Development and characterisation of copper oxide nanoparticle–functionalized corn husk cellulose–PVA membranes for guided tissue regeneration

Abstract

Introduction

Guided tissue regeneration (GTR) membranes enable selective cell repopulation, but many existing membranes suffer from limited mechanical stability, rapid degradation, and a lack of sustainable biomaterials. Corn husk, an agricultural waste rich in cellulose, offers an eco-friendly alternative. Polyvinyl alcohol (PVA) reinforcement and copper oxide nanoparticles (CuO NPs) may further enhance strength and biological performance. This study fabricated and in vitro evaluated an electrospun GTR membrane combining corn husk–derived cellulose, PVA, and CuO NPs.

Materials and methods

Cellulose was extracted from corn husk through glycerine treatment, alkaline purification, and peroxide bleaching. CuO nanoparticles were synthesised by precipitating Cu(OH)₂ followed by calcination. A composite electrospinning solution of PVA (10 wt%), cellulose (2 wt%), and CuO NPs (5 wt%) was electrospun under optimised conditions (20 kV, 0.5 mL/h, 12 cm). The resulting membrane underwent in vitro characterisation.

Results

SEM and EDS confirmed uniform CuO nanoparticle dispersion within the PVA/Cellulose fibres; FTIR verified chemical integration. MTT assay showed >96% cell viability, with small but significant reductions at 1:1 and 1:2 dilutions (p < 0.01). The membrane exhibited hydrophilicity (contact angle ∼19°), tensile stress of 0.43 MPa, and strong antibacterial effects against E. coli (p = 0.0001) and S. aureus (p = 0.00007). Anti-inflammatory and antioxidant activity increased dose-dependently. ALP expression was significantly upregulated (3.20-, 5.40-, and 8.00-fold; p < 0.01), indicating enhanced early osteogenesis.

Conclusion

The composite membrane demonstrated excellent biocompatibility, antimicrobial activity, and osteogenic potential, supporting its promise as a sustainable GTR material.

1. Introduction

The widespread prevalence of periodontitis, which leads to the destruction of alveolar bone and the formation of bony defects, has established the foundation for periodontal regeneration.¹ And three generations of GTR membranes have been developed over time.² The first generation consists of non-resorbable membranes, the second generation includes resorbable ones, and the third generation features advanced designs incorporating antibiotics, growth factors, and innovative fabrication methods such as electrospunned multilayered membranes.^3,4Despite these advancements, the fundamental principle behind all generations remains consistent: the concept of selective cell repopulation of the defect site, leading to bone tissue formation, as proposed by Melcher's theory of compartmentalisation.⁵

However, currently available membranes often present limitations such as inadequate mechanical stability in resorbable systems,⁶ need for secondary surgery with non-resorbable materials,^7,8 and limited bioactivity to actively promote regeneration. Therefore, there remains a need for more sustainable and mechanically robust alternatives. Additionally, sustainable biomaterial alternatives derived from agro-waste, such as corn husk cellulose, remain unexplored.

Corn husk, an agricultural by-product rich in cellulose, hemicellulose, and lignin, represents a sustainable and less common biomaterial source.⁹ Its high cellulose content offers promising mechanical strength for membrane fabrication while supporting environmentally responsible biomedical innovation.

Cellulose, the major component of corn husk, consists of both crystalline and amorphous regions, and its mechanical strength depends on the balance between these two phases.¹⁰ This structural integrity makes cellulose an excellent candidate for developing biomaterials such as GTR barrier membranes, where strength and stability are critical. While bacterial cellulose has been previously utilised in similar applications,¹¹ using corn husk–derived cellulose represents a green, sustainable, and environmentally friendly approach that could address periodontal challenges while reducing carbon footprint.

Since pure cellulosic fibres in thin films may lack adequate mechanical strength to function as a GTR membrane, reinforcement with a synthetic polymer is essential. Polyvinyl alcohol (PVA), a synthetic polymer obtained through the hydrolysis of polyvinyl acetate, serves as an ideal supporting material. Its hydrophilic nature, excellent film-forming ability, and high tensile strength make it suitable for enhancing the mechanical properties of cellulose.¹² When combined, PVA and cellulose can form a synergistic composite membrane with improved durability, flexibility, and bioactivity.

