Synthesis and characterization of a collagen-based composite material containing selenium nanoparticles

Abstract

Multidrug-resistant bacterial strains represent an emerging global health threat and a great obstacle for bone tissue engineering. One of the major components of the extracellular matrix of the bone is a collagen protein, while selenium is an element that has antimicrobial potential, and is also important for bone metabolism and bone health. Here we represent the incorporation of selenium nanoparticles (SeNPs) synthesized by the green chemical reduction method into collagen gels to produce a composite material, collagen/SeNPs, with antimicrobial properties. The samples were comprehensively characterized by zeta potential measurements, dynamic light scattering inductively coupled plasma-mass spectrometry (ICP-MS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), optical microscopy, field-emission scanning electron microscopy (FE-SEM), and differential scanning calorimetry The cytotoxicity of the SeNPS, as well as collagen/SeNPs, was tested on the MRC-5 cells. It was revealed that collagen/SeNPS expressed a lower cytotoxic effect. Collagen/SeNPs showed significant antibacterial activity against all tested Gram-positive strains, the major causative agents of orthopedic infections as well as Candida albicans. Furthermore, three-dimensional β-tricalcium phosphate (3D-TCP) scaffolds were fabricated by a well-established 3D printing (lithography) method, and afterward preliminary coated by newly-synthesized SeNPs or collagen/SeNPs. In addition, uncoated 3D-TCP scaffolds as well as coated by collagen/SeNPs were subjected to biofilm formation. The production of Staphylococcus aureus biofilm on coated scaffolds by collagen/SeNPs was significantly reduced compared to the uncoated ones.

Keywords

biomaterials composite materials selenium nanoparticles collagen antimicrobial activity scaffolds

Introduction

Many different polymers, both synthetic and natural, have been employed in making drug delivery systems, coating materials for implants, scaffolds, etc. They can also be used for the production of hydrogels, through chemical and physical methods such as physical crosslinking, chemical gelation, or self-assembly.¹ Special attention in the tissue engineering field has been given to collagen which is the major structural and the most abundant protein in the human body. Collagen has excellent biocompatibility due to its biodegradability and weak antigenicity.² Collagen gels and solutions have been used for drug delivery to accelerate wound healing or for building scaffolds or three-dimensional culture systems.² Selenium plays a critical role in a variety of physiological processes and it is necessary for bone health.³ Various studies have shown selenium deficiency can retard growth, alter bone metabolism and that insufficient selenium intakes have been associated with an increased risk of bone disease.^4,5 In addition, the antimicrobial and anticancer properties of selenium nanoparticles have also been reported in the literature.^6–8 The main idea of this work was to design and evaluate a new composite material combining collagen with immobilized selenium nanoparticles (collagen/SeNps) with antimicrobial activity intended as coatings for scaffolds in tissue engineering. In the body, an acidic state causes a lack of oxygenation at the cellular level and less than the maximum oxygen in the blood. Without proper oxygenation, microbial will prosper.⁹ For this reason, the synthesis of SeNPs was done by a modified method without using any surfactant or acidic reductant in the protocol. The prepared samples were subjected to essential characterization to analyze their structural, morphological, and antimicrobial properties. The cytotoxicity of the samples was tested on the MRC-5 cells. Additionally, 3D-TCP scaffolds were produced by a 3D printing method, and afterward preliminary coated by newly synthesized SeNPs or collagen/SeNPs. One of the major issues related to body implant devices generally is colonization by biofilm-forming microorganisms. The formation of biofilm has been reported to play a key role in the pathogenesis of implant-associated infections and provides an opportunity for the bacteria to develop drug resistance.^10,11 Thus, uncoated 3D-TCP scaffolds and those coated by collagen/SeNPs were further also preliminary subjected to biofilm formation. The study of biofilm formation was performed with two strains Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 27853).

