Neuroregenerative Potential of Conductive Alginate-Graphene Oxide Scaffolds

Abstract

Neural regeneration requires an optimal environment, including structural, chemical, mechanical, and electrical properties. Alginate (Alg) and graphene oxide (GO) are promising biomaterials for nerve tissue engineering, as Alg provides biocompatibility and hydrogel formation, while GO enhances mechanical strength and conductivity. For this study, GO was synthesized using the modified Hummer’s method, and Alg–GO scaffolds with varying GO concentrations were developed. FTIR spectroscopy confirmed the successful incorporation of GO into the Alg matrix, while UV–Vis and photoluminescence analyses demonstrated GO-induced modifications of the optical properties. Thermal analysis revealed improved stability with increasing GO content, whereas swelling tests showed enhanced water uptake and retention. Conductivity measurements indicated a clear improvement in electrical conductivity, particularly at moderate GO concentrations. SEM imaging confirmed a homogeneous distribution of GO within the Alg matrix, with structural uniformity across all samples. Cytocompatibility was assessed using SH–SY5Y neuroblastoma cells through MTT, LDH, and LIVE/DEAD assays. All composites supported cell attachment, viability, and proliferation, with GO concentrations up to 6% promoting optimal cell growth without inducing cytotoxicity. In contrast, excessive GO content (9%) resulted in reduced proliferation, although biocompatibility was maintained. These results highlight the potential of Alg–GO scaffolds as promising candidates for neural tissue engineering. The findings demonstrate the potential of Alg–GO scaffolds as advanced biomaterials for regenerative medicine. Future research should focus on in vivo evaluations to confirm their therapeutic applicability.

1. Introduction

Tissue engineering and regenerative medicine (TE–RM) aim to develop biological substitutes that can support, restore, or regenerate damaged tissues and organs [1,2]. In the context of neural regeneration, several interrelated parameters–including structural organization, biochemical signaling, mechanical properties, and electrical activity–play pivotal roles in functional recovery. Biomaterial-based strategies have therefore attracted growing attention for the treatment of neural injuries, particularly in the peripheral nervous system (PNS) and spinal cord [3,4].

Unlike the central nervous system (CNS), the PNS retains a limited intrinsic capacity for regeneration. Nevertheless, in cases of severe injury associated with large nerve gaps, spontaneous repair is inadequate and autologous nerve grafting remains the clinical gold standard. Despite its effectiveness, this approach is hindered by serious drawbacks, including donor-site morbidity, limited tissue availability, and size mismatch, which have driven the exploration of alternative strategies such as neural guidance conduits (NGCs) and hydrogel-based delivery platforms [5].

An ideal biomaterial for neural regeneration must combine several essential characteristics: high biocompatibility, controlled biodegradability, low immunogenicity, and non-toxic, non-teratogenic, and non-carcinogenic behavior [6,7]. Over the past two decades, both synthetic and natural polymers have been extensively investigated as scaffolding materials. Synthetic polymers–including polyglycolic acid (PGA) [8,9], polylactic acid (PLA) [10], poly(lactide-co-glycolide) (PLGA) [11,12,13,14], poly(ε-caprolactone) (PCL) [15,16], and polyurethane (PU) [17]–offer tunable mechanical properties and processing versatility, yet often suffer from hydrophobicity and acidic degradation products. Natural biomaterials such as collagen [18,19], gelatin [20], silk fibroin [21], chitosan, and Alg have demonstrated superior biocompatibility and bioactivity, providing favorable environments for cell adhesion and proliferation [22,23,24,25]. In addition, extracellular matrix (ECM)-derived materials such as fibrin, laminin, and hyaluronic acid have been successfully applied in neural tissue engineering (NTE) [26,27,28].

Among natural biomaterials, Alg shows promising outcomes in PNS-TE when compared to other available options [29]. This polysaccharide occurs naturally and is composed of repeating units of (1,4)-α-L-guluronate (G) and (1,4)-β-D-mannuronate (M) [30]. Extracted from seaweed, Alg is widely used in the pharmaceutical and food industries due to its biocompatibility and biodegradability [31]. One of its key features is its ability to form porous gel matrices with mucoadhesive properties, making it useful for various biomedical applications [32,33]. A particularly important characteristic of Alg is its ability to interact with divalent cations such as Ba²⁺, Ca²⁺, and Sr²⁺, leading to gel formation. This process occurs through crosslinking, where the cations become embedded between polymer chains, creating a stable network structure.

In parallel, graphene (G) and its derivatives have been increasingly recognized as innovative materials for neural repair [34,35]. While G offers several advantages over other carbon-based materials, it does have some drawbacks, including an unstable chemical structure and a limited number of active sites, which reduce its ability to interact with biomolecules. Chemical modifications are often recommended to address these challenges. One of the most effective derivatives is GO, which can absorb biomolecules more easily. This is due to the presence of carboxyl (-COOH) groups at its edges, along with epoxy (-O-), carbonyl (=C=O) and hydroxyl (-OH) groups on its surface. Incorporating graphene-related materials (GRMs) has been shown to enhance the mechanical properties of bio-composites, with higher concentrations leading to improved and tunable characteristics suited for specific tissues [36,37,38]. Additionally, as the amount of G increases, the hydrophobicity of NTE structures rises, influencing nerve cell attachment, growth, and development. To maximize the therapeutic potential of GRMs, further research is needed to determine the optimal concentration for achieving the best possible outcomes [39,40,41].

