Biomaterial composed of chitosan, riboflavin and hydroxyapatite for bone tissue regeneration

doi:10.21203/rs.3.rs-3148266/v1

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Biomaterial composed of chitosan, riboflavin and hydroxyapatite for bone tissue regeneration

https://doi.org/10.21203/rs.3.rs-3148266/v1

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published 09 Oct, 2023

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Bone tissue engineering is an ongoing field of research due to the existing of burning needs in restoration and reconstruction of damaged bone. Numerous studies have shown the development of the biomaterials based on the hydroxyapatite, major component of bones. Biomaterials engineering approaches involve using a combination of miscellaneous bioactive molecules which may promote cell proliferation, and thus, forming a scaffold with the environment which favor the regeneration process. Chitosan, naturally occurring biodegradable polymer, possess some essential features, i.e biodegradability, biocompatibility, and in solid phase good porosity, which may be contributed to promote cell adhesion. Moreover, doping the materials with other biocompounds, will create a unique and multifunctional scaffold useful in regenerative medicine. Riboflavin is an essential water-soluble vitamin, which participates in numerous biological process, such as transport, cell development and reproduction. Therefore, this study is focused on the manufacturing of the composite materials based on the hydroxyapatite, chitosan and riboflavin. Scanning electron microscopy showed the porosity of the composite biomaterial, important factor which can affect cell ingrowth and new bone formation. The infrared spectroscopy demonstrated chemical interlinking between hydroxyapatite and chitosan phases as well as no evidence for chemical interaction between RF and the CS-HAP scaffold. This may alter physical and chemical properties of the scaffold towards better performance in potential regenerative applications, particularly, when the matrix is supplemented with RF. Indeed, in vitro experiments showed that the riboflavin increased the cell proliferation and migration of the fibroblasts and osteosarcoma cells. Due to the urgent need of development of material with a potential to prevent of implant-associated infections, the antimicrobial and antioxidant activity of the composite were determined. The composite material showed the inhibitory effect on Staphylococcus aureus and exhibited higher antioxidant activity compare to pure chitosan. The antibacterial effect may be due to the generation of ROS level. Moreover, the riboflavin photochemical treatment with blue LED light enhanced the ROS level, which could be a more accessible and safe practice to treat the implant-associated infections. All things considered, incorporating riboflavin into the biocomposite scaffolds may accelerate new bone regeneration.

Chitosan

riboflavin

hydroxyapatite

bone regeneration

Porous surface of composite materials as a scaffold for bone cell proliferation
Riboflavin-doping materials enhance cellular proliferation and cell migration
Riboflavin light-activated for wound healing and regeneration process
Composite materials inhibit mircrobial growth

Bone naturally possesses the intrinsic ability for regeneration during the development of the skeleton or remodeling through adult life, but also in response to various types of injuries [1]. In comparison to soft tissues, which heal predominantly through the scar tissue formation, bones heal through the generation of new bone. Therefore, bone repair can be described as regenerative process [2, 3]. In the clinical settings, the most common form of bone regeneration is fracture healing [3]. The biology of bone healing is very complex, involving many types of cells and intracellular and extracellular molecular signaling pathways to regenerate the skeleton and restore its function. It is comprised of well-orchestrated series of events; including hematoma formation, fibrocartilaginous callus formation, bone callus formation, and bone remodeling [4]. However, there are cases of bone fractures in which bone regeneration is impaired; there may be delayed union or non-union of the bones. This is due to major injuries, infections, certain diseases such as osteoporosis or other surgical interventions that led to the excision of the bone. In such cases, a bone graft is essential [5].

Biodegradable materials have been at the forefront of cutting-edge research and offer a truly viable option in the designing and manufacturing of composite in biomedical engineering [6]. Chitosan (CS) is a polycationic aminopolysaccharide obtained by the N-deacetylation of chitin, the main ingredient of crustacean shells, fungi or insects [7]. It is a well-known biocompatibility, biodegradability, and cytocompatibility components of various scaffold and composites. Due to its excellent film-forming properties, many biomedical application have been considered [7–9]. Among them, biomaterial composed of chitosan and hydroxyapatite seems to be the one of the major type of material consider for bone regeneration [10]. Hydroxyapatite (HAP) is the main mineral component of bone tissue, thus it has been widely used such as bone tissue engineering or skeleton implant [11, 12]. Moreover, it has been proved that chitosan/HAP scaffold may act as a proliferative support for bone cells [13].