To further enhance the biological functionality of the membrane, metal oxide nanoparticles can be incorporated. Among these, copper oxide nanoparticles (CuO NPs) exhibit antimicrobial, anti-inflammatory, and angiogenic properties.^13,14 Their inclusion in GTR membranes can thus provide valuable biological enhancement.

Although bacterial cellulose and polymer-based membranes have been investigated^11,15 limited attention has been given to plant-derived cellulose from agricultural waste sources for periodontal regeneration. To date, the application of corn husk–derived cellulose in periodontal guided tissue regeneration (GTR) membranes remains largely unexplored. Furthermore, no previous studies have reported the integration of corn husk cellulose with polyvinyl alcohol (PVA) and copper oxide nanoparticles to fabricate a multifunctional barrier membrane. This study addresses this gap by developing and characterising a novel composite membrane and evaluating its physicochemical, mechanical, and biological properties, thereby exploring its potential as a sustainable and bioactive alternative for the management of periodontal intrabony defects.

2. Materials and methods

2.1. Fabrication of the GTR membrane

2.1.1. Cellulose extraction

Cellulose was extracted from corn husk powder using a glycerine-assisted alkaline treatment. The powder was heated with 70% glycerine at 120–150 °C for 2–3 h, washed, and treated with 2% NaOH at 80 °C. Bleaching was performed with 5% hydrogen peroxide at 70 °C. The material was washed by centrifugation, dried at 80 °C, and ground into fine powder for further use.

2.1.2. Preparation of CuO nanoparticles

Copper(II) oxide (CuO) nanoparticles were synthesised by precipitation followed by thermal decomposition. A 0.1 M copper(II) acetate solution was prepared by dissolving 1.996 g of Cu(CH₃COO)₂·H₂O in deionised water and diluting it to 100 mL. A 0.2 M NaOH solution was prepared by dissolving 0.800 g of NaOH pellets in 100 mL of deionised water. The NaOH solution was added dropwise to the copper acetate solution under vigorous stirring until a blue Cu(OH)₂ precipitate formed (Cu²⁺: OH⁻ = 1:2). The precipitate was collected by vacuum filtration, washed with deionised water until neutral pH, and dried at 100 °C for 12 h. The dried Cu(OH)₂ was then calcined at 450 °C for 3 h (heating rate 5 °C/min) to obtain CuO nanoparticles, which were ground and stored in a desiccator.

2.1.3. Preparation of electrospun PVA/cellulose/CuO membrane

A 10 wt% polyvinyl alcohol (PVA) solution was prepared by dissolving 10 g PVA (Mw 89–98 kDa, 99% hydrolysed) in 100 mL deionised water at 80 °C with stirring for 2 h, followed by cooling and degassing. Cellulose (0.2 g; 2 wt% relative to PVA) was dispersed in water and ultrasonicated for 15 min, while CuO nanoparticles (0.5 g; 5 wt% relative to PVA) were dispersed by bath sonication for 10 min. Both dispersions were added to the PVA solution and stirred for 1 h, followed by 10 min sonication to ensure uniform distribution.

The resulting solution was electrospun using a 10 mL syringe with a 21-gauge needle at a flow rate of 0.5 mL/h, an applied voltage of 20 kV, and a tip-to-collector distance of 12 cm. Fibres were collected on aluminium foil to form a uniform non-woven membrane, which was stored in a desiccator until further characterisation.

2.1.4. Characterisation of the formulated GTR membrane

The fabricated GTR membranes were prepared under standardised laboratory conditions, cut into uniform specimens, and randomly allocated to the respective experimental analyses before physicochemical and biological testing.

2.1.5. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS)

Surface morphology was analysed using FE-SEM (FEI Quanta FEG 200F). Samples were gold-sputtered and examined at 5–20 kV. Elemental composition and CuO distribution were assessed by EDS.¹⁶ The analysis was conducted at an accelerating voltage of 15–20 kV. Spectral data were processed using ZAF correction for semi-quantitative evaluation of elemental distribution.

