Surfactant-assisted synthesis of polyvinylpyrrolidone-hydroxyapatite composites as a bone filler

Immersion in SBF

Nanocomposites were immersed in 30 mL of SBF at 37°C for 3, 7 and 14 days under static conditions for the evaluation of their bioactivity. Appropriate amounts of reagent grade chemicals were dissolved in deionized water and buffered with Tris-HCl to pH 7.4 at 37°C to obtain the SBF solution. SBF ion composition is very close to that of human plasma (15, 16) as shown in Table I. After immersion of samples in SBF, they were filtered, washed with deionized water and dried.

Dried samples were studied before and after soaking in SBF with Fourier transform infrared (FT-IR) spectroscopy (Thermo Nicolet Nexus 870), X-ray diffraction (XRD; Seisert Argon 3003 PTC) and scanning electron microscopy (SEM; Philips). The KBr pellet method was used for FT-IR sample preparation. For this, 20 mg of samples was mixed well into 200 mg of dried KBr powder, pulverized and put into a pellet-forming die under pressure to form transparent pellets for recording the spectra. For XRD, samples were smoothed and put in the sample holder to record patterns at 2θ ranging from 2 to 80 degrees. Composite powders were fixed on aluminum stubs, gold sputter-coated (Sputter Coater S150B, Edward) and used for SEM observation at 10 kV.

In vitro biocompatibility test

The in vitro cell compatibility of the nanocomposites was evaluated in terms of proliferation of the unrestricted somatic stem cells (USSCs). These multipotent stem cells were isolated from human umbilical cord blood. They are a more immature cell type than bone marrow mesenchymal stem cells (BMSCs), which have better potential for proliferation and differentiation into osteoblasts (17). The stem cells were obtained from the Pastor Institute (Iran). Cells were defrozen and transferred into culture flasks containing Dulbecco’s modified Eagles medium (DMEM), 20% fetal bovine serum and 1% antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin). The medium was changed every 3 days. Samples for cell tests were sterilized with 70% ethanol for 3 hours, washed with phosphate-buffered saline (PBS) solution 3 times and incubated in the culture media before cell seeding. Cell suspensions of USSCs (15 × 10³ cells/cm²) were seeded onto the composites via direct pipetting, then samples were incubated in 1 mL of cell culture medium into 96-well cell culture plates at 37°C under 5% CO₂. The cell culture medium was changed every 3 days. The cell proliferation of USSCs cultured with and without composites (control group) was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-2H-tetrazolium bromide (MTT) assay. USSCs were incubated in 100-μL MTT solution (0.5 mg/mL, 37°C and 5% CO₂) at each time point (1, 3 and 7 days) for 3 hours. After the removal of supernatants, 100 μL/well of dimethyl sulfoxide (DMSO) was added and mixed. The absorbance of each well content was measured at 575 nm with a spectrophotometer (Perkin Elmer Co.).

Statistical analysis

Student’s t-test was conducted to determine statistical significance among groups, and statistical significance was considered to be a p value <0.05.

Physicochemical characterization

The dominant band in the FT-IR spectrum of PVP at 3,439 cm^-1 (Fig. 1A) was due to the stretching vibration of OH and the spectral width was related to the formation of inner and outer hydrogen bonds. Stretching vibration of CH and CH₂ presented the peaks at 2,955 and 2,929 cm^-1. The bands at 1,666 cm^-1 were due to the stretching vibration of N-C and C = O functional groups. The peaks at 1,494, 1,462, 1,424 and 1,374 cm^-1 probably involved angle deformation of C-H bonds of CH₂ groups in the ring. Stretching vibration of C-N and amid was reflected by the appearance of peaks at 1,288 and 1,274 cm^-1, respectively. The bands of amid V and IV were observed at 734 and 649 cm^-1 (18-19-20).

In the HA spectrum, the characteristic bands of υ₄ (PO₄^3-) can be observed at 560-604 cm⁻¹. The poor band at 470 cm^-1 is due to ʋ₂ of phosphate. The bands at 960 cm^-1 and in the range of 1,030-1,100 cm^-1 were due to ʋ₁ and ʋ₃ of (PO₄^3-), respectively. The bands around 600 and 1,049 cm⁻¹ are related to bending and stretching modes of P–O vibrations. Vibration of OH presented a broad band at 3,450 cm^-1, and the bands of water (H–O–H) absorption in the products can be observed at 3,400 and 1,629 cm⁻¹ (2, 21). Small peaks in the range between 1,411 and 1,460 cm⁻¹ (ʋ₃) and 876 cm^-1 (ʋ₂) are signs of the presence of carbonate groups in the composite and formation of carbonate apatite due to the replacement of phosphate groups in the HA structure by CO₃^2- (1).

Spectra of composites (S-1, S-2, S-3 and S-4) show the main peaks of HA (Fig. 1A). It seems that the use of PVP-SDS as a template facilitates the precipitation of HA in situ. As PVP is water soluble, it can create the proper conditions for the crystallization of HA with SDS. In fact, polar groups of SDS can provide the active sites for electrostatic interaction with the calcium ions and lead to HA nucleation (22). There is a slight shift in the position of absorption bands in the composites which is indicative of the interaction of SDS with the nucleating crystals. As can be seen, the ratio of PVP/H₂O had no effect on the FT-IR spectra of composites.

