Abstract
The present study aimed to synthesize and characterize polymer films at different graphene oxide (GO) concentrations for use in advanced cell therapy. Chitosan-xanthan (CX) films were prepared and combined with GO and then assigned to the following groups: G1 – CX; G2 – CX GO 0.5%; G3 – CX GO 1.0%; and G4 – CX GO 1.5%. The films were analyzed for their structural, mechanical, and biological properties by Raman and Fourier-Transform Infrared (FTIR) spectroscopy, Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), Confocal Laser Scanning Microscopy (surface roughness measurement), Tensile Strength, and in vitro Bioactivity assay. Tensile strength and surface roughness data were analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s test (α = 0.05). FTIR spectroscopy showed the presence of amide I and II bands (characteristic of chitosan) and carboxyl group (characteristic of xanthan). CO bands characteristic of GO were observed in groups containing these particles. Raman spectroscopy revealed D and G bands in the GO-containing groups. Film surface morphology exhibited a homogeneous, compact, and pore-free surface. The CX group had higher tensile strength (5.89 ± 1.62 KPa) (p < 0.05). No statistically significant difference was observed in the other GO-containing groups (p > 0.05). The films induced the deposition of apatite crystals on their surfaces, exhibiting activity under biomineralization conditions. Graphene oxide added into chitosan-xanthan films enhanced surface roughness and bioactivity but decreased tensile strength. The synthesized CXGO films are promising for advanced cell therapy because of their physicochemical, morphological, and mechanical properties, which seem ideal for tissue regeneration.
Introduction
Therapeutic and rehabilitation approaches to managing patients with bone tissue defects are still limiting factors for successful tissue regeneration. Owing to different etiologic factors, such as early tooth losses, cystic diseases, benign/malignant tumors, and mechanical and physical trauma, cell therapies targeted at the specificity of each remnant tissue are an urgent necessity. 1
Accordingly, guided bone regeneration seeks to replace lost bone tissues, allowing for new bone formation. Moreover, the site has to be isolated through a barrier for its maintenance during bone tissue formation and maturation until dimensional stability is obtained. Therefore, it is necessary to have a film that permits the selective entry of nutrients and cells that are capable of tissue formation and do not lead to structural loss due to infections or invasion of mucosal or epithelial tissue.2 –4
In order to enhance and possibly speed up bone regeneration, tissue engineering combines biomaterials, growth factors, and multipotent stem cells that trigger the organic process through cell differentiation and gene and protein expression, thereby guiding biomineralization. These processes mimic the extracellular bone matrix, which allows for nutrient transport and the emission of morphogenetic signals involved in the dimension, density, and structure of the newly formed bone tissue.4 –7
In bone regeneration, films made of biomaterials are widely used and should possess the following properties: osteoinduction, osteoconduction, and osteointegration. Osteoinduction promotes the differentiation of progenitor cells into osteoblasts, osteoconduction enables the biomaterial to withstand the growth and augmentation of surrounding bone tissues, and osteointegration promotes the integration of the biomaterial into the adjacent bone tissue. 1
Chitosan-xanthan, naturally occurring polymer complexes, have been described in other studies4,7 –9 to provide the necessary structure for tissue regeneration processes. Chitosan membranes demonstrate significant promise due to their strong antimicrobial properties, biodegradability, excellent biocompatibility and functionalization, improved mechanical properties, support osteoblast proliferation and differentiation, and versatility of process into various forms.10,11 The addition of Xanthan gum, a polysaccharide derived from biological sources, further improves these membranes by increasing their mechanical strength, stability across varying pH levels, and resistance to aqueous absorption. 12
The physical attributes of these biomaterials, including roughness, porosity, and medium absorption, create an ideal environment for cell growth and periosteal tissue regeneration, which can facilitate osseointegration. 6 Therefore, their physicochemical, mechanical, morphological, and biological properties make them suitable for tissue engineering.5,10,11 Additionally, previous research 7 has demonstrated that CX scaffolds, combined with mesenchymal stem cells, effectively regulate the in vivo inflammatory process over time, exhibiting great angiogenic potential and promoting new bone formation.
