Chemical modification of poly(vinyl chloride) for blood and cellular biocompatibility

Abstract

Poly(vinyl chloride) (PVC) was modified with three different ionomers including thiosulphate, thiourea and sulphite for improving the biocompatibility of the polymer. All ionomers were prepared by nucleophilic substitution using a phase transfer catalyst method. The modified forms of PVC were characterized using ultraviolet-visible (UV-Vis) spectroscopy, Fourier Transform Infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and thermal gravimetric analysis (TGA). They were found to be less stable thermally compared to the untreated polymer. The biocompatibility of the polymers was evaluated by assessing their wettability via contact angle measurements and by performing hemolysis and thrombogenicity assays. Their cellular biocompatibility was evaluated by assessing their adhesion and proliferation, and by carrying out cytotoxicity assays and nuclear staining. The results reveal that modification of the polymer with the specified ionomers significantly enhances the bio- and blood-compatibility properties.

---

Introduction

Poly(vinyl chloride) (PVC) synthetic polymeric materials have been widely used in biomedical applications including the clinical analysis of salt, blood storage, catheters etc.¹ Its mechanical properties and excellent capability to acquire desired functional groups has made it a popular choice for research in the polymer field right from the early 19^th century.² Several reports on the improvement of the biocompatibility of PVC can be found in the literature from over the last few decades.³ In addition, several studies have investigated the relationship between the degree of hydrophobicity, surface charge and cellular adhesion to examine their influence on the attachment and spreading of cells onto the surface of a material, which ultimately determines the success or failure of a biomaterial.^4–6

In view of the given structure–property relationship of a biomaterial, there is motivation to modify PVC for the alteration of its properties and hence for developing its biocompatible forms. The modification of PVC leads to changes in its surface properties such as, surface chemistry, surface energy, surface topography, etc. that could be critical in determining the biocompatibility. Therefore, such modifications of the polymer play a crucial role in determining their antimicrobial efficacy and thus their selection for consideration in medical applications.^7,8 One of the important factors to consider regarding the biocompatibility of PVC is its blood compatibility, which can be improved by the adsorption of biological molecules, such as heparin,⁹ PEG¹⁰ or fibronectin,¹¹ forming a self-assembled hemocompatible coating on its surface. In addition, various reported methods¹² showing surface modification by specific chemical groups¹³ reveal an enhancement in the hydrophilicity of the PVC surface that is vital in governing its biocompatibility.

The main principle behind the modification of PVC is a nucleophilic substitution reaction that provides an opportunity for the steady replacement of chlorine atoms through substitution with desired atoms or groups without any side reactions, resulting in modification of the surface charges that dominate at the interface between the biomaterial surface and biological environments.¹⁴ Here, we demonstrate a simple process to formulate a PVC resin with thiosulphate, thiourea and sulphite. To identify the characteristics of the newly synthesized polymers, we have examined the thermal stability, surface morphologies, hydrophilicity and antibacterial activity. Finally, the biocompatibility of the modified polymers has been assessed through hemolysis and thrombosis tests as well as using cell-based assays.

Experimental

Materials

Poly(vinyl chloride) was obtained from Ottokemi Mumbai, India. Sodium thiosulphate, thiourea and sodium sulphite were obtained from Merck Ltd., Mumbai, India. Tetrahydrofuran (THF) was obtained from Glaxo Ltd. Mumbai, India.

Modification of PVC

PVC was dissolved in THF and its prepared film was used as a control. For obtaining the modified PVC films, 10 g of PVC was dissolved in an aqueous solution of various solutes viz. 3 M sodium thiosulphate, 7 M thiourea and 7 M sodium sulphite at room temperature. The solution was heated at 60–65 °C and then tetrabutylammonium hydrogen sulphate (TBAHS) (0.15 M) was added pinch wise. The reaction mixture was kept at the same temperature for 5 h under continuous stirring. After 24 h, the solution was filtered and washed with double distilled water followed by methanol and dried under vacuum.

Henceforth, notations of PVC, PVC-TS, PVC-TU, and PVC-S will be used for the pure polymer and the modified polymers, respectively.