Riboflavin is one of the vitamin necessary in the human diet. It can be provided primarily from plant sources [14]. In industry, it is most often produced synthetically, while it is also synthesized by various types of microorganisms [15]. Endogenous riboflavin synthesis pathway may be the target of the development of antimicrobials and exogenous riboflavin against infectious diseases caused by microorganisms. Vitamin B2 is used for the prevention and treatment of migraines [16], it has excellent antioxidant properties, which is why it is needed by the animal body to protect against oxidative stress [17]. In nanotechnology, riboflavin is used for targeted drug delivery, in optoelectronics or biosensors [18]. Riboflavin is also being investigated for applications in photodynamic therapy [19, 20]. Moreover, a potential use of riboflavin as adjuvants to improve bone metabolism is also suggest [21].

In this study, the chitosan-based composites, incorporated with riboflavin and hydroxyapatite, were fabricated and their properties were evaluated (Fig. 1). The physical, structural and biological properties of nanocomposite film and the riboflavin as the naturally occurred ingredient, were characterized. The spin trapping technique in combination with electron spin resonance (ESR) spectroscopy was used to demonstrate generation of hydroxyl radicals by irradiation of RF. The extracted riboflavin was tested on eukaryotic cells to assess its cytotoxicity and proliferative potential. Furthermore, the antioxidant and antibacterial capacity of these films were assessed. Our hypothesis is that the combination of naturally occurred chitosan and riboflavin with hydroxyapatite results in a material with unique characteristics, which can be further use as the bone regeneration material.

1.1 Strain and cell lines, media, chemicals, and culture conditions

Riboflavin (RF), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue tetrazolium bromide (MTT), antibiotic solution (Antibiotic Antimycotic Solution), chitosan with medium molecular weight (190–310 kDa), aqueous solutions of acetic acid (99.8%), dimethyl sulfoxide, silver nitrate, sodium citrate tribasic and α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were supplied from Sigma-Aldrich. For the cell culture experiment Dulbecco’s Modified Eagle Medium High Glucose (DMEM, Corning), fetal bovine serum (FBS, Biowest), phosphate buffered saline w/o magnesium and calcium (PBS, Corning), 0.25% trypsin (PAA) were used. Yeast strain and all cell lines were obtained from the University of Rzeszow, Institute of Biotechnology collection. Any human or animal were not involved in the study. Engineered strain Candida famata (AF-4/SEF1/RIB1/RIB7) was used through the study. YPD (BTL, Poland) medium containing yeast extract (10 g/L), peptone (10 g/L) and dextrose (20 g/L) was used to prepare the precultures. For cytotoxicity effect, the human bone osteosarcoma epithelial cells (U2OS) and mouse fibroblasts (NIH3T3) were assessed. Standard techniques were used for mammalian cell cultures. Cells were cultured in DMEM medium supplemented with 1% of antibiotics solution and 10% of fetal bovine serum at 37°C in an atmosphere containing 5% CO2; subcultured with typical trypsin treatment.

1.2 Composite preparation and characterization

Chitosan-acetic acid solution was prepared as described elsewhere [22]. Composites, in the form of films, were obtained using the casting/solvent evaporation method. 7,5 mL of 2% chitosan-acetic acid solution was taken and mixed with the 3,5 mL of riboflavin (60 µg/mL) and 5 mL of 1% of hydroxyapatite. To simplify further sample nomenclature, abbreviation was used, where CH stands for chitosan film, CH/RF stand for chitosan mixed with riboflavin, and CH/RF/HAP stands for chitosan mixed with riboflavin and hydroxyapatite. 10 mL of each liquid composite was poured into the teflon surface and dried in 22°C for 48 h.

1.3 Composite surface morphology examination by scanning electron microscope (SEM)

Composite materials in a liquid form were placed onto the aluminium grid and were dried at room temperature for 24 h. Prior to SEM observation, the samples were sputtered with a 4 nm gold layer to provide them with electronic conductivity and avoid electronic charging during SEM imaging. The SEM analysis was carried out using a TLD electron detector in the secondary electron (SE) mode with the following operating parameters: voltage 3 kV, sample current 50 pA and magnification 1–50 kx.