Materials and Methods

Synthesis of SeNPs and collagen/SeNPs

In the synthesis of SeNPs with glucose as the reducing agent, first 30 mg of Na₂SeO₃ (Sigma–Aldrich, St Louis, MO) was dissolved in 10 mL distilled water. Then, this solution was added to an aqueous solution of glucose (0.25 g glucose in 25 mL distilled water), continuously being homogenized at 300 r/min and 130°C for 60 min, with the resulting solution becoming orange. The formation of SeNPs was immediately visualized by a color change of the reactant solution from colorless to orange. During the synthesis, the volume of the suspension has been reduced due to evaporation. Such prepared SeNPs were incorporated into the networks of collagen (collagen type I, Serva, Feinbiochemica GmbH&Co., Germany) to prepare novel collagen/SeNPs composite by a simple one-pot green method. Samples were prepared by adding 5 mL of a heated solution containing SeNPs to collagen (600 mg) under vigorous agitation. After homogenization, the mixtures were poured into petri dishes and left in a freezer overnight (t = 15 h, T = −50°C). After that, for the next 24 h, the samples were lyophilized in the freeze dryer (Christ, alpha 1-2 LDplus, Osterode am Harz, Germany) at the temperature of −57°C (temperature of ice condenser) and under the pressure of 0.37 Mbar.

Characterization of the samples

The samples were characterized by inductively coupled plasma-mass spectrometry (ICP-MS), X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), zeta potential analysis, dynamic light scattering (DLS), field-emission scanning electron microscopy (FE-SEM), and optical microscopy.

ICP-MS (iCAP Qc, Thermo Scientific, UK) as a quantitative chemical analysis was performed to determine the concentration of selenium in the samples. The solutions of samples were produced by diluting the corresponding stock solutions with 2.5% nitric acid. Quantitative analysis was performed using calibration standard solutions (MES-21-1 (AccuStandard, USA)) in the range of 10ppb–2ppm of every analyte referred to the spectral emission line of Se 196.026 nm.

X-ray diffraction spectra were obtained on an X-ray diffractometer, Philips PW 1050 diffractometer with Cu-Kα radiation (Ni filter). The samples were scanned in the 2θ range of 10°–80°, with a scanning step width of 0.05°, and 2 s per step.

The presence of specific functional groups was determined by FTIR spectroscopy. FTIR measurements were done on a spectrometer Nicolet iS10 (Thermo Fisher Scientific, Waltham, MA, USA), equipped with Smart iTX accessory (Thermo Fisher Scientific Inc. USA) at 4 cm⁻¹ spectral resolution and 32 scans. FTIR spectra were collected in reflection mode with a built-in diamond Attenuated Total Reflectance (ATR) sampling technique. The OMNIC software was used for the acquisition, processing, analyzing, and managing of FTIR data in a graphical environment.

The thermal behavior of the samples was analyzed by differential scanning calorimetry (DSC, SETARAM Instrumentation, France). The accurately weighed samples were placed in 30 μL aluminum pans, sealed and heated/cooled from room temperature up to 250°C with a heating rate of 10°C/min and using nitrogen as a purge gas. An empty aluminum pan was used as a reference.

The hydrodynamic particle sizes and zeta potential of the SeNPs were measured using the Zetasizer Nano ZS (Malvern Instruments, UK) instrument with a 4 mW HeNe laser (633 nm) and a light scattering detector positioned at 90^oat 25°C, setting a minimum of 10 and a maximum of 100 runs per measurement. The analyses were performed in triplicate.

To define the microstructure of SeNPs, field-emission scanning electron microscopy (FESEM) analysis was carried out, on a FESEM, TESCAN MIRA three XMU instrument. A carbon coating was used to prevent their charging.

The microstructure of the collagen/SeNPs was observed by OPTICA B-500MET light microscope (Optica SRL, Italy) by transmitted polarized and ordinary (not polarized) light. Images were collected with OPTIKAM PRO 8LT—4083.18 camera equipped with a scientific-grade CCD sensor.

Determining the cytotoxicity of SeNPs and collagen/SeNPs-MTT assay

Cell culture

MRC-5 cells were incubated at 37^oC and 5% CO₂ in Dulbecco’s Modified Eagle Medium supplemented with 10% bovine serum and 1% of penicillin/streptomycin mix. Trypsinization was used to passage the cells upon reaching 80% confluency.

