Polylactic acid (PLA) (MW: 60,000), polyethylene glycol (PEG) (MW: 4,500), and chlorhexidine (CHX) (≥99.5%) were obtained from Sigma-Aldrich, United States. Chloroform (Chl) was supplied by EMPARTA ACS, United States, and Golden misri hen eggs were purchased from the local market in Peshawar, Pakistan.

Eggs were cracked open and eggshells (ES) were collected in a separate utensil. The ES was cleaned in a sink under running water at 35°C for 5 min to remove impurities. ES were laid in a disinfected tray and air dried for 24 h to facilitate the process of grinding. Dried eggshells were then manually crushed before grinding in a grinder into fine powder. The powder was then passed through a number 200 stainless steel laboratory sieve (74 µm diameter) to obtain a fine powder of 74 µm grain size. It was then collected in an airtight glass jar and stored in a fridge at 4°C to be utilized for sample preparation (Shiferaw et al., 2019).

The solvent casting method was used to prepare five groups of the GTR membranes. Group 1 (C) was prepared by mixing PLA pellets and PEG flakes in 70% and 30% ratios and dissolving in 99.9% pure Chl solvent to prepare a 5% solution. Group 2 (C-10) and group 3 (C-30) were prepared by mixing 10% and 30% ES powder in the polymer mixtures respectively. Groups containing drug group 4 (C-10CHX) and group 5 (C-30CHX) were prepared by adding 0.25% and 0.5% CHX powder in the above mentioned polymers and ES powders. The mixtures were stirred on a magnetic stirrer heated to 50 °C at 800rpm until the polymers and CHX were dissolved into a homogenous solution. The solutions were then poured into glass Petri dishes and covered with an inverted funnel plugged with loose cotton for slow evaporation for 24 h. After 24 h, the membranes were placed in a hot air oven at 60°C. After 4 h, the Petri dishes were taken out from the oven and the dried membranes were peeled off from the Petri dish and stored in a sterile zip lock bag for further analyses.

The mechanical properties (tensile strength, Young’s modulus, and elongation at break) of the five groups of GTR membranes were evaluated by using a universal testing machine (UTM) (electrodynamics fatigue testing machine, Walter + bai AG, Switzerland). Three specimens (n = 3) from each group were cut into rectangular strips (30 mm × 5 mm) and tested. The crosshead speed was set at 1 mm/min. The results were calcuted as average value of the triplicate (He et al., 2019).

Mass change as a result of the water sorption capacity of the GTR membranes was measured in triplicate. Three samples from each of the five groups (n = 3) were prepared by cutting the GTR membranes into (10 × 10) mm dimensions. The dry weight of the GTR samples was measured by digital balance and noted down. After measuring the dry weight, the membranes were immersed in PBS for different time intervals (30 min, 1h, 3h, 6h, 24 h, and 10 days) to determine the percent mass change using the formula W a t e r   i n t a k e   % = W t − W d / W d × 100

The in vitro antibacterial effect of the fabricated GTR membranes was examined against Enterococcus. faecalis (Enterococcus faecalis ATCC 29212) by “disk diffusion method.” With the help of a sterile swab, E. facecalis inoculum was taken and spread over a nutrient agar (NA) plate by “Surface Streaking Method” to form colonies of the strain and incubated at 37°C for 24 h. Using an inoculation loop, colonies of E. faecalis were picked up from the NA plate and transferred to a sterile test tube containing distilled water. The bacterial suspension was then visually compared to the 0.5 McFarland standards estimating the bacterial density. Mueller Hinton Agar (MHA) media was made in distilled water and sterilized at 121 ℃ for 25 min. The MHA broth was poured into Petri dishes. The experiment was performed in triplicate (n = 3). Three small pieces from each group of the five samples of GTR membrane were cut and placed on the prepared Petri dishes and incubator at 37°C. After 48 h of incubation, Petri dishes were taken out and the mean inhibition diameters around each disk of the samples were carefully measured (Joy Sinha et al., 2017).

Sessile drop method analysis was performed to measure the wettability of the five groups of the GTR membranes. A droplet of distilled water (3 μL) was horizontally dropped on the surface of the GTR membrane with the help of a micropipette. Images were taken and the software ImageJ (plugin Contact Angle) was used to determine the contact angle of the water drop formed at the interface with the sample (Grundke et al., 2015).