Graphene oxide (GO), a graphene derivative, is known for its high surface area, mechanical strength, excellent physicochemical properties, good electrical conductivity, and remarkable biocompatibility. These characteristics make GO a promising additive for enhancing biomaterials used in tissue engineering.13,14
Despite the advancements, there is still a gap in understanding how incorporating graphene oxide (GO) into chitosan-xanthan (CX) films can enhance their properties beyond what is achieved with natural polymer complexes alone. Previous researches have shown the potential of CX scaffolds/membranes and GO.4,7 –9,13,14 However, the combined effects of GO with CX to improve bone regeneration have not been thoroughly investigated. Therefore, the present study aims to address this gap by analyzing the impact of different GO concentrations on the properties of chitosan-xanthan films for use in cell regenerative therapies.
Materials and methods
Study design
Chitosan-xanthan (CX) films were initially synthesized at different GO concentrations. The specimens were assigned to the following groups: (1) CX; (2) CX GO 0.5%; (3) CX GO 1.0%; and (4) CX GO 1.5%. The films were chemically and morphologically characterized by Raman spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), confocal laser scanning microscopy (roughness and topography), and scanning electron microscopy (SEM). Tensile strength and in vitro bioactivity were also assessed.
Preparation of CX and CX+GO polymer films
The following solutions were used: 1% xanthan (w/v) in deionized water (Milli-Q system, Millipore; pH = 7.7) and 1% chitosan (w/v) in an aqueous solution of lactic acid (v/v), using a chitosan-to-xanthan mass ratio of 1:1.7,15 For CX + GO films, GO was synthesized using liquid-phase exfoliation of graphite via the chemical route, as described by Lopes et al. 16 , and added at different concentrations (0.5%, 1.0%, and 1.5%) to the 1% xanthan solution (w/v).
Two hundred milliliters of chitosan solution were added to 200 mL of xanthan solution using a Gilson peristaltic pump (Minipuls 3) at a 10 mL/min flow rate. Mixing was performed in a jacketed flat-bottomed cylindrical stainless-steel reactor with an internal diameter of 10 cm and a height of 20 cm. Upon addition of the chitosan solution to the xanthan solution, the system was kept under agitation at 1000 rpm with the aid of a Quimis Q-251 D mechanical stirrer with marine-style impeller and inclined blades and a radius of 3 cm positioned at approximately 4 cm from the reactor base. The system’s temperature was controlled at 25°C using a thermostatic bath (Q214M2, Quimis).
After adding the chitosan solution to the xanthan solution, the rotation speed was increased to 1200 rpm and kept at that level for 10 min. 12 Thereafter, the mixture was evenly distributed in terms of mass onto 15 cm diameter polystyrene Petri dishes and allowed to ventilate (model 410D, Nova Ética) at a constant temperature (37°C) for approximately 48 h.7,12
Raman spectroscopy
Raman spectroscopy was performed on a LabRAM HR Evolution spectrophotometer (HORIBA, Villeneuve d’Ascq, France) equipped with a CCD detector and red laser. The excitation wavelength ranged from 1100 to 2100 cm−1 with a 40-s acquisition period and 1% power.
Fourier-transform infrared (FTIR) spectroscopy
The functional groups were identified by FTIR spectroscopy (FT-IR; Thermo Scientific, Nicolet 6700, Madison, WA, USA). The specimens were prepared as transparent pellets using attenuated total reflectance (germanium crystal) and the Smart Omni Sampler accessory. The spectra were obtained in the wavenumber range of 4000 to 675 cm−1, with a resolution of 4 cm−1 and 128 scans.
Thermogravimetric analysis (TGA)
The thermal stability of the films was assessed by mass changes as a function of temperature using a TGA 50M thermogravimetric analyzer (Shimadzu; Kyoto, Japan). The specimens weighing approximately 1 mg were placed on aluminum pans and heated at a rate of 10°C/min from ambient temperature to 900°C. The experiments were performed in a nitrogen atmosphere (flow rate of 100 mL/min).