Cell viability assay

First, 2x10⁴ of MRC-5 cells per well were seeded in 96 well plates and incubated for 24 h at 37^oC and 5% CO2. On the next day, chosen range of concentrations (0,1–10 μg/ml) of SeNPs was obtained using fresh cell medium to dilute samples, starting from the original suspension. Cell medium was then replaced with fresh medium containing different concentrations of SeNPs samples. Collagen/SeNPs sample was previously briefly incubated at 37^oC, until transforming to the liquid state, then it was diluted to selected concentrations (0,1–10 μg/ml) of SeNPs using cell medium. Sodium selenite powder was dissolved in the sterile distilled water and then 100 μg/ml stock diluted using cell medium. Samples were prepared in the 2 mL tubes, out of which 200 μl was added to each well, after the removal of the growth medium. Samples of higher concentrations had increased viscosity due to the amount of collagen and were incompatible with this method of treating cell culture. The treatment lasted for the next 24 h, under the same conditions of incubation. The medium was then replaced with 180 μl/well of fresh medium and 20 μl/well of MTT dye (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) from the stock of 5 mg/mL. Following 3 h incubation with the dye and formation of formazan crystals, the medium was discarded carefully. Crystals were then dissolved with dimethyl sulfoxide (DMSO), followed by light shaking of the plate. Absorbance was recorded with a Multiskan plate reader (Thermo Scientific), at 570 nm. Results of cell viability are expressed in proportion to the average absorbance of control cells, +/− standard error.

Coating of three-dimensional β-tricalcium phosphate scaffolds

Three-dimensional β-tricalcium phosphate (3D-TCP) scaffolds were fabricated by lithography-based additive manufacturing.¹² The utilized 3D printer is based on DLP technology, where a bitmap pattern corresponding to the individual cross-sections of the scaffold is projected onto a photosensitive, ceramic-filled resin. The scaffolds were printed using a commercial material (Lithabone TCP 300, Lithoz GmbH) and dried, de-binded, and sintered using the temperature schedule suggested by the manufacturer. Afterward, the scaffolds were preliminary coated by collagen/SeNPs (Figure 1). The coating was performed according to the modified protocol previously described in the literature for coating Bioglass®45S5 scaffolds.³ This was done by soaking/immersing the 3D-TCP scaffolds into SeNPs solution or collagen/SeNPs gel. The scaffolds were immersed for 30 min and after that were left to dry at room temperature and ambient pressure.

Antimicrobial activity

The colony counting method was employed for the evaluation of the antimicrobial activity of SeNPs and collagen/SeNPs. The following strains were included in the study: S. aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 13883, Salmonella enterica subsp. Enterica serovar Typhimurium ATCC 14028, P. aeruginosa ATCC 27853, and Candida albicans ATCC 10231. Microbial suspensions at approx. 10⁵ CFU/mL, prepared from fresh overnight cultures, were inoculated into Mueller–Hinton broth (HiMedia, India) for bacteria, or Sabouraud dextrose broth (Torlak, Serbia) for C. albicans, and incubated at 37°C for 24 h. Test groups were supplemented with SeNPs or collagen/SeNPs at concentrations 2.9 and 1.2 μg/mL, whereas control groups were left untreated. The number of grown colonies was counted by plating serial 10-fold dilutions on trypton soy agar (Torlak, Serbia) plates, on the next day before incubating for another 24 h at 37°C.

Antibiofilm assay

The effects of SeNPs and collagen/SeNPs on biofilm production were tested using the method described by Stepanović et al.¹³ Biofilm-producing strains, that is, S. aureus ATCC 6538, E. faecalis ATCC 29212, P. aeruginosa ATCC 27853, and C. albicans ATCC 10231, were selected for this assay. Briefly, strains (∼10⁶ CFU/mL) were incubated overnight at 37°C in the presence of SeNPs or collagen/SeNPs at concentration 2.9 and 1.2 μg/mL, by using trypton soy broth (Torlak, Serbia) supplemented with an additional 1% (w/v) glucose as the growth medium. Afterward, planktonic cells were removed by washing three times with sterile phosphate-buffered saline (PBS) and the amounts of formed biofilms were quantified by staining with 0.5% (w/v) safranin (HiMedia, India) for 15 min and measuring optical densities (ODs) of extracted stains using the EZ Read 400 Microplate Reader (Biochrom, Holliston, MA).