Hybrid scaffolds that integrate Alg with GO represent a promising strategy to combine the hydrogel-forming, biocompatible nature of Alg with the conductive, mechanical, and antibacterial advantages of GO. Such composites have demonstrated superior mechanical strength [42], antimicrobial activity [43,44], support for cell proliferation [45,46], drug delivery capability [47,48], and confirmed cytocompatibility [49]. Despite these advances, systematic studies evaluating the interplay between GO concentration, scaffold physicochemical characteristics, and cellular responses in the context of neural tissue engineering remain limited.

Beyond demonstrating GO-enhanced conductivity in Alg matrices, this work establishes a quantitatively supported composition window, with up to 3, 6, and 9% GO homogeneously dispersed in the alginate matrix, where electrical functionality, hydration behavior, and neural cell compatibility are concurrently optimized. By integrating spectroscopic, structural, electrical, and neurobiological evaluation under controlled preparation conditions, the study delivers a design-oriented framework that extends prior Alg–GO scaffold reports from proof-of-concept materials toward application-relevant neural scaffold engineering with further possibilities to extend their applications in electric stimulation and even (triggered) drug delivery. The results show that an intermediate GO loading provides the most effective overall performance, delivering meaningful electrical functionality while maintaining scaffold stability and cellular viability.

2. Materials and Methods

2.1. Materials

The preparation of GO employed ultra-high purity graphite (99.99%) together with potassium permanganate (99.22%) obtained from Lach-ner, Neratovice, Czech Republic. Concentrated sulphuric acid (95–97%) from Merck (Darmstadt, Germany) and hydrochloric acid solutions (36.5–38%) supplied by Silal Trading (Bucharest, Romania) were also used in the oxidation process. Additional reagents included phosphorus pentoxide (≥98%) and potassium peroxodisulfate (≥98%) purchased from Sigma-Aldrich (Steinheim, Germany), as well as 35% hydrogen peroxide provided by Silal Trading. Sodium alginate was purchased from Fisher Scientific U.K. Ltd. (Redox Lab Supplies, Bucharest, Romania). Glycerol was obtained from Honeywell (Morris Plains, NJ, USA) (mol. wt: 92.09 g/mol, boiling pt: 290 °C, melting pt: 18 °C; density 1.26 g/cm³). Calcium chloride was supplied by Silal Trading, phosphate-buffered saline (PBS) was obtained from Sigma Aldrich (Redox LabSupplies, Bucharest, Romania) and Tween 80 was obtained from Sigma-Aldrich, Taufkirchen, Germany. All reagents were used without further purification.

2.2. Synthesis of GO: Hummer’s Method

GO was prepared using the modified version of the Hummer method [50]. The synthesis of GO was made in two stages: pre-oxidation of graphite and the oxidation of pre-oxidized graphite. In the first stage, 10 g of graphite were added to 30 mL of H₂SO₄, followed by 5 g of K₂S₂O₈ and 5 g of P₂O₅. The mixture was heated at 80 °C, cooled to room temperature, and diluted with a large volume of deionised water. The resulting suspension was filtered and washed until a neutral pH value was reached. The solid product was dried at 80 °C for 24 h and used in the second stage. For oxidation, 10 g of the preoxidised graphite were dispersed in 230 mL concentrated H₂SO₄ under stirring in an ice bath (<5 °C). Subsequently, 30 g KMnO₄ were slowly added, and the suspension was stirred until it developed a dark green color. The suspension was transferred to a flask and stirred at 35 °C for 2 h. Subsequently, 460 mL of distilled water was added to the mixture and stirred for 15 min, followed by the gradual addition of 1.4 L of distilled water and 25 mL of 30% H₂O₂ to remove excess KMnO₄ and MnO₂. The supernatant was discarded, and the solid was repeatedly washed with 5% HCl followed by distilled H₂O until a neutral pH was achieved. The resulting GO suspension was dried at 60 °C for 24 h to get the GO powder. A total of 17 g of GO was collected using a watch glass [51,52,53].

2.3. Synthesis of Alg–GO Scaffolds

Upon mixing with water, the Alg particles gradually swelled and dissolved, forming a homogeneous solution. To ensure complete dissolution, the solution was continuously stirred for 24 h. Four films were prepared: a control sample and three films containing different amounts of GO, as shown in Figure 1. Both tubular and planar structures were obtained, the most evaluations being done on the planar sample, considering their suitability for analysis. The GO concentration was increased beyond 6% based on the conductivity tests described in the following sections. For the control film, 10 mL of the standard Alg solution was cast into a 9 cm Petri dish and cross-linked with 10 mL of 2 wt% CaCl₂ solution. For the GO-containing films, GO was first ground in a mortar with 10 mL of 3 wt% Alg solution and 0.5 mL of a 1 wt% Tween 80 surfactant solution. The addition of Tween 80 facilitates a better homogenization of GO before deposition into Petri dishes. Subsequently, the samples were cross-linked with CaCl₂ by spraying, ensuring the formation of uniform films. In this study, crosslinking was allowed to proceed for 15 min, after which the films were carefully washed with distilled water.