2.1.6. Fourier Transform Infrared Spectroscopy (FTIR) analysis

FTIR spectroscopy was employed to characterise the functional groups and molecular composition of the fabricated membranes. The analysis validated the presence of each component and confirmed their successful incorporation into the composite membrane.¹⁷

2.1.7. Tensile strength measurement

Membranes (10 × 50 mm) were tested using a universal testing machine at 5 mm/min until failure. Tensile strength was recorded in MPa.

2.1.8. Contact angle measurement

Surface wettability was measured using a goniometer (K100 force tensiometer). Advancing and receding angles were recorded, and mean values calculated.

2.2. Biological evaluation

2.2.1. Antimicrobial activity assay

Antimicrobial activity was evaluated by agar well diffusion against E. coli and S. aureus. Mueller-Hinton agar plates were inoculated with bacterial suspensions, and 6 mm wells were filled with 50 μL membrane eluate. Gentamicin and distilled water served as positive and negative controls. Plates were incubated at 37 °C for 24 h, and zones of inhibition were measured in millimetres mm (n = 3).

2.2.2. Anti-inflammatory activity (BSA method)

The anti-inflammatory potential was assessed using the Bovine Serum Albumin (BSA) denaturation assay. The reaction mixture consisted of 1 mL of the test sample at different concentrations (20, 40, 60, 80, and 100 μg/mL) and 1 mL of 1% BSA solution prepared in phosphate-buffered saline (PBS, pH 6.4). The samples were incubated at 37 °C for 20 min and then heated at 60 °C for 15 min to induce denaturation. After cooling to room temperature, the absorbance of each sample was measured at 570 nm using a UV–visible spectrophotometer. Salicylic acid served as the standard reference.

2.2.3. Antioxidant activity (2,2-diphenyl-1-Picrylhydrazyl [DPPH] assay)

The antioxidant potential was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay.¹⁸ Different concentrations of the composite membrane (20, 40, 60, 80, and 100 μg/mL) were prepared in methanol. Each reaction mixture consisted of 1 mL of the test sample, 2 mL of methanol, and 1 mL of 0.1 mM DPPH solution. The mixtures were incubated in the dark at room temperature for 30 min. After incubation, the absorbance was recorded at 517 nm using a UV–visible spectrophotometer. Vitamin C (ascorbic acid) was used as the standard.

2.2.4. Membrane preparation and elution and periodontal ligament (PDL) cell culture

Sterile membrane discs were UV-sterilised and incubated in serum-free DMEM (0.1 g/mL, 24 h) to obtain eluates. Primary human periodontal ligament (PDL) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Cells between passages 3 and 6 were used for all experiments. Before treatment, cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and allowed to attach overnight.

2.2.5. MTT assay

To evaluate cytotoxicity, the culture medium in each well was replaced with 100 μL of the prepared membrane eluates at different dilutions (1:1, 1:2, and 1:4), and the plates were incubated for 24 h. Control wells received only serum-free DMEM. After incubation, 10 μL of MTT reagent (5 mg/mL in PBS) was added to each well and incubated for 3 h at 37 °C. The medium was carefully removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to solubilise the formazan crystals. Absorbance was measured at 570 nm using a microplate reader. Cell viability was expressed as a percentage relative to the untreated control using the formula:

$$\text{Cell}\text{viability}(\%)=\frac{\text{Absorbance}\text{of}\text{treated}\text{cells}}{\text{Absorbance}\text{of}\text{control}\mathrm{s}}\times 100$$

2.2.6. Alkaline Phosphatase (ALP) gene expression

Human PDL fibroblasts were treated with membrane eluates (1:1, 1:2, 1:4) for 24 h. Total RNA was extracted using TRIzol and reverse-transcribed into cDNA. qRT-PCR was performed using SYBR Green on a StepOnePlus system. ALP expression was normalised to β-actin and calculated using the 2⁻ΔΔCt method. Experiments were conducted in triplicate.

3. Statistical analysis

Cytotoxicity, antimicrobial activity, and ALP gene expression assays were performed in triplicate, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was carried out using one-way analysis of variance (ANOVA) to evaluate differences among groups, and the corresponding p-values were calculated. A p-value <0.05 was considered statistically significant.