Further, a preliminary study regarding the formation of the biofilm on uncoated and collagen/SeNPs coated 3D-TCP scaffolds was done by using TTC (2,3,5-triphenyltetrazolium chloride, Sigma–Aldrich, St Louis, MO) biofilm staining procedure.¹⁴ The ability of S. aureus ATCC 6538 and P. aeruginosa ATCC 27853 to adhere and form biofilms on scaffolds was measured. First, uncoated scaffolds were incubated in the presence of bacteria at approx. 10⁶ CFU/mL at 37°C for 24 h, after which produced biofilms were stained by incubating the scaffolds in fresh media supplemented with 0.5% (w/v) TTC in 4:1 ratio, for 6 h at 37°C, following the washing step. Then the scaffolds were cleaned, disinfected, and sterilized by autoclaving, and the same procedure was repeated with coated scaffolds. Trypton soy broth supplemented with 1% (w/v) glucose was used as the growth medium and the quantification of biofilms was performed using the modified biofilm index, derived from computational image analysis by ImageJ 1.52a software (National Institutes of Health, Bethesda, MA).¹⁵

Statistical analysis

In the investigation of cytotoxicity (MTT assay), significance of the difference between means of summarized data for each SeNPs and collagen/SeNPs concentration was tested using a two-sample t-test in OriginPro 2018 Software. The significance level was set at p<0,05.

For statistical analysis in antimicrobial and antibiofilm experiments, each test was repeated three times. Results are presented as mean values ± standard deviations (SDs). All the groups were confirmed to exhibit normal distribution by the Shapiro–Wilk test. Experimental and control groups were compared by Student’s t-test. Calculations were performed using the SPSS Statistics, IBM SPSS Software, v24.0 (IBM, USA).

Results and discussion

The aqueous solution containing SeNPs, used for the preparation of collagen/SeNPs particles as well as collagen/SeNPs gels were analyzed by the ICP-MS method. Selenium content in the SeNPs suspension was determined to be 589 μg/ml while the selenium content in the collagen/SeNPs sample was 242 μg/g. Results were given as the mean value of three repeats on the same sample.

To determine the structure and phase composition of collagen/SeNPs, the XRD method was applied. XRD spectra of as-prepared SeNPs and collagen/SeNPs are shown in Figure 2(a). For better comparison, the diffractogram of pure commercial selenium (Sigma–Aldrich) is recorded as well. Collagen/SeNPs as an amorphous compound did not show any crystalline diffraction peaks in the presented spectra. A broad and very low-intensity peak in the 2θ range of 10–25° was observed, indicating its amorphous structure. None of the peaks that originate from sodium selenite are present in the diffractogram of SeNPs as a confirmation of complete reduction of this precursor (Supplementary Material (SM) Figure S1). It is a well-known fact that Se can exist in several allotropic forms, among which trigonal form is thermodynamically most stable. Precisely, this form is identified in the spectrum of commercial Se.¹⁶ Even more interesting is that most of the peaks recorded for commercial Se are also observed in the spectrum of Se obtained by chemical reduction with ascorbic acid (SM Figure S1), although with significantly lower intensity. However, the signal line in this sample possesses small-intense oscillations within the whole 2θ range, indicating that some of Se remained in amorphous form. Based on these data, it could be concluded that glucose serves as both a reducing and stabilizing agent since it prevents crystallization of Se in SeNPs.