CaCl₂ is widely used as a calcium ion source for ionic gelation of Alg [54,55]. Previous studies have demonstrated the effectiveness of this crosslinking method. Liling et al. [56] reported that immersing Alg films in a 2 wt% CaCl₂ solution for 2 min resulted in complete ionic crosslinking, with significant improvements in mechanical properties compared to the un-crosslinked samples. Similarly, Ibrahim et al. [57] demonstrated that immersion for 2–8 min enhanced the tensile strength and modulus of Alg films.

Tubular Alg structures were investigated for their potential application in peripheral nerve system (PNS) repair (Figure 1). The tubes were fabricated using an automated syringe-based extrusion system equipped with a coaxial spinneret, enabling controlled gelation in a CaCl₂ crosslinking bath. The spinneret consisted of a stainless-steel outer needle with an inner diameter of approximately 1.2–1.5 mm, while the inner needle diameter was 0.4–0.6 mm, allowing precise control over lumen formation. In both cases, planar or tubular experimental models, at the preliminary visual examination, it can be concluded that the Alg-GO composites are homogeneous, with the GO powder being well dispersed during the combined crushing and ultrasonation procedure.

As a control, a standard Alg solution supplemented with glycerin was used to fabricate non-conductive tubular scaffolds. To enhance electrical conductivity and promote neuronal regeneration, graphene oxide (GO) was incorporated into the Alg solution at varying concentrations.

2.4. Fourier Transform Infrared Spectroscopy

FTIR spectroscopy was employed to identify the functional groups present and to evaluate the interactions among the constituents of the composite films. Measurements were performed across the spectral interval of 4000–400 cm⁻¹ using a Nicolet iS50 FTIR instrument (Nicolet, MA, Massachusetts, USA) fitted with a DTGS detector. Spectra were collected at 4 cm⁻¹ resolution by averaging 32 consecutive scans. Data acquisition was carried out in attenuated total reflectance (ATR) configuration with a diamond crystal accessory.

In addition, two-dimensional FTIR chemical mapping was conducted to assess the spatial distribution of film components. These measurements were obtained using a Nicolet iS50R FTIR microscope (Nicolet, MA, USA) equipped with a DTGS detector, within the 4000–600 cm⁻¹ spectral range.

2.5. UV-Vis Spectroscopy Measurements

UV–Vis absorption measurements were carried out using a JASCO V560 spectrophotometer (JASCO Inc., Easton, PA, USA) fitted with a 60 mm integrating sphere accessory (ISV-469) and a dedicated film sample holder. Spectral data were collected over the 200–900 nm wavelength interval at a scan rate of 200 nm·min⁻¹.

Film opacity was determined from the relationship A₆₀₀/x = −log(T₆₀₀)/x, where A₆₀₀ represents the absorbance measured at 600 nm, T₆₀₀ corresponds to the transmittance fraction at the same wavelength, and x denotes the film thickness expressed in millimeters. Greater calculated opacity values correspond to reduced optical transparency of the films [58].

2.6. PL Spectrometry Measurements

Photoluminescence (PL) measurements were performed using a LS55 fluorescence spectrometer from PerkinElmer (Waltham, MA, USA). The excitation source was a xenon lamp operated under ambient conditions. Emission spectra were collected over the 350–600 nm wavelength range. Data acquisition was conducted at a scan rate of 200 nm·min⁻¹, using both excitation and emission slit widths set to 10 nm, together with a 350 nm cut-off filter. The selected excitation wavelength for the measurements was 320 nm.

2.7. Thermal Analysis Measurements

Thermogravimetric and differential scanning calorimetry (TG–DSC) analyses were carried out using a Netzsch STA 449C Jupiter thermal analyzer (Netzsch, Selb, Germany). Approximately 10 mg of each specimen was weighed into an open alumina crucible prior to measurement. The temperature was increased from room temperature to 900 °C at a constant heating rate of 10 K·min⁻¹. Measurements were performed under a flow of dried air maintained at 50 mL·min⁻¹. An empty alumina crucible was used as the reference.

2.8. Swelling Behavior, pH, and Conductivity Measurements

Each specimen was initially weighed to obtain its dry mass (W₀), then placed in 200 mL of phosphate-buffered saline solution (PBS, pH 7.4). Swelling ratio, pH, and electrical conductivity were monitored for every sample at fixed time points of 5, 10, 30, 45, and 60 min, as well as after 4, 6, 18, 24, and 48 h of immersion.