1.4 FTIR -ATR analysis

Infrared spectral analysis of riboflavin, chitosan and chitosan-riboflavin-hydroxyapatite composites was performed using IRSpirit-T (Shimadzu, Japan) spectrometer with attenuated total reflectance (ATR) accessory (QATR-S) at resolution of 4 cm-1 and 64 scans. The surface of the diamond crystal was cleaned with 70% ethanol to remove residuals from previous samples and a background was collected before each measurement. The spectra were baseline corrected and area normalized before the analysis using ChemoSpec [23] package in the R programming language [24].

1.5 Antioxidant properties of composite

The method for determining the scavenging ability of ABTS free radicals was based on the previously described method [25], with some modifications. Briefly, the equal amounts of liquid forms of composites were mixed with ABTS (7mM) and kept dark for further 10–30 minutes. The samples were centrifuged (5000×g, 15 minutes) and the absorbance of the supernatants was measured at 734 nm. The activity of scavenging (SA%) radicals was expressed as follow:

$$SA\%=\frac{{Abs}_{ctrl}-{Abs}_{sample}}{{Abs}_{ctrl}}\times 100$$

where Abs_ctrl and Abs_sample are the absorbances of the control and tested sample, respectively.

1.6 Antibacterial activity

For antimicrobial assay, Gram-(−) strain Staphylococcus aureus (ATCC 25923) was employed. Nutrient broth and agar (BTL, Poland) or phosphate buffered saline (Sigma-Aldrich, USA) were used for cell culturing and serial dilutions, respectively. All assays were carried out in a laminar flow hood (Thermo Scientific, MSC Advantage). The bacteria preculture was overnight incubated in aerobic conditions at 37°C, and then was diluted to give an initial concentration about 1 × 10⁶ cells/mL. Aliquots of composite material (CS/HAP/RF) was mixed with inoculum and incubated for further 24 h with shaking. After this time, the aliquots of the control and tested sample were serially diluted and poured onto the agar plate. The number of colony forming units (CFU) were counted and expressed as the CFU/mL. All experiments were done in triplicate.

1.7 Riboflavin characterization

1.7.1 Spectrum identification

Candida famata was grown for 5 days in YNB medium composed of yeast nitrogen base (1.7 g/L), glucose (10g/L) and glycine (10g/L) at 30°C on a rotatory shaker (150 rpm) in a dark. The biomass was sediment after centrifugation (10000×g, 15 minutes) and the supernatant was filtered through 0.22 µm syringe filter (Merck). As the reference riboflavin, standard of vitamin B2 purchased from Sigma-Aldrich was used. The absorption spectrum and fluorescence emission spectrum of isolated and reference RF were recorded (Infinite M200, Thermo Scientific).

1.7.2 EPR measurements of radicals

Superoxide anions were generated immediately before EPR measurements by mixing 50 µL KOH solution (25 mM) with 50 µL hydrogen peroxide (25 mM) and then 50 µL of DMSO and 50 µL of POBN (180 mM) were added. After 5 min, 100 µL of solution of isolated RF (60 µg/mL) and 100 of POBN solution (prepared as above) were mixed and placed in a quartz glass test tube in the BRUKER FT-EPR ELEXSYS E580 spectrometer (BRUKER BIOSPIN, Billerica, MA, USA) with digital registration. The following settings were used: frequency, 9.403467 GHz; central field, 348.50 G; modulation amplitude, 0.5 G; modulation frequency, 100 kHz; microwave power, 47.43mW; power attenuation 5 dB; scan range, 100 G; conversion time, 30 ms; sweep time, 30.72 s. The EPR spectra were recorded and analyzed using the Xepr 2.6b.74 software. Xepr is a comprehensive software package of the ELEXSYS series, accommodating the needs of every user with highly developed acquisition and processing tools.