FTIR spectra (Figure 2(b)) of the SeNPs, collagen, and collagen/SeNPs were recorded in the range of 400–4000 cm⁻¹ using a Thermo Scientific Nicolet iS10 Spectrometer. In addition, a spectrum obtained from commercial Se was given for comparison purposes. Analysis of the sample collagen/SeNPs revealed the presence of carboxyl, hydroxyl, and amide groups corresponding to typical collagen.¹⁷ The spectrum has a broad absorption band between 3287 and 3319 cm⁻¹ that can be assigned to –OH or –NH vibrations. There is an intact triple helical structure of collagens with the appearance of a typical amide II band of collagen at 1524–1547 cm⁻¹. Amide I band was observed at 1650 cm⁻¹ while the amide III band was found in the range 1230–1249 cm⁻¹. The change in the appearance of the amide III band in sample collagen/SeNPs indicates the change in collagen secondary structure which can be due to the interaction with SeNPs. The major difference in the spectra is observed in the fingerprint region, as a broad, intensive peak around 1050 cm⁻¹ is noticed only in the spectra of lyophilized collagen/SeNPs and SeNPs. This peak could be assigned to the vibration of C-O bonds in glucose¹⁸ (SM Figure S2). Additionally, two phenomena were noticed by comparing the spectra of collagen and lyophilized collagen/SeNPs. The first one is the absence of an amide B band for collagen/SeNPs, while the second one is the shifting of the amide II band toward higher frequency values. Since both of these bands originate from deformational vibrations of peptide NH bounds (the amide B band represents the overtones of the amide II band), mentioned occurrences can be correlated with increased hydrogen bonding. Similar behavior was reported in the past by Susi et al.¹⁹ They demonstrated that the intensity and vibrational frequency of the amide II band in collagen samples could be enhanced with an increase of hydrogen bonding. When collagen solution in water is heated the triple helix is broken and it separates into single coils, forming a solution. During gelation, the collagen chains try to regenerate the collagen triple-helix structure but can line up only in sections. The unaligned parts of the coils then align with parts of other coils. The helices interact to form a network, where the strands are held together by hydrogen-bonded junction zones and in some cases by chemical linkages. Collagen gels formed are thermos-reversible gels because they are held together by weak bonds. It is worth noting that none of the peaks characteristic for sodium selenite (observed at 715 cm⁻¹ and 445 cm-¹, SM Figure S2) were present in the spectrum of SeNPs, indicating a complete reduction of precursor. When it comes to the spectrum of pure S, as expected, none of the vibrations occurred after interaction with the incident IR beam.

The thermal analysis of collagen/SeNPs and SeNPs is given in Figure 2(c). For a better interpretation of obtained results, DSC thermograms of pure collagen and commercial selenium are shown as well. Based on the given thermograms four main events can be noticed: (i) loss of adsorbed moisture, (ii) melting of glucose (SM. Figure S3), (iii) denaturation of collagen, and (iv) a broad endothermic region that probably represents the combined effects of the previous two, along with a loss of water that was chemically bonded to the collagen chains. The thermal behavior of collagen and collagen-based hydrogels is an important property. There is a significant difference in the results obtained for hydrated and dried, especially lyophilized samples.^20,21 For the latter one, denaturation temperature is shifted toward higher values but with lower enthalpy, that is, amounts of bonded water. As collagen/SeNPs represents a composite system, the presence of glucose in SeNPs probably led to an increase in hydrogen bonding and the amount of bonded water molecules. Thus, the broad peak was observed and the three mentioned events cannot be separated. Nevertheless, the beginning of this endothermic change in the signal line is shifted compared to the onset of pure collagen denaturation. Such behavior is often observed in the crosslinking of collagen.^22,23 Conversely to SeNPs, when commercial Se is subjected to the heating regime, a clear and sharp peak is observed at 223°C due to the melting of Se crystals. Interestingly, in the thermogram of Se obtained by chemical reduction, besides this endothermic peak, an additional exothermic event occurred at 103°C (S. Figure 3). This phase transition could be assigned to the crystallization of the amorphous amount of Se in this sample. These results are in good correlation with those obtained by XRD measurements. The role of glucose in the stabilization of SeNPs is sufficiently intense to prevent the crystallization of this material.

Considering the results obtained from FTIR and DSC measurements, it is possible that in the sample of collagen/SeNPs presence of hydroxyl groups from glucose increases hydrogen bonding and crosslinking of collagen which eventually leads to entrapment of water molecules even at higher temperatures. Thus, evaporation of adsorbed water is not detected in the lower temperature region of collagen/SeNPs. This bridge-forming hydrogen bonding could be very important for hydrogels since it indicates higher stability and a possible increase in mechanical properties,²² both very important from the application aspect.