The swelling degree, S (%), was determined according to Equation (1):

$$\mathrm{S}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{W}\mathrm{t}-\mathrm{W}0}{\mathrm{W}0}}\times 100$$

(1)

All measurements were performed in triplicate (n = 3), and the results are reported as mean ± standard deviation (SD). At each interval, the experimental formulations (Alg–3%GO, Alg–6%GO, and Alg–9%GO) were statistically compared with the Alg control using Welch’s two-sample t-test with Bonferroni correction. Statistical significance was considered at p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Data analysis was conducted under the assumption of an approximately normal distribution, in accordance with established practices in biomaterials research.

2.9. Electrochemical Conductivity Measurements

Electrical conductivity of Alg–GO solutions was measured using a parallel-opposed dual-electrode cell (stainless steel cylindrical electrodes, 1.483 mm diameter × 4.823 mm length) used in conjunction with a calibrated (KCl 0.02 n solution was used) digital high resolution internal resistance/conductivity meter (RC3563, Xtester, Guangzhou, China) provided with separate paths for current injection and voltage measurements, to reduce electrode polarization and improve accuracy across a wide conductivity range [59]. By knowing the cell constant (determined during the calibration stage from the values of the known conductivity of a KCl 0.02 n solution and measured resistance) and measuring the sample’s resistance, one may now easily calculate the value of conductance and hence that of the conductivity.

2.10. SEM Analysis Measurements

The morphology and microstructural features of the specimens were examined by scanning electron microscopy using a QUANTA INSPECT F50 system fitted with a field emission gun (FEG) source (SEM images (50,000×), QUANTA INSPECT F50, FEI Company, Eindhoven, The Netherlands), offering a resolution of 1.2 nm. Elemental composition analysis was conducted with an attached energy-dispersive X-ray spectroscopy (EDS) detector with a MnK resolution of 133 eV.

Prior to imaging, both the surfaces and fractured cross-sections of the Alg-based samples were sputter-coated with a thin gold layer to improve electrical conductivity. The metallization step was performed for 60 s to obtain a uniform conductive coating.

2.11. Evaluation of Cytocompatibility of Alg–GO Scaffolds Using SH–SY5Y Cells

The human neuroblastoma cell line SH–SY5Y (CRL-2266, ATCC) was employed as an in vitro model to evaluate the cytocompatibility of Alg–GO scaffolds. A three-dimensional cell–scaffold construct was established by seeding cells onto the scaffold surface at a density of 3.5 × 10⁴ cells/cm². Samples were cultured in a growth medium composed of Minimum Essential Medium Eagle (MEM) and F12 medium (1:1, v/v), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic mixture.

Cell–scaffold constructs were maintained under standard incubation conditions (37 °C, 5% CO₂, and 95% relative humidity) for 7 days. Biocompatibility analyses were performed at 3 and 7 days after seeding. Pure Alg scaffolds were used as control samples in all biological assays.

Cell viability and proliferation were quantified using the MTT assay based on the metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Samples were incubated with MTT solution (1 mg/mL) for 4 h under standard culture conditions to allow the formation of formazan crystals by metabolically active cells. The resulting crystals were dissolved in isopropyl alcohol, and absorbance was measured at 550 nm using a FlexStation3 spectrophotometer (Molecular Devices, San Jose, CA, USA).

Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) release in the culture medium using the LDH-based in vitro toxicology assay kit (TOX7, Sigma-Aldrich, Steinheim, Germany), following the manufacturer’s instructions. Absorbance values were recorded at 490 nm with the same spectrophotometric system.

Qualitative analysis of cell viability and distribution on the scaffolds was performed using a LIVE/DEAD viability/cytotoxicity kit (Invitrogen, Life Technologies, Foster City, CA, USA). Live cells were labeled with calcein AM (green fluorescence), while non-viable cells were stained with ethidium bromide (red fluorescence). Samples were examined using a laser scanning confocal microscope (Nikon A1/A1R Confocal System, Tokyo, Japan), and images were processed with the associated acquisition and analysis software.

3. Results and Discussion

3.1. FTIR Spectroscopy and Microscopy of Alg–GO Films

The FTIR spectra of the Alg and Alg–GO materials were recorded and compared (Figure 2). The IR spectrum of the Alg sample showed characteristic absorption bands. The spectral band at 3239 cm⁻¹, along with the peak near 2900 cm⁻¹, was attributed to –OH stretching and aliphatic –CH stretching vibrations, respectively. The peaks observed at 1549 and 1406 cm⁻¹ corresponded to the asymmetric and symmetric stretching vibrations of carboxylate anions, respectively. The bands at 1151 and 1023 cm⁻¹ were assigned to C–O stretching vibrations of the pyranose ring. In the fingerprint region, the peaks at 649 and 514 cm⁻¹ were associated with the asymmetric ring vibration of α-L-glucopyranuronic acid [60,61]. Upon functionalization of Alg with GO, the FTIR spectra of the composites exhibited a noticeable shift in the peaks at 3239, 1549, and 1023 cm⁻¹, attributed to the strong interactions that occur. This observation suggests that GO was successfully integrated into the Alg matrix, by strong interactions and not only by mechanical entrapment [62,63]. The most important shift is associated with the peak from 1406 cm⁻¹, which appears at 1417 cm⁻¹ in the case of Alg-9%GO. Considering the insert from Figure 2a, it can first be seen that the main band centered at ~1580 cm⁻¹ became much larger once GO is added into the alginate matrix, meaning that a strong interaction between these components occurs. Similar important changes (shifts and peaks in intensity) are observed in the 900–1200 cm⁻¹. These are especially visible at 3% GO, while the addition of more GO practically does not consistently change this behavior, which means that GO is starting to not interact with the Alg matrix and confer the premises for the development of bridges between graphene clusters, which is important to get improved electric capabilities.