1.7.3 Raman spectroscopy

The identification of RF in extract from yeasts was performed by comparing Raman spectra of the extract and the reference RF sample in solution and in form of a powder. The i-Raman Plus (B&W Tek, Delaware, USA) spectrometer with a 785 nm diode laser as the excitation source and CCD cooled detector was utilized. The instrument was coupled to a video microscope (B&W Tek Inc., BAC151, 20X objective lens). The spectra were collected at 5 accumulations, acquisition time 15 s, and laser power of at least 20 mW. The sample solution was deposited on aluminium folia and allowed to evaporate before the measurements.

1.7.4 Biological properties of yeast riboflavin

Metabolic activity and cell migration ability after RF treatment were performed using the MTT and wound healing assay, respectively [26, 27]. Cells were seeded onto the 96- or 12-well plate at a density 3×10⁴ or 1×10⁵ cell/well. For MTT assay, 200 µL of DMEM containing different concentrations of RF were added to the wells. After 24 h of incubation, the medium was discarded and 200 µL of MTT (0.5 mg/mL) suspended in DMEM was then added per well. After 4 h of additional incubation, the supernatant was again discarded, and the purple formazans were dissolved with 100 µL of DMSO. The absorbance was read at a test wavelength of 570 nm and a reference wavelength of 630 nm and the OD results were presented as the percentage of controls values. For the wound healing assay, confluent cells at 12-well plate were scratched in the diameter of the culture well. Cells were then washed with PBS to remove non-adherent and dead cells. Wells were filled with 500 µL of DMEM supplemented with various concentrations of RF. Wound closures were periodically visualized (0, 24, and 48 h) under microscope and were compared to the etoposide treatment (well-known inhibitor of the cell migration).

Self-healing of bone from damage caused by infection or trauma is limited; thus, external intervention is needed to stimulate bone repair [28]. At present, various biomaterials are designed to develop a composite materials suitable for bone regeneration. Hydroxyapatite is often used in dental and orthopedic implants, because it can induce the formation of bone-like apatite and promote bone healing [29]. Fillers such as bioactive nano-hydroxyapatite is also used used, where nano-hydroxyapatite surface allows osteoblastic cell adhesion and growth; thus, new bone is formed by substitution from adjacent normal bone [28, 30]. However, HAP possess some limitation, i.e. its tendency to fragment and trigger inflammatory reactions [31]. It has been reported that hybrid material composed of chitosan and hydroxyapatite have synergetic actions to significantly improve the biocompatibility and osteoconductivity in living bodies of those materials [32]. In view of the requirements of bone regeneration biomaterials for biocompatibility, a hybrid composites based on the chitosan and hydroxyapatite were fabricated. Moreover, in order to develop more efficient biomaterial, doping it with riboflavin as the proliferating agent, could be of interest and desirable. Gel casting method was used to manufacture the nanocomposite. Figure 2 shows the surface morphology of the composite materials visualized by SEM. The SEM images revealed that surface of the composite materials (Fig. 2B-D) are porosity and contain pores, compare to the smooth surface of the pure chitosan (Fig. 2A). This features may contribute to the cell adhesion of the bone regeneration material, due to the phenomenon that porosity may play an important role in the osteoconductivity [33]. Some studies have demonstrated a greater degree and faster rate of bone ingrowth or apposition with percentage porosity. Sufficient porosity of suitable size and interconnections between the pores, provides an environment to promote cell infiltration, migration, vascularisation, nutrient and oxygen flow and removal of waste materials while being able to withstand external loading stresses [34, 35].

We further used vibration spectroscopy to identify the chemical composition of yeast extract prepared for the experiments and to inspect chemical structure and thus possible interlinks between compounds/phases comprising the CS-RF-HAP composite (Fig. 3).

Chitosan was characterized primarily by bands at 1643, 1553, 1410, 1378, 1310, 1250, 1153, 1072, 1028, 943 and 896 cm^-1, majority of which is consistent with reports from other studies [36–39]. The infrared spectroscopy showed substantially altered spectrum of the CS/HAP/RF composite compared to that of pure chitosan. This reflected not only presence of added HAP, enriching spectrum with bands assigned primarily to vibrations of phosphate group (strong bands located in the region between 1150 − 1000 cm^-1 [40–42], but also showed changes in the location, intensity and shape of bands attributed to CS. The most striking changes were related to the amide I band corresponding to the C = O stretching vibrations of the residual N-acetylated groups, and to the amine and hydroxyl groups distributed evenly along the chitosan chain. These groups serving putatively as interactive sides were deduces on the base of disappearance or diminishing intensity of bands at about 1650, 1378, 1310, 1153, 943 and 896 cm^-1, assignation of which was discussed in literature [43–47].