Characterizations

Fourier transform infrared (FTIR) spectroscopy. Fourier transform infrared (FTIR) spectroscopy was used to detect the functional groups and to understand the nature of the interaction between the functional groups and PVC. Thin films were prepared using a solution-cast technique in THF which was used as a solvent. PVC, PVC-TS, PVC-TU and PVC-S with THF were poured into glass Petri dishes and films were peeled off with the help of a spatula. FTIR spectra were recorded in the transmission mode at room temperature with wave numbers ranging from 400 to 4000 cm⁻¹ using a Nicolet 670 FTIR with a resolution of 4 cm.

Ultraviolet-visible (UV-vis) spectroscopy. The ultraviolet-visible (UV-vis) spectroscopy measurements were carried out by using a Shimadzu (UV-1700) Pharma Speck, operating at a wavelength range of 200–800 nm. Samples were prepared as transparent thin films by dissolving PVC, PVC-TS, PVC-TU and PVC-S in THF and all the experiments were carried out at room temperature.

Contact angle measurements. The contact angles of the pure and modified polymers were measured using a Kruss F-100 tensiometer system. For estimating the contact angles, modified and pure PVC dissolved in THF were processed to form relatively thicker polymer films (1 × 10 × 20 mm³). Estimation of the free energy was performed using double distilled water. The data represent a mean value of the contact angles obtained from three different experiments. This property is very important for a biomaterial as it signifies the wettability (i.e., hydrophobic or hydrophilic nature) of the materials.

Thermal gravimetric analysis. The thermal stability of the modified and unmodified PVC films was examined by using a thermogravimetric analyzer (TGA) (Mettler-Toledo) associated with a differential analyzer. The data were collected at temperatures ranging from room temperature up to 600 °C. All the experiments were performed at a heating rate of 20 °C min⁻¹ in a nitrogen atmosphere.

Scanning electron microscopy. The surface morphology of the particles of the PVC, PVC-TS, PVC-TU and PVC-S polymers was investigated by SEM images acquired using a Quanta 200 F.

Bacterial viability assay. For the bacterial culture, E. coli (ATCC 25922) was obtained from the American Type Culture Collection (ATCC), and their clinical strains were preserved at the Department of Microbiology, Institute of Medical Sciences, BHU, Varanasi, India. Fresh bacterial broth cultures were prepared before the screening procedure. The strain was hydrated and streaked for isolation on an LB agar. Following growth, a single isolated colony was selected and used to inoculate 3 mL of (Luria-Bertani) LB broth media.¹⁵ The bacteria culture was grown on a shaking incubator set at 150 rpm for 18 hours at 37 °C. The resulting suspension was then adjusted to have an optical density at 480 nm (OD⁴⁸⁰) of 0.42, corresponding to a bacterial density of 10⁹ colony forming units (CFU) per mL. Thereafter, the solution was serially diluted over a 3-log range to a bacterial density of 10⁶ CFU mL⁻¹.

Modified and unmodified polymer films were cut into small segments (1.0 × 1.0 cm pieces) with a sterile pinch cutter. All samples were initially surface treated to eliminate any microorganisms present. The samples were immersed in 70% ethanol for 1–3 min and then sterilized with aqueous sodium hypochlorite (4% available chlorine) for 3–5 min and finally rinsed in sterilized double distilled water. Each sample was then dried under aseptic conditions.

1 mL of the 10⁶ CFU mL⁻¹ solution of E. coli was pipetted into each well tube, while ensuring complete submersion of the sample. The well tube was then placed in a stationary incubator at 37 °C. After 24 h, samples were taken out from the well tube, washed with deionized water and then immersed in 1 mL of saline water. The samples were further vortex-mixed for a few seconds to remove all the bacteria attached on the surface. Finally, 0.02 μL of the resulting bacterial suspension was used for streaking on the culture plate.

