assessed by seeding human osteosarcoma cell line (MG-63) to determine biocompatibility, adhesion and alkaline phosphatase expression.

2 Experimental

2.1 Materials

Gelatin (M_w=40–50 KDa), (3-glycidyloxypropyl)trimethoxysilane (GLYMO, M_w=236.34 s g/mol) and glutaraldehyde (25%, d=1.058 g/cm³) were purchased from Merc Co. Ltd. (Germany). Sodium chloride (NaCl, M_w=58.44 g/mol), potassium chloride (KCl, M_w=74.55 g/mol), sodium bicarbonate (NaHCO₃, M_w=84.01 g/mol), calcium chloride dihydrate (CaCl₂·2H₂O, M_w=147.01 g/mol), magnesium chloride hexahydrate (MgCl₂·6H₂O, M_w=203.30 g/mol), sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O, M_w=137.99 g/mol), ethanol (99.8%, M_w=46.07 g/mol) and acetone (99.9%, M_w=58.08 g/mol) were purchased from Samchun Pure Chemicals Co. Ltd. (Korea). Olive oil, thiazolyl blue tetrazolium bromide (MTT, M_w=414.32 g/mol), Triton X-100 and dimethyl sulfoxide (DMSO, 1X) were purchased from Sigma Co. Ltd. (USA). Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco-BRL, Life Technologies Co. Ltd. (NY, USA). Alkaline phosphatase kit (ALP) was purchased from MAN Co. Ltd. (Iran). All chemicals were utilized directly without further purification. Aqueous solutions were prepared with deionized (DI) water.

2.2 Synthesis of gelatin-siloxane microspheres

Gelatin-siloxane microspheres were synthesized by the single emulsion method. Thus, gelatin was dissolved in DI water with a concentration of 20% (W/V) at 40°C for 12 h; 20 wt.% of the GLYMO was added to the solution and stirred for 2 h. The solution was added drop-wisely to stirred-oil phase (olive oil) at 2000 rpm (aqueous solution: oil phase=1:50). The prepared microspheres were aged for 14 days at 4°C. After that, oil top of the deposited-microspheres was removed, samples were washed by cooled acetone under 550 W ultrasonication (WUC-D10H, WISD Co., South Korea) for 15 min and finally air-dried.

2.3 Biomimetic bonelike hydroxyapatite coating

The 10X-SBF solution was prepared based on an investigation of Tash et al. (17). Briefly, all the chemical reagents except NaHCO₃ were dissolved in 900 ml DI water at ambient temperature (Table 2). Then remained DI water was added to the solution and pH received to 4.3–4.4. The prepared solution was kept at 4°C until utilization. Before addition of microspheres, 10 mm of NaHCO₃ was added to the stock solution and pH reached to 6.5.

Table 2:

Reagent concentration of 10X-SBF solution.

Reagents	Amount (g) – 1 l	Concentration (mm)
NaCl	58.443	1000
KCl	0.373	5
CaCl₂·2H₂O	3.675	25
MgCl₂·6H₂O	1.016	5
NaH₂PO₄·H₂O	0.250	3.62
NaHCO₃	0.084	10

For biomimetic mineralization, 50 mg of bioactive gelatin microspheres was immersed in 50 ml 10X-SBF solutions. The final blend was placed under two different conditions:

Group 1: Coating the microspheres by 10X-SBF in a thermoshaker (LS-100, Thermo Scientific, USA) with the rotational speed of 30 rpm and temperature of 37±0.5°C for 8 h. The media were refreshed every 2 h.

Group 2: Coating the microspheres by 10X-SBF in a microwave oven (KOR6N7RS, Daewoo Company, UK) for nine times (30 s operation and 30 s rest) at 600 W.

Finally, all the microspheres were washed with ethanol (two times) and DI water (three times), then lyophilized in a freeze dryer (FD-10, Pishtaz Engineering Co. Iran) at a temperature about −58°C and pressure 0.5 Torr for 24 h. Table 3 shows the characteristics of synthesized spheres.

Table 3:

Composition of gelatin microspheres.