The cytotoxicity of ES powder, control (C), and experimental groups (C-10, C-30, C-10CHX, C-30CHX) was determined by performing Alamar blue assay using NIH3T3 mouse embryonic fibroblasts and MC3T3 pre-osteoblasts cell lines (American Type Tissue Culture CRL-1658 and CRL 2594). The biocompatibility of a biomaterial with MC3T3 can imply their osteogenic potential in vivo conditions. The NIH3T3 fibroblasts and MC3T3 were cultured using Alpha-modified Eagle’s medium (Biosera, United Kingdom), 10% FBS, and 1% Penicillin/Streptomycin (Pen/Strep). Three samples from each group (n = 3) were first sterilized with 70% ethanol and then washed with PBS three times (15 15-min intervals) before the experiment. After 24 h, the medium was replaced by Alamar blue reagent and incubated at 37°C for 4 hours. After 4h, the absorbance was measured by the micro-plate reader at 570 nm. The cell viability results were expressed as average absorbance values of the triplicates using the formula % C e l l   V i a b i l i t y = s a m p l e   a b s o r b a n c e / c e l l   c o n t r o l   a b s o r b a n c e × 100

FTIR analysis was performed in this study to confirm the presence of ES powder and CHX in the fabricated GTR membranes (Qasim et al., 2021a). Figure 1A demonstrates the characteristic peaks of ES powder and GTR groups C, C-10, and C-30. Stretching vibrations of ES powder belonging to C–O and O–C–O linkages have been observed at 1,404 and 874 cm⁻¹ respectively. Another small sharp peak was observed at 704cm⁻¹ representing the absorbance by the Ca-O bond (Akram et al., 2021). In the FTIR graph, an increase in the intensity of the characteristic peak is indicative of an increase in the amount of the corresponding constituent. Therefore, C-10 containing 10% ES powder has lower peaks present than C-30 containing 30% ES powder while in the control group (C), they are absent altogether since there is no ES powder in group C. Similarly, in Figure 1B , it would be seen that group 1(C) did not present with a characteristic peak associated with C-N bending of CHX at 1,119 cm, ⁻¹ while these have been identified for group C-10CHX and C-30CHX (Priyadarshini et al., 2017). These findings confirm the presence of ES powder and CHX in the experimental groups of GTR membranes.

Scanning electron microscopic evaluation of the surface morphology of the five groups of GTR membranes at ×250 magnification has been shown in Figure 2A. Group 1 (C) showed the conglomerated appearance of PLA and PEG blend with numerous voids which were typical of a GTR membrane surface fabricated by the solvent casting method (Ghosal et al., 2018). With the addition of ES filler, there was a marked decrease in surface porosities in the experimental groups (Figures 2B–E). The addition of CHX as a drug also had significant effect on surface characteristics. The SEM images showed aggregates of ES filler and CHX dispersed on the surface of the GTR membranes, which contributed to the increase in the surface roughness but decreased the surface voids (Ghafoor et al., 2020). Surface roughness of a membrane plays a critical role in cell adhesion and proliferation as the osteoblasts are reported to be sensitive to roughness of the surface of a material. The reason could be explained by the increased surface area of the material for serum proteins to act for a higher percentage of cell adhesion than their polished counterparts (Li et al., 2012).

A homogenous cross sectional area structure was observed by SEM in Figure 3. Group 1 (C) showed porous and dense morphology. The experimental groups (C-10, C-30, C-10CHX, and C-30CHX) also showed similar morphology but were denser with fewer voids present which suggests that the filler components ES powder and CHX were uniformly distributed throughout the samples (Wu et al., 2022).

The XRD pattern of experimental ES powder is shown in Figure 4. Through X-ray diffraction, data from ES powder was collected at 10–70° in the 2-theta range. The ES powder showed characteristic peaks of CaCO₃ at (012), (110), (104), (006), (110), (113), (202), (024), (211), (122), (244), (300), (012) at 23.1°, 29.37°, 35.9°, 39.3°, 43.2°, 47.5°, 48.5°, 57.4°, 48.5°, 57.4°, 58.3° and 60.86°,64.77° and 67.97° respectively. The diffraction peak marked at approximately 34.30 (2-theta) indicates that the ES powder contains a calcite crystalline structure similar to the reported data of CaCO₃ (Al-Jaroudi et al., 2007). The XRD analysis of ES powder revealed the existence of CaCO₃ in the thermodynamically stable calcite phase as a major component of the powder. The presence of more than 90% CaCO₃ having rhombohedral crystal alignment was proved through this analysis. Therefore, ES powder as a natural source of CaCO₃ was utilized for the fabrication of novel GTR membranes in this study (Wei et al., 2004).