FTIR microscopy was employed to evaluate the homogeneity of the films. Four characteristic wavelengths were selected for this purpose: 1130, 1467, 1668, and 3545 cm⁻¹, corresponding to major absorption bands of the film components. The 3545 cm⁻¹ band was associated with moisture and served as an indirect indicator of uniformity, since water was adsorbed from the air in a non-homogeneous manner depending on both the film’s density and its composition (because alginate and graphene oxide exhibit different hygroscopic behaviors).

By comparing the four spectral maps, it was observed that films of pure sodium alginate displayed slight heterogeneity in Ca²⁺ retention, likely due to the absence of the reinforcing agent (GO). Without GO, alginate chains were less easily displaced, which resulted in a more compact crosslinking structure, as evidenced by the calcium alginate distribution recorded at 1668 cm⁻¹. In contrast, the Alg–3%GO sample exhibited a highly homogeneous structure, as all maps showed similar patterns regardless of the wavelength considered.

As the GO content increased, the maps obtained at 1130, 1467, and 1668 cm⁻¹ remained consistent, indicating structural homogeneity; however, the maps recorded at 3545 cm⁻¹ revealed proportional differences in moisture distribution. This suggested that at higher GO concentrations, slight heterogeneities appeared due to the differential adsorption of humidity.

3.2. UV-Vis Spectroscopy Results

The yellowish-tinted Alg transparent films exhibited an absorption maximum around 450 nm, a characteristic feature of films made from this polymer [58].

The incorporation of GO into the polymer matrix changed the color to black, resulting in strong light absorption across the visible domain. The maximum absorption for the Alg films was observed at 9%GO content (Figure 3).

Table 1. Calculated opacity for the Alg and composite Alg–GO films.

Sample Code	Thickness (mm)	Opacity
Alg	0.04 ± 0.01	0.826 ± 0.220
Alg–3%GO	0.06 ± 0.01	18.741 ± 3.212
Alg–6%GO	0.06 ± 0.01	21.654 ± 3.712
Alg–9%GO	0.06 ± 0.01	22.748 ± 3.899

presents the calculated values of opacity for the alginate and composite films, with higher values indicating greater opacity. The simple Alg film exhibited a low opacity value, consistent with its good transparency as previously reported [58,64]. The addition of 3%GO dramatically increased the opacity of the films, while further addition of GO up to 9% had only a marginal effect. At both 6 and 9% of GO, practically full absorption is obtained between 450 and 900 nm, while in the case of the sample with 3% GO, the absorption is not partial in this interval. Looking at the standard deviation of the samples, the relative standard deviation is ~17% for the composite samples, while in the case of the pure alginate, this is ~26%, which means that these samples exhibit lower heterogeneities (this factor being the most important leading to opacity).

3.3. PL Spectrometry Results

The photoluminescence (PL) spectra for Alg and Alg-based composite films are presented in Figure 4. The Alg film exhibited strong fluorescence, as is typical for polysaccharide [65,66]. The Alg fluorescence is quenched when GO was added into the composite film, due to interactions between the nanosheets and the polymer structure. The nanosheets blocked the fluorescent emission by interacting with the moieties responsible for the Alg fluorescent emission, indicating that the composite was more than a simple physical mixture. Furthermore, the fluorescence quenching could also have been attributed to the fact that GO introduced non-radiative pathways for the recombination of excited electrons and corresponding holes, likely through surface conduction.

As the percent of GO increased from 3% to 9%, the intensity of the fluorescent emission further decreased, particularly the peak at 391 nm, which nearly disappeared at Alg–9%GO sample. The other emission band in the visible spectrum (in the blue region, at 457 nm) was also strongly dependent on the GO percentage.

3.4. Thermal Analysis

The thermal analysis results, TG and DSC curves, were presented in Figure 5. The main differences between Alg and the composite films were related to the amount of residual humidity, observed as mass loss up to 200 °C. All composite films exhibited a slightly higher mass loss in the temperature range 20–205 °C, as indicated by the values from

Table 2. The principal data from the thermal analysis.