Code	Gelatin concentration	GLYMO concentration	Mineralization method
GGM	20% (W/V)	20 wt.%	–
GGM10S8	20% (W/V)	20 wt.%	Conventional
GGM10SM	20% (W/V)	20 wt.%	Microwave-assisted

2.4 Morphological observations

The microstructure of the gelatin-siloxane microspheres and hydroxyapatite-coated spheres was determined by field-emission scanning electron microscopy (FE-SEM, MIRA3, TESCAN Co., Czech Republic) at an accelerating voltage of 15 kV. All the samples were sputter coated (Emitech K450X, Ashford, UK) with a thin layer of gold at 45 mA. Energy-dispersive X-ray spectroscopy was performed to determine Ca/P ratios of HA deposited-hybrid spheres.

2.5 Chemical characterization

The infrared spectrum of dried-spheres was obtained by a Fourier transforms infrared spectrophotometer (FTIR, Nicolet Is10, Thermo Fisher Scientific, USA). Hence, the composite samples were mixed with KBr in ratio 1:50 and pelletized under vacuum. The pelletized constructs were analyzed between 400 and 4000 cm⁻¹ with a resolution of 4.0 cm⁻¹ and eight scans.

2.6 Phase analysis

The X-ray diffraction (XRD, Philips PW3710) of the mineralized samples were carried out through using Cu-Kα radiation from a rotational anode generator at 40 kV and 30 mA in the range of 2θ=5–80°.

2.7 Atomic force microscopy

The surface roughness was performed after placing gelatin-siloxane microspheres onto cleaved mica and then measured by atomic force microscopy (AFM, Proberstation 150 with DS95 SPM head, DME Co., Denmark) analysis. AFM setup was set to the resonance frequency of 50–105 kHz and force constant 0.15–1.5 N/m.

2.8 Cell culture studies

Cell culture studies were followed by culturing human osteosarcoma cell lines (MG-63, Materials and Energy Research Center) on microspheres as described in previous study (18). Briefly, 5×10⁴ cells/ml was seeded within the 20-mg sterile spheres and immersed in DMEM with phenol red supplemented with 10% (v/v) FBS, 1% penicillin-streptomycin at 37°C, 5% CO₂ and 95% humidity. During every 30 min, the cell-loaded samples shaken at 30 rpm for 2 min. The cell-microsphere interactions were assessed after 2 days. Thus, polymeric constructs were fixed with 2.5% glutaraldehyde solution for 2 h at ambient temperature and were dehydrated through ascending concentration of ethanol solutions for SEM observations.

Biocompatibility of microspheres before and after mineralization was evaluated using MTT assay. Therefore, spheres immersed in cell culture medium in contact with MG-63 cells for 2 and 6 days. The precipitated formazan dissolved in DMSO to the measured optical density of the solution at a wavelength of 570 nm. Cell culture plate introduced as a control group (100% cell viability).

ALP activity was followed as described before (19). Proliferated cells were lysed in 0.1% Triton X-100 and freeze-thawing at 37°C. ALP activity was determined at days 2 and 6 after seeding according to the MAN company instructions at a wavelength 405 nm.

2.9 Statistical analysis

Results were presented as the mean±standard deviation of at least five experiments and were processed using Microsoft Excel 2013 software. The significance of the average values was calculated using a standard software program (SPSS GmbH, Munich, Germany), and p≤0.05 was considered significant.

3 Results and discussion

Morphology and microstructure of hybrid microspheres were determined by FE-SEM micrographs and elemental characterization (Figures 1 and 2). Figure 1 indicates spherical shape, smooth, uniform and approximately monodisperse gelatin-siloxane microparticles without any wrinkles. The incoherence of particles to each other is the other point of strength in synthesized spheres. As Sun et al. (20) indicated the optimum concentration of gelatin, usage of olive oil with higher viscosity compared with other types of oil, water-to-oil ratio, stirring speed and time of aging led to the realizing these results. Gelatin spheres were modified using GLYMO to induce bioactive behavior. Providing the covalent links for cross-linking hydrophilic functional groups in a polymer led to the introduction of silanol groups in the chemical composition. Silane coupling agents induce bioactive properties to the structures, increase mechanical stability and improve adhesiveness behavior; the same results were obtained in other investigations (21), (22), (23). Silanol groups in GLYMO act as a nucleation site for more sedimentation of calcium and phosphate ions (15). Other study noted that -COOH and -NH₂ groups in gelatin affect the in situ formation of HA (24) in which carboxyl groups have shown higher activity in the attraction of calcium and phosphate ions compared with amines (25). Moreover, negatively charged functional groups on the surface of gelatin tend to attract calcium ions in 10X-SBF solution via electrostatic attraction. The changes in surface charge were followed by precipitation of phosphate ions and ingrowth of calcium phosphate layers. Obtained images after HA formation (Figure 2) represented homogeneous coating the micron-sized structures in modified 10X-SBF, whereas the degree of homogeneity and speed of mineralization increased in microwave-assisted methods (600 W) compared with conventional coating systems (8 h immersion). Generally, concentrated SBF led to rapid mineralization after 8 h that is the strength of the 10X-SBF formulation to SBF solution. Utilizing microwave energy is a fast, simple and efficient technique that agitates the biomimetic formation of mineralized layers with controllable size distribution. Additionally, biomimetic HA exhibited nanoscale morphology such as Costa et al. (26) as a result of heat transfer due to friction and collisions between molecules and reduction of Gibbs energy during microwave emission (27). However, internal heating in microwave energy enables higher spatial distribution of heating rate compared with conventional external heating (14). Pylypchuk et al. (16) represented the same results after immersion of Ti in 10X-SBF and heating the solution. In fact, thermal energy accelerated HA mineralization.