The hydrodynamic particle sizes and zeta potential of the SeNPs were measured using the Zetasizer Nano ZS. Zeta potential is the function of the surface charge which develops when any material is placed in a liquid, that is, this is the function of the dispersion/suspension pH which determines particle stability in the dispersion. Besides the size and shape of the particles, their surface charge has also a great impact on cellular uptake as well as regarding the dispersion stability of the particles. From that point of view, measurement of the electrokinetic or zeta-potential is very important. The mean zeta potential value for SeNPs was determined to be −45.2 mV, indicating the high stability of these particles. The mean measured value of the particle size for SeNPs in dispersion is 199 nm (Figure 3(a)). The polydispersity index (PDI) is an indication of the variance in the sample, and a PDI of 0.205 ± 0.019, was determined in the case of SeNPs which indicates that the sample does not have a quite narrow size distribution in dispersion but this is considered sufficient in practice for biomedical applications.²⁴

Representative FE-SEM images of as-prepared SeNPs (Figure 3(b)) were obtained using the electron microscope FE-SEM, TESCAN MIRA three XMU. SeNPs are spherical, uniform, with smooth surfaces, and with sizes of about 200 nm. The microstructure images of the collagen/SeNPs (Figure 3(c)) were obtained by the OPTICA B-500MET light microscope. The images show polymer threads forming three-dimensional collagen fibers. From the images randomly distributed spherical SeNPs can be seen which are incorporated within the collagen network.

Cytotoxicity of the SeNPs and collagen/SeNPs was investigated by MTT colorimetric assay on the MRC-5 cell line of human fetal fibroblasts. The results are presented in Figure 4. In this research phase, our attempt was to preliminary assess the biocompatibility of SeNPs and collagen/SeNPs on human cells. Due to their previous use for assessing biological activities of selenium-containing nanomaterials,^25,26 and also high sensitivity to selenium compounds, especially to Na₂SeO₃,^26,27 the MRC-5 cell line was selected for gaining the initial insight into our material biocompatibility. ISO 10993-5:2009(E), assessing in vitro cytotoxicity of medical devices, recognizes MRC-5 cell line in Part 5 (Cell lines), in the footnote stating preferred cell lines. MRC-5 is a normal human fibroblast cell line. Fibroblasts are the main constituent of the connective tissue, possess the ability to produce collagen type I, and have one of the key roles in wound healing. Because of their differentiation plasticity, they are present throughout the human organism and perform many functions.^28,29 Fibroblast cell lines express a similar toxicity response as osteoblasts.³⁰ Among fibroblast lineages, the MRC-5 cell line is widely known and used. MRC-5 are biologically similar to mesenchymal stem cells (MSC), and under passage 15 are even capable of induced differentiation into chondrocytes and osteocytes similarly to MSC.³¹

Obtained values of cell survival indicate that the presence of collagen significantly improved the viability of the cells exposed to concentrations of selenium above 0.1 μg/ml. Survival of cells treated with collagen/SeNPs did not fall under 80% at all tested concentrations, which is considered the threshold for cytotoxic effect, while survival of those treated with SeNPs gradually decreased with increasing concentrations of selenium. This is a preliminary finding showing that incorporation in collagen can alleviate toxicity of selenium nanoparticles on human fibroblasts, allowing the use of the range of concentrations necessary for antimicrobial effects.

The antimicrobial activity of SeNPs and collagen/SeNPs was investigated with S concentrations which did not significantly affect MRC-5 cells viability. The obtained results are presented in Figure 5. The growth of K. pneumoniae, S. Typhimurium, and C. albicans was significantly inhibited by both tested compounds. Collagen/SeNPs exhibited greater activity against E. coli, whereas SeNPs yielded better results regarding the inhibition of Gram-positive strains (S. aureus, E. faecalis, and B. subtilis).