Earlier studies on composites produced on the base of CS and HAP showed similar spectral features which were attributed primarily to formation of hydrogen bonds between CS and HAP via mentioned above functional groups [40, 46–49]. The examined spectra resembled also, to some extent, spectra of phosphorylated chitosan [50, 51], that may suggest also formation of covalent bonds with the participation of the phosphate groups. It should be mentioned that the FTIR-ATR technique, used in this study, reflects chemical structure only at the surface of examined samples. Obtained spectral features indicate also some heterogeneity of the composite sheets in terms of the compounds ratios and formed interlinks that was demonstrated by relatively high standard deviation of the spectral absorbances. (Fig. 3). This study provided no clear evidence that RF was bound via chemical bonds to the CS-HAP scaffold, as the contribution from RF was observed in the composite spectrum at 1740 and at 1650 cm^-1 (a small sharp peak instead of reduced broad amide I) without substantial changes of location compared to the RF reference spectra [52, 53]. Possibly, this compound is just adsorbed and thus easily available to perform its biological activity. The infrared spectroscopy allowed also to detect carbonation of HAP (a shoulder at 1443 cm^-1 and a weak band at 870 cm^-1), that made the composite matrix of less regular structure compared to HAP without the substitution [54]. This may alter physical and chemical properties of the scaffold [55] towards better performance in potential regenerative applications, particularly, when the matrix is supplemented with RF.

Development of new types of antioxidants biomaterial is of utmost importance due to increasing problems with pathogens transmission. Hence, in the next study the antioxidant and antimicrobial potential of active biomaterial was examined. In general, the incorporation of RF increased the antioxidant activity compare to pure and hydroxyapatite –doped chitosan (Fig. 4A). Numerous studies have investigated the antioxidant properties of some vitamins such as vitamin E, vitamin C and carotenoids [review in 17] and their effects on human health. However, the riboflavin is the one neglected antioxidant vitamin, which in fact acts as a coenzyme for redox enzymes in FAD and FMN forms. The results of the reviewed studies indicate that the antioxidant nature of RF is due to protection the body against oxidative stress, especially lipid peroxidation and reperfusion oxidative [17]. The researchers already have shown the development of the biodegradable material incorporated with riboflavin, however there is no evaluation its antioxidant potential [56]. Antimicrobial results showed the inhibitory effect on Staphylococcus aureus; the number of colony forming units was significantly reduced (Fig. 4B). Thus, the results of this work are very valuable, in the field of the using material to bone regeneration and impact on the oxidative injuries as well as the material with enhancement of antibacterial potential, i.e in food packaging industry.

This antibacterial performance of the composite doped with riboflavin may be the results of the photosensitizer action of RF. It is known that irradiated riboflavin generates significant intracellular ROS and induces oxidative stress [57]. Thus, we further examined the nature of the RF isolated from the yeast, which was the component of the composite materials.

The riboflavin production was determined, during a fermentation period extended for 5 days. During this experiment, we noticed that the color of the fermentation broth gradually turned yellow and emitted fluorescence when exposed to UV light (Fig. 5 inset). To characterize ribflavin, its UV-visible and excitation/emission spectra were recorded with a fluorescence spectrophotometer. The absorption spectrum and fluorescence emission spectrum of isolated and reference riboflavin are presented in Fig. 5A and Fig. 5B, respectively. Over the wavelength range of 300–800 nm, the absorption spectrum of both riboflavin demonstrated two absorption peaks at 370 and 440 nm (Fig. 5A). In the fluorescence scans (Fig. 5B), the emission maximum is reached at 533 nm for the fluorescence emission spectrum, with excitation at 450 nm. The data obtained for the riboflavin isolated from yeast overlapped with the spectrum of the reference riboflavin. Indeed, the identification of the riboflavin has been determined previously, where the highest peak was reached at ~ 370 and ~ 440 nm or at ~ 530 nm, for UV-Vis and fluorescence spectrum respectively [58, 59].