Scanning electron microscopy (SEM)
A single calibrated operator morphologically analyzed the films under a scanning electron microscope (JSM 5600 LV; JEOL, Tokyo, Japan) at an accelerating 15 kV and 2000x magnification. For the SEM analyses, the specimens were gold sputtered in a metallizing machine (Bal-tec SCD050, Furstentum, Liechtenstein).
Surface roughness and topography
Surface topography at the nanometric level and roughness (Ra) in µm were analyzed under an Olympus LEXT OLS 4000 3D confocal laser scanning microscope (Olympus Corp, Tokyo, Japan).
Tensile strength analysis
The tensile mechanical properties of the films were assessed following ASTM D-882. 4 Ten specimens (20.0 mm high × 10.0 mm) were tested on an Instron 4411 universal testing machine (Illinois, Chicago, USA) using a 50 N load cell, a gauge length of 5 cm, and a speed of 1 mm/min.
In vitro bioactivity assay
The bioactivity assay was introduced by Kokubo and Yamaguchi 17 and consists of the immersion of the inorganic material in simulated body fluid (SBF), whose ionic concentration is similar to human blood plasma. Changes in surface morphology, specifically, the presence of apatite (bone apatite) on the biomaterial surface, is a positive finding in a bioactivity assay. Cell-free culture media have been used to bridge the divide between the results of in vitro and in vivo experiments and to evaluate the behavior and bioactivity on the surface of biomaterials. 18
The bioactivity assay was conducted in a Panasonic CO2 incubator (model COM-19AIC-PA) at 37°C and 5% CO2 at 3, 7, and 14 days. The specimens were immersed in 20 mL of McCoy medium for SEM assessment of spontaneous precipitation of bone apatite on the biomaterial surface. A single calibrated operator morphologically analyzed the films under an SEM (JSM 5600 LV; JEOL, Tokyo, Japan) at an accelerating 15 kV, WD between 14.2 and 20.4 mm, and 3500x magnification. For the SEM analyses, the specimens were gold sputtered in a metallizing machine (Bal-tec SCD050, Furstentum, Liechtenstein).
Statistical analysis
The data on tensile strength and surface roughness were assessed by one-way analysis of variance (ANOVA) and Tukey’s post-hoc test at a significance level of 95%.
Results and discussion
The presence of GO on experimental films was assessed by Raman spectroscopy. Figure 1 shows the presence of this particle with specific bands in all specimens at different GO concentrations (D band = 1355 cm−1 and G band = 1577 cm−1). Nevertheless, these bands were not observed on the CX film, indicating the absence of these particles in this experimental group.4,9,16
Raman spectra of the chitosan-xanthan (CX) film, CX-graphene oxide at 0.5% (CX GO 0.5%), CX GO 1.0%, and CX GO 1.5%. Presence of D and G bands in the GO-containing groups.
Figure 2 shows the FTIR spectroscopy results. All films exhibited amide I (peak at 1016–1025 cm−1) and amide II (1560 cm−1) bands, which are characteristic of chitosan, in addition to the carboxyl group (1410 cm−1), characteristic of xanthan.16,19 GO-containing groups showed CO bands at 1414 and 1070 cm−1.20,21 According to Aguiar et al., 21 the 1240–1405 cm−1 range corresponds to the overlapping of CH2-OH, CH3, CN-NH, and C-O stretching bands and deformation of C-OH in carboxylic acid, which are expected to all films.
FTIR spectroscopic analysis of the chitosan/xanthan (CX), CX/graphene oxide (CXGO) at 0.5%, 1.0%, and 1.5%. CO bands were observed in GO-containing groups.
The thermogravimetric analysis evaluates the mass of the specimen as a function of temperature. As temperature rises, the materials lose weight (Figure 3). Mass initially decreases the polymer mass loss from 90°C to 340°C. Mass loss may be associated with chitosan depolymerization and degradation of xanthan gum, which occurs at higher temperatures.4,15 Loss of mass can also be related to the structural loss of the particle, which may undergo degradation as temperature rises. 22
Thermogravimetric analysis (TGA) of the CX, CX GO 0.5%, CX GO 1.0%, and CX GO 1.5%. Films lose weight as the temperature increases. The mass loss decreased as follows: CX > CX GO 1.0% > CX GO 1.5% > CX GO 0.5%.