Biocompatibility

Hemolysis assay. The hemolytic activity of the various polymers was investigated according to the standard procedure described by Kapusetti et al.¹⁶ using acid citrate dextrose (ACD) human blood. ACD blood (5 mL) was prepared by adding 4.5 mL of fresh human blood to 0.5 mL ACD. The ACD solution was prepared by mixing 0.544 g of anhydrous citric acid, 1.65 g of dehydrated trisodium citrate and 1.84 g of dextrose monohydrate in 75 mL of distilled water. The polymer films were cut into 0.5 × 0.5 cm pieces and equilibrated in a phosphate buffered solution for 30 min at 37 °C in desiccators. For the positive and negative controls, distilled water and a buffer solution were used, respectively. Thereafter, 0.2 mL of ACD blood was added to each test tube and they were finally kept for 1 h in an incubator at 37 °C. The test tubes were centrifuged for 8 min at 800 rpm. The optical density of the supernatant was measured at 545 nm. The percentage of haemolysis was calculated as follows:

Thrombogenicity assay. The polymer films were hydrated by equilibrating them with saline water, and they were kept at 37 °C in Petri dishes. ACD human blood (0.2 mL) was placed onto each film. Blood clotting was initiated by adding 0.02 mL of a 0.1 M KCl solution followed by proper mixing with a Teflon stick. The clotting process was stopped by adding 5 mL of distilled water after 30 min. The clot formed was fixed in 5 mL of a 3.6% formaldehyde solution for 5 min. The fixed clot was washed with distilled water, blotted between tissue papers and weighed.

Cell culture studies. The mouse mesenchymal stem cell (mMSC) line, C3H10t1/2, was used for all the experiments. The cells were cultured in 25 cm² flasks at 37 °C in a humidified atmosphere with 5% CO₂. Dulbecco’s modified Eagle’s medium (DMEM)-high glucose medium, in combination with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution, was used for culturing the cells. The cells were seeded onto the samples at an equal density of 2 × 10³ cells per surface (10 × 10 mm²) for all cell-based assays.

Specimen for cell culture studies. The films of PVC and its various derivatives were prepared by a solution casting method in Petri dishes. The prepared films were placed between two Teflon sheets and clamped for 10 min to obtain the plane surface of the materials. The cured specimens were removed from the molds and their edges were smoothed with an emery paper. The specimens were stored at room temperature. A specimen size of 10 × 10 mm² was selected for the in vitro cell culture studies. Before performing the cell-based studies, the specimens were washed with isopropanol to remove the attached debris. For surface sterilization, each specimen was washed thrice with phosphate buffered saline (pH ∼ 7.2), and exposed to UV light for 8 h.

Cell adhesion. The ability of the samples to support cell adhesion was determined by staining the cells adhered to their surface with crystal violet. The cells were seeded on to the surface of the samples at an equal density and incubated at 37 °C in a humidified atmosphere with 5% CO₂ for 4 h. Prior to the addition of a dye, the culture medium was aspirated and the cells were washed twice with cold phosphate buffered saline (PBS) at pH 7.2, and fixed using a 4% formaldehyde solution. After the addition of the dye, the cells were incubated at room temperature for 30 min and then washed three times with cold PBS. The endogenous crystal violet was then extracted using absolute methanol and the absorbance of the solution was measured at 544 nm using a Fluostaroptima (BMG Labtech, Germany) microplate reader. Cells adhered to the surface of the samples were quantified using the following formula:

Cell viability. The MTT assay is a colorimetric test for measuring the activity of enzymes that reduce 3-[4,5-dimethylthiozol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) to formazan, giving a purple color appearance. The cytotoxicity of the samples was assessed using the MTT assay as described previously.¹⁷ The samples were cut into small pieces (10 × 10 mm) and placed into a 12-well tissue culture plate (Corning, Germany), followed by their sterilization. 2 × 10³ cells in 20 μL of medium were seeded onto the samples and cultured for three different amounts of time. On the 1^st, 3^rd and 5^th days following culture, the cells grown on each sample were assayed by the addition of and incubation with 5 mg mL⁻¹ MTT for 4 h at 37 °C. Only viable cells have the ability to reduce the yellow water-soluble MTT tetrazole complex into the dark blue crystals of formazan, which is insoluble in water. After 4 h, the MTT-containing medium was aspirated and 1 mL of ethanol–DMSO (Himedia, India) (1 : 1) was added to lyse the cells and solubilise the water-insoluble formazan. Viable cells on the surface of the samples were quantified spectrophotometrically by measuring the absorbance of the lysates at 570 nm, using a Fluostaroptima (BMG Labtech, Germany) microplate reader. The percentage of live cells on each sample was evaluated by comparing the absorbance of the samples to that of a control well where cells were seeded onto the surface of a polystyrene tissue culture plate.