Oxidative stress plays an important role in homeostasis and disease in most tissues. Reactive oxygen species (ROS) are continuously generated as byproducts of normal cellular metabolism [60]. Moreover, ROS are increasingly being recognized as a key component of the bone repair paradigm [61]. RF has been reported to be one of the photosensitizers, which can be activated in certain wavelengths to produce reactive oxygen species with strong oxidation, so as to inactivate pathogenic microorganisms. This unique properties is directly related to its chemical structure, which creates great potential for making RF as a mediator to prepare polymer functional materials. The critical structure that determines RF to belong to the flavin family is a tricyclic structure 7,8-dimethyl-10-alkylisoalloxazine. This essential fragment is responsible for the redox process, subsequent catalytic activity, UV absorption, and photosensitivity [62]. Indeed, some studies have been conducted on the role of RF in combination with biopolymers in tissue engineering [63]. The absorbance and fluorescence spectrum of isolated RF, may suggest its well behavior as the photosensitizer after irradiation. The next experiment presents an EPR study on the free radicals formed by exposing riboflavin to blue light. POBN has been used as spin-traps for the short-lived free radicals formed during this process. The concentration of radicals after irradiation of RF is shown on Fig. 6. The blue-light irradiation increased the initial concentration of radicals in RF, up to the 15 minutes time of illumination. While, after switching off the light, the constant level (no further increasing) of radicals is observed. This study shows the possibility of using RF as the component of materials for bone regeneration, which may interact after irradiation resulting in increasing radicals. Sel et al reported on impact of UVA irradiation of riboflavin, which indeed, generated oxygen-dependent hydroxyl radicals [64]. Moreover, it is noticed that the vitamin under visible light can also generate reactive oxygen species (ROS), including superoxide anions, and singlet oxygen [65]. Based on these facts, we can conclude that photoilluminated riboflavin renders the redox status of bacterial cells into a compromised state leading to significant membrane damage ultimately causing bacterial death. Although, we showed the antibacterial potential of composite, the more effective material after its irradiation would may have occurred. This study aims to add one more therapeutic dimension to photoilluminated of composite doped with RF, as it can be effectively employed in targeting bacterial biofilms occurring after biomplantation.

Examination of the riboflavin presence in the yeast extract was performed by means of vibration spectroscopy. Initially applied the infrared spectroscopy provided only poor results since the extract deposited on a diamond crystal of the ATR device produced low and thus noisy absorbance. This resulted from the fact that only low amount of the dissolved compounds remained on the ATR crystal surface after diluent evaporation. More plausible information was achieved using Raman spectroscopy along a microscope device. The spectral features of the extract highly resembled those of the RF standard in solution (Fig. 7). Particularly, high conformity in the location and shape was observed for the most prominent and specific band at around of 1347 cm^-1, assigned to the in-plane vibrations of the isoalloxazine ring [66]. Moderate absorbances at low frequency region (around 1180, 973, 878, 542 and 441 cm^-1), possibly assigned to C-H and O-H deformations vibration of ribityl chain and out-of-plain vibrations of the rings [67, 68] further confirmed high performance of the extraction process.

Riboflavin is well known component of flavo-coenzymes important in numerous reactions. But also other biological properties have been ascribed to riboflavin. Among them, its additive effect on osteoblast differentiation of MC3T3-E1 cells seems to be promising and may be a therapeutic approaches for dealing with osteoporosis [21]. Moreover, the development of biomaterials with unique features has lastly attracted great attention for bone regeneration, wound healing, and medical purposes [69]. Thus, we further performed the experiments to assess the biological activity of RF as a future component of composite material. The potential effect of RF on cell metabolic activity was tested using MTT assay which measures the cell mitochondrial activity through NAD(P)H-dependent cellular oxidoreductase enzyme [70]. The metabolic activity of various concentrations of the riboflavin toward two different cell lines: mouse embryonic fibroblasts (NIH 3T3) and human osteosarcoma (U-2OS) is shown on Fig. 8. We found that riboflavin treatment significantly increased the cell metabolic activity, especially when exposed to the vitamin isolated from microorganism. The results show that this metabolite does not have a toxic impact on the tested cell lines. Indeed, our data are in line with the Chaves Nato et al. who reported on the riboflavin and its irradiated version which did not affect osteoblast viability [21]. Moreover, the protective effect of riboflavin as a component of other material has also been suggested. Xizhe Li et al. manufactured ultrasmall riboflavin-protected silver nanoclusters (RF@AgNCs) that can effectively kill or suppress the growth of pathogen. At the same time, they were found to be non-toxic to human red blood cells and mammalian cells [71].