The micrographs of experimental films (Figure 4) show smooth, pore-free, and thick surfaces in the GO-containing groups. The density and homogeneity of this surface allow for its use as a protective barrier and guidance for bone regeneration. These characteristics of surface quality support the prevention of connective tissue from invading defective spaces and allow for osteoblast adhesion and blood clot stabilization.4,23–25
SEM micrographs (2000x) of CX and GO-containing films.
Adding GO particles to the polymer matrix increased the surface roughness compared to the CX group (p < 0.05). Moreover, the different concentrations of GO were not significantly different in terms of roughness (p ⩾ 0.05) (Figures 5 and 6). Cell proliferation requires that the cells be adhered to a surface, and surface characteristics such as roughness are closely related to the proliferative potential.26,27 Li et al. 27 incorporated graphene and carbon particles and noted an increase in the surface roughness of a polymer matrix, which sped up the metabolism of bacterial cells. Regarding the regenerative process, the higher the proliferation process, the higher the gene and protein expression and, consequently, the larger the biomineralization and new bone formation.
The roughness of CX, CX GO 0.5%, CX GO 1.0%, and CX GO 1.5% films. Different lowercase letters indicate statistical differences between the groups (p < 0.05). GO-containing groups showed a higher roughness surface compared to the CX group.
Confocal images of CX (a), CX GO 0.5% (b), CX GO 1.0% (c), and CX GO 1.5% (d) films.
The experimental groups containing GO particles exhibited lower tensile strength than the CX group (p < 0.05), as shown in Table 1. The lower value after the incorporation of the particles could be explained by interference of the particles in the crosslinking capacity of the polymer complex. 28 Despite the reduction in tensile strength, all groups showed adequate mechanical properties to act as a barrier in guided bone regeneration, holding significant promise compared to other benefits of GO incorporation. 4
Mean (standard deviation) of tensile strength of films in the dry state.
Different lowercase letters indicate a statistical difference in the columns (p < 0.05).
The micrographs of the films at 3, 7, and 14 days grown in McCoy medium are shown in Figure 7. Amorphous and irregularly shaped agglomerates were precipitated on the films. However, CX films exhibited areas without such deposits at 3 and 7 days. Calcium- and phosphate-rich precipitates were observed on the films after 14 days, indicating apatite deposition and the bioactivity potential of these materials. The presence of higher concentrations of GO contributed to bioactivity. In contrast, the presence of the CX polymer complex alone led to smaller calcium/phosphate precipitates or no precipitation at all, which could be related to promoting osteoconduction.4,29
Micrographs of the films (3500x) showing the in vitro bioactivity of (a-c) CX at 3, 7, and 14 days, respectively; (d-f) CX GO 0.5% at 3, 7, and 14 days; (g-i) CX GO 1.0% at 3, 7, and 14 days; (j-l) CX GO 1.5% at 3, 7, and 14 days. The blue arrow shows irregular and amorphous agglomerates precipitated on the films, except for CX at 3 and 7 days.
Based on these findings, the film has physicochemical properties that allow maintaining the copolymer elements for guided bone regeneration via advanced cell therapy. Wet film tests and assays mimicking the moist oral environment, in addition to different sterilization methods, may complement and validate clinical use in a more reliable fashion.
Conclusion
Incorporating graphene oxide into chitosan-xanthan films improves their surface roughness and biomineralization capability, enhancing their suitability for tissue regeneration. Although GO addition reduced the tensile strength of the films, the CX with the addition of GO films showed promising bioactivity by supporting apatite formation. Therefore, CXGO films have the potential for advanced cell therapy applications, particularly for improved guided bone regeneration outcomes.
Footnotes
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
This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (grant number #304493/2014-7) and from Technological Development and Innovative Extension (DT) category 2 of the CNPq (grant number # 303054/2023-9).