Nuclear staining. The ability of the samples to support the proliferation of cells was assessed by staining the cells with 4′,6-diamidino-2-phenylindole (DAPI, Sigma) after an incubation period of 24 h. The cells were seeded on to the surface of the samples at an equal density and incubated at 37 °C in a humidified atmosphere with 5% CO₂. Prior to the addition of a dye, the culture medium was aspirated; the cells were washed twice with cold phosphate buffered saline (PBS) at pH 7.2, and fixed using a 4% formaldehyde solution. The cells were then permeabilized using a 0.1% solution of Triton X100 (Himedia, India) for 45 seconds and incubated with the dye at 37 °C for 5 min. Images of the intact cellular nuclei stained with the dye were captured with a fluorescence microscope.

Statistical analysis. Statistical analyses were performed on the means of the data obtained from three independent experiments by using GRAPH PAD PRISM for Windows software. The results are expressed as mean values (±SE). The analysis of variance followed by a post hoc Dennett’s test was performed for the contact angle measurements, hemolysis assay and cell adhesion assay for one-way analysis of variance (ANOVA). In addition, Bonferroni’s method was used to analyse the cell viability data for multiple comparison tests in ANOVA. In all cases, a p value was obtained from the ANOVA analyses; the conventional value of 0.01 was considered to express statistical significance.

Microscopic fluorescence image system. Cells were cultured on the polymeric material surface under standard conditions. The cells were stained with a DAPI dye for the nuclei and observed using a Zeiss, Axiovert 25 inverted fluorescence microscope equipped with an objective of 100× magnification.

Results and discussion

Spectroscopic analysis

Fig. 1(a) shows the FTIR spectra of polymeric PVC and the functionalized PVC materials. A number of characteristic peaks can be observed: stretching of C–H of CHCl at 3200–2700 cm⁻¹, the wagging of methylene groups at 1430 cm⁻¹, stretching of C–H of CHCl at 1258 cm⁻¹, C–C stretching at 1065 cm⁻¹, rocking vibration of CH₂ at 966 cm⁻¹ and, vibration stretching of C–Cl bonds of syndiotactic and isotactic structures of PVC at 614 and 695 cm⁻¹. A similar structure has been reported previously in the literature.¹⁸

Fig. 1 (a) FTIR spectra of pure and functionalized forms of PVC. (b) UV-Vis spectra of PVC and its derivatives.

The structure of the modified polymers was established on the basis of the replacement of chlorine atoms in the polymer chain. The presence of the nucleophile was confirmed by the FTIR spectra and UV-vis spectroscopy. Fig. 1(a) shows the IR spectra of the pure and modified forms of PVC; thiosulphate (S₂O₃²⁻) and sulphite (SO₃²⁻) groups show the S₂O₃²⁻ stretching at 1017 cm⁻¹ and 960 cm⁻¹, respectively. Strong stretching of C–S at 690 cm⁻¹ with weak stretching of C–S–S–C¹⁹ at 540 cm⁻¹ was observed. For PVC-thiourea, NH stretching was observed at 3315 cm⁻¹ and 3180 cm⁻¹. The band at 1619 cm⁻¹ may be due to N–H bending, while a band at 1425 cm⁻¹ was observed for N–C–N stretching in thiourea substituted PVC. For PVC–sulphite a band at 3420 cm⁻¹ was observed due to the C–OH group. Thus, the data indicate that PVC was successfully modified with the different types of functional groups by a nucleophilic substitution reaction. Kameda et al.²⁰ have shown substitution of the chlorine ion by I⁻, SCN⁻, OH⁻, N₃⁻ and pthalamide anions in PVC resins using a nucleophilic solution and thus developed various forms of polymers with enhanced conductive properties and substantial antibacterial activity.