4. Results

4.1. Scanning Electron Microscopy (SEM)

The surface morphology of the PVA/Cellulose/CuO (P/C/CuO) composite membrane was examined using scanning electron microscopy (SEM) at magnifications of 10,000× and 200× (Fig. 1). At higher magnification, the micrograph showed spherical CuO nanoparticles uniformly embedded within the fibrous PVA/Cellulose matrix, indicating strong interfacial interaction and good compatibility between the polymeric components. The smooth and continuous fibre arrangement suggested effective nanoparticle incorporation without visible agglomeration. At lower magnification, the membrane exhibited a dense, interconnected fibrous network with bright spots corresponding to the well-dispersed CuO nanoparticles across the surface. This homogeneous morphology confirmed successful composite formation, contributing to enhanced structural integrity of the membrane.

Fig. 1.

Scanning electron microscopy images for Corn husk cellulose- PVA- CuO Nanoparticles membrane at (a) 10,000× showing spherical CuO nanoparticles uniformly embedded within the fibrous PVA/Cellulose matrix and (b) 200× showing dense, interconnected fibrous network with bright spots corresponding to the well-dispersed CuO nanoparticles across the surface.

4.2. Energy Dispersive X-ray Spectroscopy (EDS)

EDS analysis was calculated at an accelerating voltage of 15–20 kV, and the spectra were processed with ZAF correction for semi-quantitative evaluation. The EDS spectra showed that carbon and oxygen dominated the membrane composition (C 68.22 wt%, O 31.78 wt%; spot 2: C 57.22 wt%, O 18.34 wt%), consistent with the PVA/cellulose matrix. Copper was detected only in the CuO-treated regions, with measured CuK signals of 24.44 wt% (6.11 at%) at one region and 43.21 wt% (13.94 at%) at another, confirming the presence of copper species within the membrane (Fig. 2, Table 1). The atomic per cent of copper was lower than the weight per cent, as expected for a heavier element. No additional elemental contaminants were observed above the detection limit. Elemental mapping corroborated these point analyses by showing Cu signal distributed across the membrane surface, indicating successful incorporation and reasonably uniform dispersion of CuO nanoparticles within the PVA/Cellulose matrix.

Fig. 2.

EDS spectra and elemental mapping of PVA/Cellulose membranes (a) EDS spectra of untreated membrane, (b) EDS spectra of CuO-treated regions, and (c) Elemental mapping of CuO-loaded membrane.

Table 1.

Energy-dispersive X-ray spectroscopy (EDS) analysis showing the elemental composition of three regions of the sample. Tables present weight percentage (Wt%) and atomic percentage (At%) of carbon (C), oxygen (O), and copper (Cu) with ZAF matrix correction applied. Table (a) represents the untreated region, Table (b) indicates the copper-treated region, and Table (c) corresponds to the copper-rich area.

Element	Wt%	At%
CK	57.22	75.68
OK	18.34	18.21
CuK	24.44	06.11
Matrix	Correction	ZAF
CK	31.28	53.38
OK	25.51	32.69
CuK	43.21	13.94
Matrix	Correction	ZAF
CK	68.22	74.09
OK	31.78	25.91
Matrix	Correction	ZAF

4.3. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

The FTIR spectrum of the PVA/Cellulose/CuO composite membrane exhibited characteristic absorption peaks confirming the successful incorporation of all components (Fig. 3). The broad band at 3308 cm⁻¹ corresponded to the O–H stretching vibration of hydroxyl groups present in both PVA and cellulose, indicating hydrogen bonding interactions between the polymer chains. The peak at 2919 cm⁻¹ was attributed to C–H stretching vibrations of the aliphatic groups in PVA. The band observed at 1418 cm⁻¹ represented C–H bending vibrations, while the strong peak at 1090 cm⁻¹ corresponded to C–O–C stretching vibrations of cellulose and PVA, confirming their intermolecular blending. The absorption band at 840 cm⁻¹ was related to C–O–H bending vibrations, and the distinct peak appearing around 587 cm⁻¹ was assigned to the Cu–O stretching vibration, confirming the successful incorporation of copper oxide nanoparticles within the polymer matrix. The overall spectral features indicated strong intermolecular interactions between PVA, cellulose, and CuO, suggesting the formation of a stable and well-integrated composite membrane suitable for guided tissue regeneration applications.