Figure 1:

Spherical shape, homogeneous, uniform, and monodisperse microspheres were synthesized via single emulsion method.

FE-SEM micrographs of gelatin-siloxane microspheres (A, B).

Figure 2:

10X-SBF solution accelerated the biomimetic formation of HA. Moreover, utilizing microwave energy resulted in homogeneous coating with nanoscale HA.

FE-SEM micrographs of biomineralized microspheres [GGM10S8 (A, B) and GGM10SM (D, E)] and elemental characterization of synthesized HA by energy-dispersive X-ray spectroscopy [GGM10S8 (C) and GGM10SM (F)].

Elemental composition of hybrid microspheres is illustrated in Figure 2C and F for GGM10S8 and GGM10SM platforms. Ca/P ratio in stoichiometry composition of HA (Ca₁₀(PO₄)₆(OH)₂) is the most important parameter in the regeneration of bone (14). Observation of Ca and P elements proved the formation of calcium phosphate layers and resemblance of Ca/P ratio to a mineral constituent of natural bones confirmed the formation of proper biomimetic HA for the repair process. This ratio was 2.01 for GG10S8 samples and increased to 2.18 for GGM10SM species (bone Ca/P wt.% ratio=2.15). The results indicated the positive effect of microwave irradiation to deposition of carbonate ions and more similarity of the synthesized composition to carbonated-HA in the human bone. Creation of carbonated HA was influenced by the presence and concentration of elements such as Mg, Na, K and Cl. The concentration of each mentioned element in 10X-SBF formulation and stimulation using microwave radiation help to rapid absorption of required ions to simulate physiological HA chemical structure. The same results were obtained in other studies (26), (28).

The formation of biomimetic layers on the microspheres was determined by FTIR spectrum (Figure 3A). For GGM sample, vibration peaks at 1649 cm⁻¹ and 1530 cm⁻¹ are related to amide I band and II bands, respectively (5). The characteristic peak at 1233 cm⁻¹ is assigned to amide III (coupling of stretching vibration of CN and bending vibration of NH). The absorption peaks of Si-C at 1275 cm⁻¹, Si-O-Si at 1160 and 1081 cm⁻¹, and Si-OH at 921 cm⁻¹ is related to cross-linking of gelatin with GLYMO (29). The interaction between amino groups in gelatin and oxirane groups of GLYMO leads to the formation of coupling agents. After that, the trimethoxy groups of GLYMO hydrolyze to create silanol groups. Si-O-Si bonds fabricate after condensation reaction to provide more stable structure. The mineralization of bioactive microspheres in 10X-SBF led to the appearance of a peak at 560 owing to precipitation of phosphate ions. Characteristic peaks at 869 and 1392 cm⁻¹ are correlated with sedimentation of carbonate ions (15). Hydroxyl groups of biomimetic HA were shown at 1630 cm⁻¹. FTIR spectrum indicated that microwave radiation terminated to a higher amount of mineralized layers in lowest time due to the enhancement of peak intensities.

Figure 3:

Rapid and homogeneous biomineralization of hybrid spheres in 10X-SBF solution under microwave irradiation resulted in appearing and increasing the intensity of HA peaks.

FTIR (A) and XRD (B) characterization of the hybrid organic-inorganic structure.