The absorbance of UV-Vis light by polymeric materials is mainly attributed to electron transitions among the σ, π and n energy levels from the ground state to higher energy states. The UV-Vis spectra in the wavelength range of 200–400 nm of PVC and its derivatives are shown in Fig. 1(b). One absorbance peak observed for PVC near 206 nm is due to an n–π* transition. Another absorbance peak, observed in the PVC-TS samples at 209–249 nm is credited to a π–π* transition due to conjugation. As can be seen, there are sharp absorption peaks at 218 nm for thiosulphate, 249 nm for thiourea and 209 nm for sulphite. Safyan et al.²¹ have used sodium thiosulphate and sodium sulphite for the identification of polysulfide and oxidized sulphur species together and observed similar results for thiosulphate and the sulphite anion. In addition, Mushtari et al.²² have found such a transition peak due to the C=S chromophore in the derivatives of pyridylthiourea. Similarly, Madhurambal et al.²³ have observed comparable results while analyzing urea and thiourea with urea–thiourea–zinc chloride crystals. The peak representing a π–π* transition showed a red shift in modified PVC with respect to pure PVC due to the presence of different functional groups.

Thermal gravimetric analysis

The thermal gravimetric analysis results for pure PVC and functionalized PVC are shown in Fig. 2. Two transition steps can be observed from the thermogram of pure PVC of which the first step corresponds to the weight loss caused by the dehydrochlorination of PVC that begins at a temperature of 240 °C, while the second transition step represents the total weight loss resulting from the degradation of the dehydrochlorinated residues.¹⁸ However, in the case of PVC-TS, PVC-TU and PVC-S, the first transition step starts at the onset of 200 °C, 218.7 °C and 190 °C, respectively, while the second transition step of all functionalized PVCs is similar to that of pure PVC. The thermal degradation temperature of functionalized PVC shifts slightly to a lower temperature in comparison to pure PVC. Thus, the outcome clearly shows significant differences in the range of thermal degradation temperatures of pure and functionalized PVC resins. This shows that the existence of functional groups in the polymer chain significantly promotes the degradation of functionalized PVC (i.e. lowers the thermal stability).

Fig. 2 Thermograms of pure and functionalized PVC analyzed in a nitrogen atmosphere.

However, there have been contrasting reports regarding the thermal stability of PVC upon chemical modification. One study indicates an increment of around 50 °C in the degradation temperature when PVC is incorporated with polyethylene glycol.¹⁰ Thermal stability is generally expected to increase upon chemical crosslinking in the polymer. In some cases, however, the literature reveals that it may also decrease.²⁴

Fig. 3(a) shows the relative hydrophilicity and hydrophobicity of the materials, evaluated by contact angle measurements of the synthesized polymers in contact with water. The influence of chemical modification on the wettability property of the materials was examined and the results are represented in Fig. 3(a). The chemical modification of PVC results in a significant decrease in the contact angles, indicating that the modified polymers are more hydrophilic. This is an important factor in governing the wettability of a biomaterial, and it promotes cell growth and proliferation and thereby influences the biocompatibility property of a biomaterial. The results show that the average values of the water contact angles of pure PVC, PVC-TS, PVC-TU and PVC-S are around 82°, 65°, 55° and 60°, respectively, within the accuracy level²⁵ of ±1°. Previously, James et al.¹⁰ showed a similar improvement in the hydrophilic property of plasticized PVC by modifying its surface with thiocyanate. Furthermore, they found that the hydrophilic property of their modified material was not supportive to bacterial adhesion, typically observed for S. epidermidis and S. aureus.¹⁰ Similarly, Lakshmi et al. showed an enhancement in the degree of hydrophobicity of the plasticized PVC upon surface modification with thiosulphate and found that the modified PVC exhibited significantly greater hemolytic activity as well as lower cellular adhesion with fibroblast cells.¹⁹

Fig. 3 (a) Contact angle measurements of pure PVC and functionalized PVC resins and (b) the hemolysis percentage of pure PVC and functionalized PVC polymers.