Fig. 3.

FTIR spectroscopy for the PVA/Cellulose/CuO composite membrane exhibiting characteristic absorption peaks corresponding to hydroxyl (OH⁻) and Copper oxide (CuO) functional groups, confirming the successful incorporation of all components.

4.4. Tensile strength analysis

The mechanical properties of the PVA/Cellulose/CuO composite membrane showed a maximum force of 0.61 N, corresponding to a tensile stress of 0.40 MPa and a tensile stress at break of 0.43 MPa (Fig. 4). A tensile strain at break of 23.42% indicated good ductility and flexibility without brittle failure, supporting its suitability for biomedical applications.

Fig. 4.

Tensile strength measurement showed a maximum force of 0.61 N, corresponding to a tensile stress of 0.40 MPa and a tensile stress at break of 0.43 MPa.

4.5. Contact angle measurement

The membrane exhibited contact angles of 18.6° (left) and 19.4° (right), with an average of 19° (Fig. 5). As this value is well below 90°, the P/C/CuO membrane demonstrated hydrophilic behaviour, indicating excellent surface wettability and suitability for tissue-contact and regenerative applications.

Fig. 5.

Contact angle measurement of the membrane using an OSSILA goniometer, showing the droplet profile on the right and left sides used to assess surface wettability.

4.6. Antimicrobial activity assay

The antibacterial activity of the P/C/CuO composite membrane was evaluated using the agar well diffusion method. The membrane demonstrated effective inhibition against both Gram-negative and Gram-positive bacteria (Fig. 6).

• •Escherichia coli: 17 ± 0.4 mm
• •Staphylococcus aureus: 15 ± 0.3 mm

Fig. 6.

Agar well diffusion plates showing zone of inhibition of the control (gentamicin) and the membrane against a)Escherichia coli,b)Staphylococcus aureus.

The positive control (gentamicin) produced larger inhibition zones (22 ± 0.5 mm for E. coli and 21 ± 0.4 mm for S. aureus), whereas the negative control (distilled water) showed no inhibition. Statistical analysis confirmed significant differences between the membrane and the controls (p < 0.05), with p = 0.0001 for E. coli and p = 0.00007 for S. aureus (Table 2).

Table 2.

Antimicrobial Activity of the P/C/CuO composite membrane, representing the zone of inhibition in the positive, negative control, and P/C/CuO groups. Values are presented as mean ± standard deviation (n = 3). Statistical significance was analysed using one-way ANOVA, with ∗p < 0.05 considered significant.

Microorganisms	Positive Control (Gentamicin)	Negative Control (Distilled Water)	P/C/CuO Membrane	p-value
Escherichia coli (Gram –)	22 ± 0.5 mm	0 ± 0 mm	17 ± 0.4 mm	0.0001∗
Staphylococcus aureus (Gram +)	21 ± 0.4 mm	0 ± 0 mm	15 ± 0.3 mm	0.00007∗

Although gentamicin exhibited higher potency, the membrane showed substantial broad-spectrum activity, with slightly greater effectiveness against E. coli, likely due to enhanced interaction of CuO with Gram-negative cell walls.

4.7. Anti-inflammatory activity (BSA method)

The anti-inflammatory activity assay revealed that both samples exhibited a dose-dependent increase in the percentage inhibition of protein denaturation as the concentration increased from 20 to 100 μg/mL (Fig. 7).

Fig. 7.

Anti-inflammatory activity of the P/C/CuO composite membrane compared with salicylic acid. The X-axis represents concentration (μg/mL), and the Y-axis represents percentage inhibition of protein denaturation (%).

At 20 μg/mL, the P/C/CuO membrane showed an inhibition of approximately 22%, while salicylic acid demonstrated about 28% inhibition. When the concentration was increased to 40 μg/mL, the inhibition rose to around 38% for P/C/CuO and 48% for salicylic acid. At 60 μg/mL, the P/C/CuO composite achieved nearly 52% inhibition compared to 65% for the standard. Further increases to 80 μg/mL resulted in 64% inhibition for P/C/CuO and 80% for salicylic acid. Finally, at 100 μg/mL, the P/C/CuO membrane reached about 75%, while salicylic acid attained 90% inhibition.