Sample	Δm (20–205 °C)	Δm (205–300 °C)	Residual Mass (900 °C)	T5% °C	T10% °C	T15% °C
Alg	12.92%	31.53%	11.49%	97.5	159.5	214.4
Alg–3%GO	14.11%	31.88%	9.98%	84.5	133.7	206.8
Alg–6%GO	15.88%	30.10%	7.89%	76.2	118.3	196.3
Alg–9%GO	16.14%	29.50%	8.52%	80.7	126.7	195.8

. This mass loss increased with higher GO content, most likely due to water molecules remaining trapped between the GO sheets. Between 205 and 300 °C, the polymer chains fragmented and were partially oxidized, corresponding to the principal mass loss recorded for all samples (~29–32%). Complete oxidation of the carbonaceous mass occurred at different temperatures for Alg and the composite films, as evidenced by the strong and sharp exothermic effect observed on the DSC curves. For Alg, this peak appeared at 513.7 °C, whereas for Alg–3%GO it shifted to a lower temperature of 483.6 °C. With further increases in GO content, the exothermic peak shifted to 465–467 °C and was not significantly influenced. This means that the addition of the GO is favoring the degradation of the alginate matrix. The most important change is observed at the addition of 3% GO (the shift being ~27 °C), while practically for 6 and 9 percent, just ~16 °C shift was observed. Practically, the peaks associated with the samples with 6 and 9% GO appear at the same values.

The mass loss also increased for the composite samples compared to the Alg film during this oxidation process, indicating a direct relationship with the presence of GO. Therefore, it could be assumed that GO was completely oxidized between 450 and 500 °C [67], together with the residual carbonaceous mass originating from the Alg chains.

The final mass loss, occurring after 600 °C, was attributed to the decomposition of CaCO₃ formed during the degradation of Alg chains that had been cross-linked with Ca²⁺ ions. The residual mass was lower for the composite films, as the Alg proportion was smaller.

3.5. Swelling Behavior

The swelling profile of Alg hydrogels containing different concentrations of GO was evaluated in Figure 6a,b over a short period of 60 min and extended periods up to 120 h. All formulations had identical starting swelling values (0%), ensuring comparable baseline measurements.

As shown in Figure 6a, the swelling ratio increased notably within the first 5 min, especially in the GO-loaded samples. Alg–6%GO exhibited the highest swelling ratio at 5 min (220%), followed by Alg–9%GO (180%), Alg–3%GO (139%), and the control (64%). This trend continued at 30 and 60 min, where the swelling ratios increased significantly with GO concentration. At 60 min, Alg–6%GO showed the highest swelling capacity (893%), followed closely by Alg–9%GO (878%), Alg–3%GO (809%), and the Alg control (524%).

To further investigate the hydrogel behavior over extended periods, the weight of the swollen hydrogels was monitored at 4 h, 6 h, 24 h, 48 h, and 5 days (Figure 6b).

All formulations demonstrated a rapid increase in weight during the first 24 h, indicating strong water absorption. This trend continued, and by 48 h, the Alg–6%GO hydrogel showed the greatest weight gain (2387%), outperforming all other samples. Interestingly, by day 5 (120 h), a slight decrease in weight was observed in all samples, potentially due to water loss or network degradation. Despite this, the Alg–6%GO hydrogel maintained high retention (1989%), while Alg–3%GO and Alg–9%GO retained 2177% and 2016%, respectively.

The findings demonstrated that the incorporation of GO into the Alg matrix enhanced water uptake and swelling behavior, attributable to the increased hydrophilicity and porosity introduced by GO. Among the formulations, Alg–6%GO exhibited the most favorable performance in both short- and long-term water absorption, which was attributed to improved matrix integrity and water-binding capacity conferred by GO.

3.6. pH Variation

The pH of the PBS solutions started from ~7.6 and gradually decreased throughout the monitored period (Figure 7a,b). The decrease in pH over time suggests a noticeable acidification after introducing Alg matrices in PBS. During this process, calcium phosphates precipitate and alginic acid is released into the solution, slightly altering the pH [68,69].

During the first 60 min (Figure 7a), all samples showed a slight decline in pH. The Alg control showed the most pronounced drop, while the GO-containing scaffolds displayed improved stability.

Over the extended period of 120 h (Figure 7b), all samples continued to show a gradual decrease in pH. The Alg control sample experienced the most pronounced decline, indicating a significant acidification over time because the alginic acid was not adsorbed on the samples. In contrast, GO-loaded samples demonstrated improved pH stability, with alginic acid being better adsorbed on the GO by hydrogen bonds.

Overall, the addition of GO to Alg scaffolds slightly reduced the acidification over time. While all samples, including the control, experienced a gradual decrease in pH, the final values for GO-enhanced Alg remained slightly higher than the control. This suggested that the incorporation of GO, regardless of concentration, helped maintain a more stable pH environment compared to the pure Alg scaffold. These findings supported the potential use of GO-enhanced Alg scaffolds in biomedical applications where pH stability was important, even if some acidification occurred.

3.7. Conductivity Variation in Solution

The initial conductivity of the PBS solution for all samples was 15.97 × 10⁻³ Ω⁻¹⋅cm⁻¹. By the first hour, the GO-containing samples displayed slightly lower conductivity compared to the control.