Fig. 4 shows SEM images of PVC residues modified with thiosulphate, thiourea and sulphite. No significant difference in the surface morphology of the pure and modified PVC particles was observed in the SEM images. Irregular and uneven particle morphologies were prominently observed in all cases. However, a notable difference in the wettability property of the pure and synthesized PVC resins was revealed by contact angle measurements of the polymer films. The modified PVC surface was found to be more hydrophilic as demonstrated by a significant decrease in the water contact angles. Similarly, their surface charge varies quite distinctly though the surface morphology of the pure PVC particles appears similar to that of the treated PVC particles (Fig. 4). The modified PVC particles show a highly charged surface due to the presence of ionic groups. Thus, the results indicate that nucleophilic substitution with ionomers viz. thiosulphate, thiourea and sulphite, does not alter the morphology of the PVC surface, yet significantly affects the wettability of the PVC resins.

Fig. 4 Scanning electron micrographs of PVC and the derivatives of PVC resin after the chemical modification. (a) & (b) PVC, (c) & (d) PVC-TS, (e) & (f) PVC-TU and (g) & (h) PVC-S.

Bacterial adhesion is a complex process whose numerous aspects to date have not been well understood due to the involvement of a number of physicochemical factors in this process.²⁶ While measurement of bacterial adhesion is important itself, it alternatively serves as a basis to characterize the antibacterial property of biomaterials.²⁷ The degree of antibacterial activity based on bacterial adhesion on the polymeric samples (over 24 h) is presented in Fig. 5. Although bacterial adhesion is reportedly a dynamic process, the observation was performed after 24 h of incubation for a better assessment of the adhesion. The data reveal, in all cases, no decrease in the colonies of the plated bacteria that were pre-adhered to the surface of the pure and modified samples; this implies the inefficiency of the modifications in reducing the adherence of E. coli to the polymer surface.

Fig. 5 Antibacterial activity of PVC and its functionalized polymers; colonies of E. coli grown on (a) PVC, (b) PVC-TS, (c) PVC-TU and (d) PVC-S.

The hemolysis phenomenon of blood is a major concern associated with bio-incompatibility.²⁸ Hemolysis occurs when red blood cells come in contact with water and it is an important parameter to ensure biocompatibility of a material. The data show that the recorded level of hemolysis is less than 5% in all cases;²⁹ suggesting that the modified forms of PVC are advanced biomaterials and could be used as alternatives to the pure form of PVC. However, an attempt is in progress to further improve the polymers.

Thrombogenicity evaluation

The weight of the blood clots obtained after incubation of blood with PVC, PVC-TS, PVC-TU and PVC-S for 30 min was 1.9, 1.3, 1.6 and 1.1 mg, respectively. These results are consistent with the previous studies. Reported literature suggests that³⁰ the surface properties play a vital function at a molecular level in governing surface-induced hemolysis. Notably, increased hydrophilicity of the materials directly corresponds to their improved biocompatibility. In addition, several studies suggest that a biomaterial with a positively charged surface promotes thrombogenesis when exposed to blood, while negatively charged biomaterials tend to suppress the thrombogenesis process,³¹ most likely due to the fact that blood cells and platelets have a net negative charge on their surface.

Cell culture studies

All forms of polymers supported cellular adhesion under the standard conditions. Fig. 6 shows the percentage of mMSCs adhered to the PVC, PVC-TS, PVC-TU and PVC-S polymers after 4 h. A polystyrene tissue culture Petri dish (without sample) was used as a control in all cases. The total set of modified polymers shows a significantly higher adhesion percentage compared to the pure form of PVC. The level of cellular adhesion was found to be notably reduced on PVC-TS surfaces compared to the other modified polymers. There was no significant difference observed between PVC-TU and PVC-S as both showed a relatively similar range of cellular adhesion on their surface. A previous study suggests that the functional groups present on the surface of a biomaterial directly influence biocompatibility. Curran et al.³² have investigated the importance of functional groups in governing cellular adhesion using human mesenchymal stem cells. They have demonstrated the adhesion behavior of cells with methyl, amino, silane, hydroxyl and carboxyl groups and shown that all surfaces maintained viable cellular adhesion throughout the test period.