Overall, the results confirmed that both samples exhibited increasing inhibition of protein denaturation with concentration, indicating dose-dependent anti-inflammatory behaviour. Although salicylic acid consistently showed higher inhibition percentages, the P/C/CuO composite membrane demonstrated significant anti-inflammatory potential, suggesting its effectiveness in stabilising proteins and preventing denaturation.

4.8. Antioxidant activity (2,2-diphenyl-1-Picrylhydrazyl [DPPH] assay)

The antioxidant assay results revealed that at 20 μg/mL, the P/C/CuO membrane showed a scavenging percentage of approximately 32%, while vitamin C exhibited about 45%, indicating moderate initial antioxidant potential (Fig. 8). When the concentration increased to 40 μg/mL, scavenging improved to nearly 50% for P/C/CuO and 62% for vitamin C. At 60 μg/mL, the membrane achieved around 63% inhibition, closely approaching the 72% observed for vitamin C. Further increases in concentration to 80 μg/mL resulted in 74% scavenging for P/C/CuO and 88% for vitamin C. At the highest tested concentration of 100 μg/mL, the P/C/CuO membrane reached about 82%, while vitamin C achieved 92% inhibition.

Fig. 8.

Antioxidant activity of the P/C/CuO composite membrane compared with vitamin C using the DPPH radical scavenging assay. The X-axis represents concentration (μg/mL) and the Y-axis represents DPPH scavenging activity (%).

Overall, the P/C/CuO composite exhibited strong antioxidant potential that increased with concentration, though slightly lower than the standard. The observed trend confirms the dose-dependent antioxidant effect of the membrane, suggesting its ability to donate hydrogen atoms or electrons to neutralise DPPH radicals.

4.9. MTT assay

4.9.1. Cytotoxicity evaluation

The cytotoxic potential of the fabricated membrane was assessed using the MTT assay on human periodontal ligament fibroblasts, and the results are statistically significant (Table 3).

Table 3.

One-way ANOVA summary for cytotoxicity (%) at different concentrations of membrane eluates. Values are presented as mean ± standard deviation (n = 3). Statistical significance was analysed using one-way ANOVA, with ∗p < 0.05 considered significant.

Group	Mean ± SD (%)	N	p-value (vs Control)	Significance
Control	100.00 ± 0.37	3	1.000
1:1 Dilution	96.47 ± 0.71	3	0.0039∗	Significant (p < 0.01)
1:2 Dilution	98.27 ± 0.36	3	0.0055∗	Significant (p < 0.01)
1:4 Dilution	99.10 ± 0.56	3	0.0796∗	Not significant (p > 0.05)

4.9.2. Cytotoxicity assessment (MTT assay)

The cytotoxicity of the membrane eluates at dilutions of 1:1, 1:2, and 1:4 and the results are presented in Fig. 9. The control group exhibited a mean cell viability of 100 ± 0.37%, while the 1:1, 1:2, and 1:4 dilutions recorded 96.47 ± 0.71%, 98.27 ± 0.36%, and 99.10 ± 0.56%, respectively.

Fig. 9.

Cytotoxicity assessment of the P/C/CuO composite membrane on periodontal ligament cells using the MTT assay. The X-axis represents the experimental groups (Control, 1:1 dilution, 1:2 dilution, and 1:4 dilution), while the Y-axis represents cell viability (%) of periodontal ligament cells.

The eluates demonstrated high cytocompatibility across all dilutions. Although the 1:1 (p = 0.0039) and 1:2 (p = 0.0055) dilutions showed minimal but statistically significant reductions in viability compared to the control, the 1:4 dilution (p = 0.0796) showed no significant difference. Importantly, all values remained well above the accepted cytocompatibility threshold of 70%, confirming that the membrane is non-toxic and compatible with human periodontal ligament fibroblasts.