XRD test was determined by the crystalline structure of the coating layer. The observation of a broad peak in XRD spectrum of GGM sample (Figure 3B) confirmed its amorphous structure. By contrast, hybrid organic-inorganic constructs after HA formation ended to the reduction of amorphous peak intensity and appearance of sharp diffraction peaks related to apatite. HA is an anisotropic crystal that crystalline peaks were apparent at 2θ angles of 31.59, 40.83 and 45.35 (based on JCPDS#76-0694 card) correspondence to the reflection of (211), (310) and (222) crystals (6). As mentioned above and observed in the spectrum, silanol coupling agents are good choice to support nucleation and growth of calcium phosphate layer. In addition, the high and narrow peaks in microwave-assisted technique indicated that this method could be able to produce high purity components (14) and improve mimicking bone ECM owing to the capability of attraction more ions for rapid synthesizing HA through enhancement of atomic movements. However, the role of modified SBF and similarity of ion concentration to the human body plasma in the simulation of the natural bone composition should not be ignored.

Surface topography of samples was studied by AFM test, as shown in Figure 4. According to the results, the uniform and smooth surface of GGM samples changed after mineralization. Surface roughness significantly increased after coating the HA on the surface of microspheres so that the roughness in GGM constructs gained from 246.8 nm to 2.12 and 3.6 μm for GGM10S8 and GGM10SM, respectively. Microwave-irradiated spheres showed a great influence on changing the roughness due to energy transfer and capability to the attraction of more ions from 10X-SBF.

Figure 4:

Surface roughness of gelatin-siloxane microspheres before and after mineralization.

[GGM (A), GGM10S8 (B) and GGM10SM (C)].

The gelatin-siloxane microspheres showed biocompatibility in terms of MG-63 cells culture. Figure 5A–D represents cell adhesion, MTT assay, ALP activity of GGM and GGM10SM cell-cultured samples. SEM micrographs of seeded cells on the surface of spheres demonstrated cell attachment, whereas MG-63 cells spread significantly on the surface of HA-coated samples. MTT assay illustrated that the viable number cells increased with time and the rate of enhancement was higher in GGM10SM constructs. This phenomenon may be affected by higher values of releasing Si from GGM hybrid samples. Moreover, low affinity of spheres to interact with water molecules owing to hydrophobic nature of GLYMO and cross-linking of gelatin (29) lead to absorption of the more moderate amount of supplemented media for cellular growth and postpone the adhesion, growth and spreading. By contrast, the surface of the GGM10SM construct is generous of hydroxyl functional group owing to HA coating that can promote cellular spreading by providing required nutrients through absorption of media. Furthermore, the presence of HA layer may improve cell proliferation by postponing the rate of Si release. Therefore, GGM10SM showed spread cells with many pseudopodia. Based on these results, both samples are biocompatible because of ability to proliferate as a function of increasing the time, adhesion and spreading of cells to the spheres, a viability of more than 87% cells.

Figure 5:

A cell culture study via MG-63 cells.

Cellular adhesion and spreading on GGM (A) and GGM10SM (B) constructs. The cellular viability (C) and ALP activity (D) of cultured cells in interaction with GGM and GGM10SM platforms.

The ALP activity of the MG-63 cells as an osteogenic marker after culturing the cells on the gelatin-GLYMO microspheres indicated that the activity increased up to 6 days. According to the results, hybrid microspheres carried out osteogenic functions. However, ALP activity of apatite-contained spheres with better cell attachment exhibited higher values that mean their better osteogenic capacity and support mineralization. Also, the role of biomimetic coating the platform with HA in simulation bone ECM and more expression of ALP should be mentioned.

4 Conclusion

Herein, a modification process with silane coupling agents was used to fabricate hybrid gelatin-GLYMO microspheres via single emulsion technique to mimic bone ECM. Presence on siloxane network in the chemical structure of microspheres led to the bioactivity of prepared constructs. Immersion the spheres in 10X-SBF solution terminated to rapid biomimetic HA formation within 8 h while the mineralization accelerated through emission of microwave energy. Microwave irradiation resulted in the formation of approximately monodisperse nano-HA on the surface with the nearest Ca/P ratio to natural bone. Besides, biocompatibility of GGM10SM samples suggested their ability for biomedical applications. Enhancement ALP activity in microwave emitted spheres confirmed the potential gelatin-siloxane spheres for bone regeneration.

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Received: 2017-9-22

Accepted: 2017-12-2

Published Online: 2018-1-25

Published in Print: 2018-5-24

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https://doi.org/10.1515/epoly-2017-0196

Keywords for this article

10X-SBF; gelatin microsphere; microwave; mineralization; siloxane

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