Fig. 6 Biocompatibility evolution of PVC and its derivatives. The percentage value of mesenchymal stem cell adhesion on PVC and its functionalized forms was evaluated using crystal violet. The absorption values were taken at the wavelength of 544 nm. *P < 0.05, **P < 0.01 and ***P < 0.001.

To determine the effects of the functional polymers on metabolic activity, the MTT assay was performed. The cytotoxicity of the polymeric materials after their incubation with cells for 1, 3 and 5 days was observed in a culture medium. The cytotoxicity was measured by determining the cellular viability using an MTT assay. Fig. 7 represents the plot for the viability percentage of mMSCs and shows that significantly lower levels of cytotoxicity are observed in the case of the functionalized polymeric materials. The viability of the cells seeded on a bare tissue culture-grade polystyrene Petri dish was considered as a control. The cell viability was found to be ∼43% for PVC after 1 day of culture while it increased significantly by another 77% (P ≤ 0.001), 86% (P ≤ 0.001) and 80% (P ≤ 0.001) for PVC-TS, PVC-TU, and PVC-S, respectively. Similarly, after 3 days of culture, the viability was noted to be around 42% for PVC and was increased further by 49% (P ≤ 0.01), 62% (P ≤ 0.001) and 49% (P ≤ 0.01) for PVC-TS, PVC-TU, and PVC-S, respectively. Also, a similar trend was observed following 5 days of culture, ∼1% for PVC while it was increased by another 61% (P ≤ 0.001), 71% (P ≤ 0.001) and 62% (P ≤ 0.001) for PVC-TS, PVC-TU, and PVC-S, respectively. In summary, the cell viability was found to be significantly higher in the case of functionalized PVC polymers in comparison to its pure form.

Fig. 7 Cell viability of mouse mesenchymal stem cells seeded on the surface of PVC, PVC-TS, PVC-TU and PVC-S. Cells were seeded directly onto the polymeric biomaterial surface and cultured for 1, 3 and 5 days in a growth medium. *P < 0.05, **P < 0.01 and ***P < 0.001.

Nuclear staining

Fig. 8 shows the nuclei of adhered mesenchymal stem cells adhered on PVC and functionalized PVC. The nuclear staining indicates that the cells adhered on the modified forms of PVC were significantly higher in comparison to that of the control PVC. The microscope images further reveal that pure PVC does not support cellular adhesion at all while PVC-TS, PVS-TU and PVC-S assist adherence of cells to a significant extent compared to the pure material. Thus, these results suggest that modification of the PVC resins with different functional groups leads to an enhancement in their biocompatibility properties.

Fig. 8 Nuclear morphology of mMSCs grown on different polymeric surfaces for 24 h. Cells were cultured in direct contact with various samples and analyzed with a fluorescence microscope. (a) PVC; (b) PVC-TS; (c) PVC-TU; (d) PVC-S.

Conclusions

This work demonstrates the influence of different functional groups on the characteristics of the PVC surface and the resulting biocompatibility property. For this purpose, functionalized forms of PVC using thiosulphate, thiourea and sulphite have been fabricated through a nucleophilic substitution reaction using a phase transfer catalyst. The outcome reveals that the functionalized polymers are hydrophilic in nature, show reduced hemolytic activity, and support bacterial and cellular adhesion significantly. Further research, including in vivo testing for improving the biocompatibility of the surface modified PVC polymers, is needed to fully validate their potential uses in biomedical-related applications. We anticipate that the fabricated functionalized PVC polymers could be useful for recreating tissue-engineered implants, designing medical devices and developing drug delivery systems.