FT-IR spectra of S-1, after soaking in SBF at different times (Fig. 1B), presented similar peaks to those before immersion. The bands related to OH, PO₄^3-, N-C, C = O and carbonate groups are shown in the FT-IR spectra. The presence of carbonate groups and the formation of carbonate apatite can be a sign of composite bioactivity (2).

Similar XRD patterns for HA in the absence of PVP-SDS and composite samples (S-1, S-2, S-3 and S-4) are observable in Figure 2A. The XRD patterns of all samples showed 3 main peaks at 26.2, 32.5 and 39.8° (2θ) assignable to the [002], [211] and [310] planes of crystalline HA, respectively, which means that the use of PVP and surfactant had no effect in changing the HA crystallite structure. In the sample S-1, which contained more PVP and a higher PVP/H₂O ratio, a broader peak was observed due to the slower growth of HA caused by the slower reaction between the calcium and phosphate ions. When the PVP/H₂O ratio was low (S-4), the peaks were sharper and distinguishable; this could have been due to the rapid diffusion of calcium and phosphate ions into the PVP gel.

There were no changes in XRD patterns of S-1 after soaking in SBF (Fig. 2B) even after 21 days, which means that the crystal structure of the HA was not destroyed by the ions of the SBF solution. A gradual intensity increase and a reduction of the width of HA peaks by increasing the immersion time in SBF could have been a sign of apatite growth on the surface of samples, and a proof of composite bioactivity (2).

Total morphology of HA in the absence of PVP-SDS, in the SEM micrograph (Fig. 3A) shows spherical particles, whereas HA morphology has been changed to a mixture of nano rod and spherical particles in composite samples (Fig. 3B-C-D-E). This morphology change was due to the HA formation mechanism. Bonding between calcium ions and negative charge site of SDS can form micelles; the formation of HA takes place by diffusion of calcium and phosphate ions in such micelles. The PVP-SDS template shape and the ratio of PVP/H₂O play an important role in the HA crystallite shape and the rate of crystal growth, respectively. When the template has a cylindrical structure, rod-like HA crystallites will be formed (1, 23, 24). It seems that by reducing the ratio of PVP/H₂O from S-1 to S-4, the diffusion rate of ions was increased, and the template shape tended to be more cylindrical in form, which led to rod-like HA formation, so that in S-4, more particles are in rod shape a with longer size.

It seems that HA composite preparation via micelle-templated precipitation by PVP-SDS is more favorable compared with the approach of Weeraphat et al (1), which used a polyvinyl alcohol (PVA)-SDS template. Our micelles allowed better control of HA morphology and led to more rod-shaped particles being formed, whereas with PVA-SDS, HA particles grew in an irregular manner. This can be explained by considering the difference between PVP and PVA capability in hydrogel formation, due to their chemical structure (14). The lesser hydrogel strength of PVP can help so that SDS-active sites react properly with calcium ions, and diffusion takes place easily, leading to better-controlled morphology of HA. In most works using a cationic surfactant to obtain rode-shaped HA, a hydrothermal treatment was needed, whereas in this work, just a 30-second microwave radiation was applied (9-10-11).

SEM images of S-1 after immersion in SBF (Fig. 4A-B-C-D) show redistribution of HA crystallites and formation of rod-like nHA particles. It seems that by increasing the immersion time from 1 to 3 weeks, the size and amount of HA crystallites increased, and a new apatite-like phase was formed. This inorganic crystallite growth can be the sign of composite bioactivity. So, It is expected that after implantation of composites in the body (in vivo condition), this new apatite phase will be able to nucleate and grow on their surface.

Cell experiments

The cytotoxicity effects and cell proliferation of composites were investigated through MTT assay. As can be seen in Figure 5A, cell numbers increased with culture time in all tested groups. There was no significant difference between cell viability of S-1 and control (p>0.05) after 7 days. Other composites were biocompatible, but there was a significant difference between their cell viability and that of controls (p<0.05) at all time points. USSCs cultured on S-1 showed much more cell proliferation compared with PVP and HA after 1 and 3 days (p<0.05), whereas the other samples did not follow this trend. Among the composites, It was expected that S-4 would show better cell viability and proliferation due to the presence of rod-shaped HA particles, which is similar to the inorganic phase in the natural bone, but S-1 showed better results, and there was a significant difference in its cell viability (p<0.05) at all time points. This result could be due to smaller size and more regular distribution of HA crystallites in S-1.

Investigation of cell proliferation is an important technique to evaluate the biocompatibility of biomaterials in vitro (22). Figure 5B presents phase-contrast micrographs of cell attachment on the S-1 sample after culture for 1 and 7 days. At the first day, recognition of elongated fusiform-shaped USSCs is difficult. At 7 days, a cell colony is observable due to a large quantity of cell proliferation that is attached to the composite. Obviously, the composite sample has no negative effect on cell morphology, viability and proliferation.