4.9.3. Live and dead cell assay

Live/dead staining images (Fig. 10a–d) further confirmed membrane biocompatibility. Cells in the control group displayed elongated, spindle-shaped morphology with uniform distribution. Comparable morphology, confluence, and minimal dead cell staining were observed across all eluate dilutions (1:1, 1:2, and 1:4). The predominance of viable cells and lack of membrane disruption support the MTT findings, verifying that the membrane does not induce cytotoxic effects.

Fig. 10.

Live and dead cell assay for the membrane (a control, b- 1:1 dilution, c- 1:2 dilution, and d- 1:4 dilution). Cells in the control group displayed elongated, spindle-shaped morphology with uniform distribution. Comparable morphology, confluence, and minimal dead cell staining were observed across all eluate dilutions (1:1, 1:2, and 1:4).

4.9.4. Alkaline Phosphatase (ALP) expression

The ALP gene expression analysis revealed a clear and progressive dose-dependent upregulation in response to treatment with the P/C/CuO membrane extracts (Fig. 11). The control group demonstrated baseline expression (fold change = 1.00 ± 0.15). Treatment with the 1:1 concentration produced a substantial increase, reaching 3.20 ± 0.30-fold, while the 1:2 concentration further elevated ALP levels to 5.40 ± 0.40-fold. The highest upregulation was observed at the 1:4 concentration, showing 8.00 ± 0.50-fold expression relative to the control.

Fig. 11.

Bar graph showing ALP gene expression in periodontal ligament cells treated with different concentrations of the membrane. The X-axis represents the experimental groups (Control, 1:1, 1:2, and 1:4 combinations), and the Y-axis represents the fold change in ALP expression relative to control.

Statistical analysis using a one-way ANOVA test confirmed that all treatment groups exhibited significantly higher ALP expression compared with the control. The p-values were 0.0026, 0.0003, and 0.0001 for the 1:1, 1:2, and 1:4 groups, respectively, indicating strong statistical significance (p < 0.05) (Table 4).

Table 4.

Alkaline Phosphatase (ALP) gene expression across different concentrations of P/C/CuO membrane extracts. Values are presented as mean ± standard deviation (n = 3). Statistical significance was analysed using one-way ANOVA, with ∗p < 0.05 considered significant.

Groups	Mean ± SD	N	p-value (vs Control)
Control	1.00 ± 0.15	3	1.000
01:01	3.20 ± 0.30	3	0.0026∗
01:02	5.40 ± 0.40	3	0.0003∗
01:04	8.00 ± 0.50	3	0.0001∗

The consistent increase in ALP activity with rising concentrations of the membrane extracts suggested that the P/C/CuO composite effectively stimulated early osteogenic differentiation of periodontal ligament fibroblasts. Since ALP is a key early marker of osteoblast maturation, these results supported the membrane's potential to enhance bone-forming activity. Overall, the findings demonstrated that the composite membrane induced a significant and dose-responsive enhancement of osteogenic gene expression, underscoring its promise as a biomaterial for periodontal regeneration and bone tissue engineering.

5. Discussion

The present study developed a corn husk–derived cellulose membrane reinforced with polyvinyl alcohol (PVA) and copper oxide nanoparticles (CuO NPs), converting agricultural waste into a sustainable biomaterial with potential applications in guided tissue regeneration.

Corn husk–derived cellulose provided a biodegradable and hydrophilic scaffold supporting cellular attachment and mechanical stability, while PVA improved elasticity and polymer integration. Similar reinforcement effects of PVA in cellulose-based scaffolds have been reported previously, where polymer blending enhanced structural stability and flexibility. The incorporation of CuO nanoparticles further contributed antimicrobial, antioxidant, and osteogenic properties, consistent with earlier reports describing the multifunctional behaviour of metal-oxide nanoparticle–reinforced polymer matrices.