On contrary, the biofilm of Gram-positive strains (S. aureus and E. faecalis) was more affected by collagen/SeNPs, which inhibited its production by 79–88% when applied at 2.9 μg/mL of selenium content. Significant inhibition of P. aeruginosa and C. albicans biofilm production was not observed, instead, the production even seemed to be stimulated in some cases (Figure 6). In accordance with this observation, P. aeruginosa produced more biomass when cultivated on collagen/SeNPs coated scaffolds compared to control (Figure 7). A possible explanation for this phenomenon could be that collagen, although as mentioned above, possesses many important beneficial properties, especially in tissue engineering,² it can also promote bacterial adhesion and growth.³² However, collagen/SeNPs coated scaffolds were significantly less prone to S. aureus biofilm attachment compared to uncoated scaffolds (Figure 7). This represents a very important finding since the surgical site infections are primarily caused by S. aureus, by far the most common surgical wound pathogen.³³ Since we examined the influence of SeNPs and collagen/SeNPs on the early stage of biofilm formation, which is strongly affected by the ability of surface attachment, we used 24 h of incubation.^34–36 Longer periods of incubations are used when the aim is to investigate the influence on later stages of biofilm maturation or the activity in biofilm eradication.³⁷

Although SeNPs can be very effective against versatile microbial species, their utilization remained a matter of debate from a toxicity aspect, for a long period.³⁸ Based on presented cell viability results, usage of higher concentrations of SeNPs within collagen/SeNPs for achieving the higher and broader antibacterial and antibiofilm activity could be potentially risky on the other side. Thus, considering all these facts, here presented collagen/SeNPs can be considered as a good and safe candidate for the coating of ceramic scaffolds, with the ability to efficiently suppress the formation of S. aureus biofilm. Achieving higher and broader antibacterial and antibiofilm activity of the collagen/SeNPs will be certainly a challenge of our future investigations.

Conclusion

Spherical SeNPs with sizes of about 200 nm have been synthesized by a simple, green chemical reduction method, and additionally incorporated into collagen gels to produce a composite material, collagen/SeNPs, to examine its therapeutic potential in regenerative medicine and wound healing. The quantitative chemical analysis of the samples, their amorphous structure and phase composition, thermal properties, and the stabilization effect of SeNPs solution on the triple helix structure of collagen, morphology, and stability were confirmed by ICP-MS, XRD, FTIR, DSC, SEM, optical microscopy, DLS, and zeta potential measurements. The cytotoxicity of SeNPs, as well as collagen/SeNPs, was examined toward MRC-5 cells. The viability of cells increased after the treatment with collagen/SeNPs in comparison to the cells treated with SeNPs. The samples significantly reduced growth of all tested microbial strains, except P. aeruginosa. Biofilm production of Gram-positive strains (S. aureus and E. faecalis) was also significantly inhibited by SeNPs and collagen/SeNPs. Furthermore, the production of S. aureus biofilm on collagen/SeNPs coated TCP scaffolds was significantly reduced compared to the uncoated TCP scaffolds. Our data suggest that collagen-based composite material containing selenium nanoparticles is a very promising candidate for applications in the tissue engineering field. A future aspect of this research will include the examination of composite materials with different ratios of SeNPs and collagen gels to improve their influence on inhibition, that is, the suppression of P. aeruginosa biofilm formation and other pathogens on coated scaffolds, to investigate the cell biology response to these composite material as well as in-vivo studies.

Supplemental Material

sj-pdf-1-jba-10.1177_08853282211073731 – Supplemental Material for Synthesis and characterization of a collagen-based composite material containing selenium nanoparticles

Supplemental Material, sj-pdf-1-jba-10.1177_08853282211073731 for Synthesis and characterization of a collagen-based composite material containing selenium nanoparticles by Magdalena M Stevanović, Nenad Filipović, Maja Kuzmanović, Nina Tomić, Dušan Ušjak, Marina Milenković, Kai Zheng, Juergen Stampfl and Aldo R Boccaccini in Journal of Biomaterials Applications

Footnotes

Acknowledgments

We thank Dr Malte Hartmann for the support in printing the 3D-TCP scaffolds.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Agreement on realization and financing of scientific research work of the Institute of Technical Sciences of SASA in 2021 (Record number: 451-03-9/2021-14/ 200175), the bilateral project between Serbia and Austria (project No: SRB 24/2018, project title: Scaffolds with controlled 3-D architecture designed by photopolymerization), and bilateral project between Serbia and Germany (DAAD project 57514776).

ORCID iDs

Magdalena M Stevanović

Nina Tomić

Supplemental material

Supplemental material for this article is available online.

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