Over the extended period (Figure 8), conductivity increased steadily in all samples, indicating ongoing ion release. Alg–3%GO and Alg–9%GO reached the highest values, surpassing the control, whereas Alg–6%GO plateaued at a lower level, suggesting reduced ion release at this concentration, probably due to an optimal ratio between Alg and GO.

All GO groups showed statistically significant differences compared to the control, with most comparisons reaching *** (p < 0.001). These results indicated that the incorporation of GO strongly and consistently modified the conductivity of the solution over time. Statistical significance was not shown on the graph for clarity.

The overall rise in conductivity demonstrated that the scaffolds interacted with the PBS solution, likely through ion release or modification of the ionic environment. The enhancement in conductivity, particularly at higher GO concentrations, highlighted the potential of these scaffolds to improve the electrical environment and support neural cell communication and function.

3.8. Electrochemical Conductivity Results

The conductivity increased with the addition of GO. The Alg control sample (0%GO) exhibited minimal conductivity, while samples with 3%, 6%, and 9%GO showed a general increasing trend. The 3%GO sample displayed a conductivity in the range of 5.99 × 10⁻¹⁰ Ω⁻¹·cm⁻¹, which was relatively low compared to the other samples. The 6% GO sample showed a slight decrease in conductivity (4.22 × 10⁻¹⁰ Ω⁻¹.cm⁻¹), suggesting that at this concentration, GO may still have been dispersed within the insulating Alg matrix, thereby limiting charge transfer efficiency. Relative conductivity values (dimensionless), calculated as the ratio of each measured conductivity to the lowest average conductivity, were also determined (

Table 3. Conductivity values of Alg–GO solutions with varying GO concentrations. The relationship between GO content and conductivity was analyzed.

No.	GO Content (%)	Conductivity Ω−1.cm−1	Average Conductivity	Relative Standard Deviation	Relative Conductivity
1	3	6.05 × 10−10	5.99 × 10−10	3.54	1.42
		6.16 × 10−10
		5.75 × 10−10
2	6	4.50 × 10−10	4.22 × 10−10	5.98	1.00
		4.15 × 10−10
		4.01 × 10−10
3	9	3.45 × 10−9	3.38 × 10−9	3.44	8.01
		3.44 × 10−9
		3.25 × 10−9

). The results showed that, for all GO concentrations, the values remained within one order of magnitude compared with the lowest conductivity but comparable with the native neural tissues, that is, according to the literature, from µS/cm to mS/cm [70].

The 6% GO scaffold provided a balanced combination of moderate conductivity and biocompatibility, reducing the risk of excessive current flow that could damage cells. In contrast, the 9% GO solution demonstrated a marked increase in conductivity, which made it more suitable for applications requiring enhanced electrical stimulation, such as neural regeneration or bioelectronic interfaces.

A significant jump in conductivity was observed in the 9%GO sample, which reached 3.38 × 10⁻⁹ Ω⁻¹.cm⁻¹ an order of magnitude higher than the 3% and 6%GO samples. This sharp rise indicated that at 9%GO, the percolation threshold for electrical pathways had been reached, enabling more effective charge transfer. According to percolation theory, conductivity in composite materials increases abruptly once the critical concentration of conductive filler (in this case, GO) is reached, enabling efficient electron transport across the network [71].

3.9. SEM Analysis Results

Alg materials often have a porous microstructure, which may be visible in SEM images. These pores can vary in size and distribution, influencing properties such as swelling behavior and permeability [72].

SEM analysis of the Alg control film revealed a granular-like surface morphology, resulting from the collapse and rearrangement of the Alg gel network during the drying process. The surface consisted of granules with heterogeneous size and morphology, ranging from small, nearly spherical particles to irregularly shaped aggregates. Higher-magnification images enabled a detailed examination of the microstructure, revealing polymer organization characterized by a subtle hill–valley topography, which can be attributed to the crosslinking-induced restructuring of the initially flowing gel (Figure 9a).

In contrast, the surface of Alg–GO films exhibited a rough and textured appearance due to the interaction with GO, which appeared as flakes or thin sheets with irregular shapes. The edges of the sheets seemed to be rough or crumpled due to the oxidation process and functional groups attached to the G layers (Figure 9b–d). Depending on the concentration and distribution of GO within the Alg matrix, the composite became more brittle, increasing the likelihood of crack formation in the Alg–GO composite. The distribution of GO throughout the Alg matrix was homogeneous, indicating the potential for enhanced electrical conductivity properties. Importantly, no crystals can be observed on the surface (CaCl₂), resulting from the crystallization of the salts used in the gelation-crosslinking process.

3.10. Cytocompatibility Assessment of SH–SY5Y Cell Culture in Contact with Alg–GO Scaffolds

Cell viability and proliferation, evaluated via quantitative MTT assay (Figure 10a), indicated an overall good interaction between SH–SY5Y cell culture and the tested Alg–GO materials. At 3 days post-seeding, a normal rate of cell viability was registered across all tested materials, with no significant differences between the four composites. After 7 days of in vitro culture, the composites enriched with 3% and 6% GO showed similar viability values, with no significant changes between them. However, the addition of more GO up to 9% led to a significant decrease in cell viability, in comparison to plain Alg and the other GO-loaded Alg materials (p < 0.05). Interestingly, a significant (p < 0.001) proliferation rate was found on all tested composites from 3 to 7 days of cell culture.