SEM analysis revealed a smooth and uniform surface architecture, indicating good compatibility among cellulose, PVA, and CuO nanoparticles. Comparable structural uniformity has been reported in PVA/CuO nanocomposites, where CuO incorporation improved surface morphology and mechanical performance.¹⁹ Compared with commonly used collagen membranes, which often display heterogeneous fibrillar structures, the membrane in the present study demonstrated greater structural uniformity.²⁰ EDS analysis confirmed the homogeneous elemental distribution of CuO nanoparticles without impurities, consistent with previous reports of effective CuO dispersion within polymeric matrices.²¹

FTIR analysis confirmed strong intermolecular interactions and stable polymer–nanoparticle integration. Similar findings have been reported in CuO-reinforced PVA composites, where characteristic Cu–O vibrational peaks and hydrogen bonding interactions contribute to improved structural stability.²² Ananya R et al. also demonstrated successful component integration in EHA–HA Gelatin membranes through FTIR analysis.²³

Biocompatibility is essential for GTR membranes. The high cell viability observed in this study is consistent with previous reports demonstrating favourable fibroblast compatibility in PVA/CuO nanocomposites.²⁴ The live/dead assay further confirmed viable spindle-shaped fibroblasts with intact morphology, similar to observations in CuO-containing nanofibrous systems that promoted fibroblast proliferation and wound healing.²⁵

Mechanical integrity is also important for maintaining membrane stability during tissue regeneration. The tensile strength observed in the present study aligns with previous findings, indicating that CuO nanoparticle incorporation improves cross-linking interactions and mechanical properties of polymeric membranes.¹⁹ Contact angle analysis demonstrated the strong hydrophilicity of the composite membrane. Ananya R et al. reported moderate wettability in EHA-based GTR membranes supporting osteoblast adhesion and fibroblast attachment.²³ Similar reductions in contact angle have been reported in nanoparticle-reinforced PVA and cellulose-based composites following metal-oxide incorporation.²⁶

The antimicrobial evaluation demonstrated effective bacterial inhibition, likely due to copper ion release and nanoparticle-mediated membrane disruption. Similar antimicrobial activity has been reported in CuO-incorporated PVA films, showing broad-spectrum inhibition against Gram-positive and Gram-negative bacteria.²⁷ Sheersha Pramanik et al. reported that chitosan–gelatin composites enhance cellular adhesion and reduce inflammatory responses.²⁸ However, the incorporation of CuO nanoparticles in the present membrane provides additional intrinsic antimicrobial bioactivity.

The composite membrane also exhibited concentration-dependent antioxidant activity. This observation is consistent with previous studies reporting significant free-radical scavenging activity in CuO-reinforced biopolymer films due to the redox-active nature of copper oxide nanoparticles.²⁹ The osteogenic potential observed in the present study, evidenced by dose-dependent upregulation of ALP expression, aligns with findings by Linya Zeng et al., who reported enhanced osteogenic differentiation in biomimetic GTR membranes through upregulation of osteogenic markers such as RUNX2 and OCN.³⁰ These findings suggest that the membrane may promote early osteogenic differentiation essential for periodontal and bone regeneration.

Overall, the corn husk cellulose–PVA–CuO composite membrane represents a sustainable and multifunctional biomaterial for guided tissue regeneration. Its favourable microstructure, hydrophilicity, cytocompatibility, antimicrobial activity, antioxidant capacity, and osteogenic potential support its promise as an environmentally sustainable alternative to conventional GTR membranes.

6. Conclusion

This study demonstrated that corn husk–derived cellulose reinforced with PVA and CuO nanoparticles can be developed into a bioactive membrane with favourable structural properties and biocompatibility. The composite utilised an agricultural waste resource while exhibiting characteristics desirable for guided tissue regeneration membranes. Within the limitations of this study, the material showed potential as a sustainable biomaterial platform. However, further in vitro optimisation and in vivo investigations are required to confirm its regenerative efficacy and clinical applicability.

Declaration of ethical clearance

The conducted study was an in-vitro study, hence ethical clearance not required.

Patient's/Guardian's consent

The authors declare that the above-mentioned manuscript has no need of Patient's/

Guardian's consent since, it is a non-clinical research manuscript.

Limitations and future scope

This study was limited to in vitro evaluations conducted under controlled laboratory conditions, which may not fully represent the complex biological environment in vivo. Factors such as enzymatic degradation, immune response, and tissue dynamics were not assessed. Therefore, further studies, including in vivo models, long-term degradation analysis, and comparisons with commercially available guided tissue regeneration (GTR) membranes, are required to validate the membrane's regenerative potential and clinical applicability.

Sources of funding

The authors declare that this is a self-funded study.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.