References

1. G. Khang, H. B. Lee and J. H. Lee, Polymeric Biomaterials, in The Biomedical Engineering Handbook, Second Edition. 2 Volume Set, ed. Joseph D. Bronzino, CRC Press, 2000.
2. D. Braun, J. Polym. Sci., Part A: Polym. Chem., 2004, 42, 578–586.
3. Z. Xiaobin, C. M. James, Y. Q. Hua, W. H. Robin and G. D. O. Lowe, J. Mater. Sci.: Mater. Med., 2008, 19, 713–719.
4. G. Speranza, G. Gottardi, C. Pederzolli, L. Lunelli, R. Canteri, L. Pasquardini, E. Carli, A. Lui, D. Maniglio, M. Brugnara and M. Anderle, Biomaterials, 2004, 25, 2029–2037.
5. H. Miguel, N. Rodrigo, R. Helmut and M. Carmen, Polym. Degrad. Stab., 2006, 91, 1915–1918.
6. K. G. Theo, S. T. Hetty and B. J. Henk, Biomaterials, 2004, 25, 1735–1747.
7. K. Tomohito, O. Masahiko, G. Guido, M. Tadaaki and Y. Toshiaki, J. Polym. Res., 2011, 18, 945–947.
8. Z. Zhengbao, M. Yan, Y. Xiuli, L. Meng and D. Zhifei, J. Appl. Polym. Sci., 2009, 256, 805–814.
9. B. Biji, D. S. Kumar, Y. Yoshida and A. Jayakrishnan, Biomaterials, 2005, 26, 3495–3502.
10. J. R. Nirmala and A. Jayakrishnan, Biomaterials, 2003, 24, 2205–2212.
11. Y. Jian-Lin, J. Staffan and L. Asa, Biomoterials, 1997, 18, 421–427.
12. S. Moulay, Prog. Polym. Sci., 2010, 35, 303–331.
13. J.-G. Joseph, React. Funct. Polym., 1999, 39, 99–138.
14. M. T. Khorasani and H. Mirzadeh, Radiat. Phys. Chem., 2007, 76, 1011–1016.
15. S. K. Amit, G. Mayank, T. Ragini, N. Gopal, S. K. S. Akhoury, B. T. Yamini and K. Dharmendra, Pharmacogn. J., 2012, 4, 35–40.
16. K. Govinda, M. Nira, S. Vakil, K. K. Rajni and M. Palay, J. Biomed. Mater. Res., Part A, 2012, 100, 3363–3373.
17. M. Tim, J. Immunol. Methods, 1983, 65, 55–63.
18. S. Narumon, W. Jatuphorn and T. Chanchana, J. Appl. Polym. Sci., 2013, 130, 2410–2421.
19. S. Lakshmi and A. Jayakrishnan, Biomaterials, 2002, 23, 4855–4862.
20. K. Tomohito, O. Masahiko, G. Guido, M. Tadaaki and Y. Toshiaki, Polym. Degrad. Stab., 2009, 94, 107–112.
21. K. A. Safyan, Int. J. Electrochem. Sci., 2012, 7, 561–568.
22. M. A. Nurwahyuni and Y. M. M. Sukeri, Prosiding Seminar Kimia Bersama, 2009.
23. G. Madhurambal, M. Mariappan and S. C. Mojumdar, J. Therm. Anal. Calorim., 2010, 100, 763–768.
24. M. Fiaz and M. Gilbert, Adv. Polym. Sci., 1998, 17, 37–51.
25. Z. Xiaoxian, L. Yaoxin, H. M. Jeanne and C. Zhan, Phys. Chem. Chem. Phys., 2015, 17, 4472–4482.
26. D. Pavithra and D. Mukesh, Biomed. Mater., 2008, 3, 1–13.
27. S. David, W. S. Daniel, L. R. Thomas and B. H. Robort, Cathet. Cardiovasc. Diagn., 1996, 39, 287–290.
28. G. K. Kamal, M. K. Pradeep, S. Pradeep, G. Mayank, N. Gopal and M. Pralay, Appl. Surf. Sci., 2013, 264, 375–382.
29. A. John, in Polymer Science and Technology, ed. K. L. Richard, O. Zale and E. Martin, 1975, vol. 8, pp. 181–203.
30. I. Kazuhika, I. Yasuhiko, E. Sayaka, S. Youichi and N. Nobuo, Colloids Surf., B, 2000, 18, 325–335.
31. B. Jonathan, Biological Performance of Materials, Fundamental of Biocompatibility, 3rd edn, Revised and Expanded, 1999, ch. 9.
32. C. M. Judith, C. Rui and H. A. John, Biomaterials, 2006, 27, 4783–4793.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra03362d

---