LDH assay indicated the cytotoxic potential of Alg–GO enriched materials upon the SH–SY5Y cell culture (Figure 10b). After 3 days, relatively low LDH levels were detected in the culture medium, with no significant differences among the composites. These levels remained stable after 7 days post-seeding, without causing significant toxicity in the neuroblastoma cell line. Nonetheless, a slight increase in LDH release was observed as the GO content increased.

LIVE/DEAD confocal microscopy (Figure 10c) results were consistent with those obtained from MTT and LDH assays. At 3 days post-seeding, only a small number of cells adhered to the material surfaces. However, these cells exhibited an elongated shape and formed small groups of cells. All captured images revealed green-labeled viable cells in contact with the Alg–GO composites. After 7 days of in vitro culture, the LIVE/DEAD staining showed a significant increase in cell density, confirming that cells proliferated in the presence of Alg–GO composite materials. Nevertheless, proliferation on Alg–9%GO was less pronounced compared with the other samples, pure Alg or composite ones.

Overall, these findings indicate that the presence of GO, regardless of concentration in the 3–9%, supports proper cell development. However, incorporation of up to 6% GO appears to provide optimal stimulation for cell proliferation, without inducing cytotoxicity.

3.11. Structure–Property–Biological Correlations Defining an Optimal GO Loading

Relationships between electrical conductivity, swelling degree, and cell proliferation were examined across the entire composite series. Electrical conductivity increased nonlinearly with GO loading, with a marked rise at 9% GO (≈one order of magnitude higher than 3–6%), consistent with a percolation-type transition. In contrast, the swelling degree reached its maximum at 6% GO (≈2387% at 48 h), exceeding both 3% GO and 9% GO formulations. Cell proliferation (MTT, day 7) followed a different trend, showing the highest values for 3% and 6% GO, and a significant reduction at 9% GO (p < 0.05). A multi-parameter comparison matrix (

Table 4. Multi-parameter comparison used for the Alg-GO formulations.

Sample	Conductivity	Swelling 48 h	MTT Day 7	Rank
Alg	low	medium	medium	✗
Alg–3%GO	medium	high	high	✓
Alg–6%GO	medium+	highest	high	✓
Alg–9%GO	highest	high	lower	✗

) further illustrates that the 6% GO formulation provides the best combined performance across electrical, hydration, and biological criteria.

The 6% GO scaffold lies at the intersection of these trends, combining elevated conductivity with the highest swelling capacity and preserved cell proliferation, thereby quantitatively supporting its selection as the optimal compromise formulation. While this enhanced electrical permissiveness is advantageous for charge transfer at the cell–material interface, it does not by itself guarantee electrically driven neural regeneration. Active modulation of neurite outgrowth and functional maturation generally requires externally applied electrical stimulation with carefully controlled parameters, including field strength, frequency, and duration, tailored to the target neural tissue. Accordingly, in the present work, scaffold conductivity should be regarded as an enabling property that supports electrically active and stimulation-compatible environments, rather than a standalone regenerative stimulus.

4. Conclusions

The principal contribution of this work is the controlled, multi-parameter determination of an intermediate GO loading that balances electrical functionality, hydration behavior, and neuro-cytocompatibility under identical fabrication conditions. This approach provides a design-relevant composition window for conductive Alg-based neural scaffolds. An important milestone in developing composite materials loaded with graphene-related materials was achieved by developing highly loaded composites, where the inherent biocompatibility of Alg, combined with the distinct physicochemical characteristics of GO, provides a synergistic platform that promotes neural cell adhesion and electrical conductivity. FTIR spectroscopy confirmed the successful incorporation of GO into the Alg matrix and revealed strong intermolecular interactions between the two phases. UV–Vis and photoluminescence analyses further indicated GO-induced modifications in the optical behavior of the composites. Electrical conductivity measurements demonstrated a marked improvement in conductivity, a property of particular relevance for electrically active neural tissues.

Among the tested formulations, Alg–GO scaffolds containing 6% GO exhibited the most favorable balance between structural stability, swelling capacity, and conductivity, suggesting their suitability for neuroregenerative applications. FTIR microscopy and SEM analysis confirmed a homogeneous distribution of GO within the Alg network, ensuring structural uniformity. Biological assessments using SH–SY5Y neuroblastoma cells revealed robust viability and proliferation across all samples. While scaffolds with higher GO content (9%) remained non-cytotoxic, they exhibited slightly reduced proliferation compared to lower GO concentrations.

These findings highlight the biocompatibility and multifunctional performance of Alg–GO composites and support their potential use in peripheral nerve regeneration. Future work will focus on integrating electrical stimulation protocols with the proposed scaffold system and evaluating neuron-specific responses, including differentiation, network formation, and electrophysiological behavior, in order to fully assess its potential for neural tissue engineering applications.