Many authors usually report on the cytotoxicity or biocompatibility of the tested compound toward cells based on the only one method used for the analysis. Therefore, in this study either the MTT assay, or wound healing assay have been performed to evaluate the metabolic activity or the motility capacities of the cells, respectively. Cell mobility plays a crucial role in many physiological and pathological processes. Cell migration takes place during embryo development, wound healing or immune response. The motility capacities has been also implicated in many diseases like cancer or inflammation [72]. Cell mobility could therefore be used as a parameter to assess the physiological state of cell [27, 73]. In order to evaluate the NIH3T3 migration response, cells were exposed to different RF concentrations and allowed to migrate for 48 hours (Fig. 9). Etoposide was used as the chemical which exhibits negative impact on the wound-healing ability of the cell [74].

After 48 h exposure of the cells toward the riboflavin, the scratch closure for NIH3T3 cells was close to 100%, for each tested concentrations (Fig. 9). Whereas, the etoposide acts here as the chemical which inhibited the cells migration. Based on these two biological experiments, we concluded that riboflavin had no negative effect on cell migration and its metabolic activity. What more, it may play a role as the compound which activates the proliferation of the cell.

Developing non-cytotoxic composite material with unique additional functions is crucial for bone regeneration and tissue engineering field. The present study demonstrated the effective multifunctional composite. The material can be prepared into scaffold using the chitosan as a matrix, and hydroxyapatite and riboflavin as a phase filler. The porous surface morphology and possible interlinkage between the components, after doping of the pure chitosan, were confirmed through the SEM and FTIR analysis, respectively. Compared to existing technologies of manufacturing the chiotsan/HAP scaffold, doping it’s with riboflavin exhibited the novel features; i.e antimicrobial potential and antioxidant activity. Moreover, the noncytotoxic RF significantly enhanced cellular proliferation and migration, and is light-activated producing the free radicals. Therefore, this study demonstrated the new material which could be useful for bone tissue regeneration.

AUTHOR INFORMATIONS

Corresponding Author

Małgorzata Kus-Liśkiewicz, Institute of Biotechnology, College of Natural Sciences, University of Rzeszow, Pigonia 1, 35‑310 Rzeszow, Poland; https://orcid.org/0000-0003-2564-6820, [email protected]

Author

Justyna Gaweł, Institute of Biotechnology, College of Natural Sciences, University of Rzeszow, Pigonia 1, 35‑310 Rzeszow, Poland

Justyna Milan, Institute of Biotechnology, College of Natural Sciences, University of Rzeszow, Pigonia 1, 35‑310 Rzeszow, Poland; https://orcid.org/0000-0002-8361-6833

Jacek Żebrowski, Institute of Biotechnology, College of Natural Sciences, University of Rzeszow, Pigonia 1, 35‑310 Rzeszow, Poland; https://orcid.org/0000-0001-8424-857X

Dariusz Płoch, Institute of Material Engineering, College of Natural Sciences, University of Rzeszow, Pigonia 1 St, 35‑310 Rzeszow, Poland; https://orcid.org/0000-0001-6255-1279

Ireneusz Stefaniuk, Institute of Materials Engineering, College of Natural Sciences, University of Rzeszow, 1 Pigonia Street, 35-310 Rzeszow, Poland https://orcid.org/0000-0003-2616-9595

Author Contributions

The manuscript was written through contributions of all authors. MK-L designed the original idea and wrote the manuscript; MK-L, JG, JM conducted the biological experiments; JZ performed the FTIR-ATR and Raman analysis; DP performed SEM imaging; IS conducted the EPR measurements. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT

The authors are grateful to prof. Andriy Sybirnyy and prof. Justyna Ruchała for enabling experiments on the Candida famata strain.

DATA AVAILABILITY

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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Biomaterial composed of chitosan, riboflavin and hydroxyapatite for bone tissue regeneration

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