The mean diameters of the zone of inhibition (mm) after 48 h are represented in Figure 6 . The highest value for the zone of inhibition was observed in group 5(C-30CHX) (17.3 ± 0.58 mm) followed by group 4 (C-10CHX) (10 ± 1 mm as tabulated in Table 1). There were no zones of inhibitions found in Groups 1(C), group 2 (C-10), and group 3(C-30). Statistical analysis showed a significant difference (p ≤ 0.05) between groups containing CHX; group 4(C-10CHX0 and group 5(C-30CHX) and other groups without the drug; group 1(C), group 2 (C-10) and group 3 (C-30). In the experimental groups containing CHX, group 5 showed a significant difference from group 4 (p < 0.000).

Oral cavity harbours a plethora of microorganisms like bacteria, viruses, fungi and protozoa Contamination by bacteria is one of the most significant factors leading to a reduced outcome in alveolar bone regeneration using the GTR technique. Enterococci particularly E. faecalis has shown to play an important role in the progression of periodontal diseases which might negatively impact the progress of the regenerative therapy (Bhardwaj et al., 2020) CHX is a broad spectrum antimicrobial effective against Gram positive bacteria, Gram negative bacteria and fungi. Therefore, imparting antibacterial property to a GTR membrane could be extremely beneficial for successful GTR therapy (Bhardwaj et al., 2020; Abdo et al., 2023). In this study, the antibacterial activity of the specimens against E. faecalis was studied in vitro using disc diffusion methods. After 48 h of incubation, the maximum zone of inhibition was seen around group 5 (C-30CHX) containing the highest amount of CHX (0.5%) followed by group 4 (C-10CHX) containing 0.025% of CHX while there were no zones of inhibition was noticed around groups that did not contain CHX (C, C-10, and C-30). These results indicated that increasing the concentration of CHX increased the antibacterial potential of the GTR membrane which is following a study in which increasing the concentration of CHX doubled the antibacterial activity of a collagen based GTR membrane (Khan et al., 2022a).

The mean percent mass change after 1 h as shown in Figure 7 was highest for group 3 (C-30) which is consistent with literature that composites with higher loading of ES powder show more water sorption. The presence of a high level of CaCO₃ in the ES powder can absorb more water, so it increases the water absorption as the filler increases (Farahana et al., 2015). This is in contrast to a study in which hydroxyapatite was used as a filler material. Swelling decreased as the amount of HA increased in the samples (Qasim et al., 2021b). Group 5 (C-30CHX) containing the same amount of ES filler as group 3 (C-30) but also containing CHX has the lowest percent mass increase among the groups after 1 h. Water sorption depends mainly on two factors; the hydrophilicity/hydrophobicity of the material and the extent of porosity. Hence, the reduction may be due to CHX filling up any voids present making it more impermeable and hence less water sorption and mass increase (Vasudevan and Chan Wei, 2020) Or it could also be attributed to CHX forming a surface coating on the ES powder due to its adsorbent property. Since CHX is significantly less hydrophilic than CaCO₃, the mass change was lowest for C-30CHX. Group 5 (C-30CHX) containing the maximum amount of drug CHX has the lowest water absorption. This could be explained by the less porous structure ofgroup 5 (C-30CHX) resulting in lesser swelling or CHX forming a surface coating on the ES powder due to the adsorbent nature of the ES powder particles (Khan et al., 2022b).

The results of the sessile drop method technique showed that the contact angle of the fabricated GTR membrane was lowest for group C-30 at 22.29° (tabulated in Table 2). Contact angles recorded for all groups were below 90 indicating hydrophilic surfaces (Latthe et al., 2014).

GTR membranes should be fairly hydrophilic for water since it is the conveying medium to carry nutrients to the bony area that needs to be regenerated. Hydrophilic surfaces are also reported to enhance cellular attachment, proliferation, and elongation of the osteoblasts and other cells (Brum et al., 2021). However, too high water levels in the GTR membrane can soften the membrane and compromise its mechanical properties (Fernandes et al., 2020).

The results of this study showed that all five groups of GTR membranes were hydrophilic (contact angle <90°) (Ahmad et al., 2018). Experimental group 3(C-30) had the smallest contact angle (22.2°) indicating very strong hydrophilicity as shown in Figure 8. This can be explained by the presence of high amount of CaCO3 in the sample. CaCO₃ is a hydrophilic mineral and non-toxic substance that has been widely used as a functional filler in biomaterials for improving aspects like mechanical properties and bioactivity (Shamsuri and Sumadin, 2018). These results are consistent with studies in which bioglass and hydroxyapatite were used as the inorganic filler. Increasing the amount (weight percentage) of bioglass and hydroxyapatite in a polymeric matrix increased the wettability of the GTR membranes (Soltani Dehnavi et al., 2018; Zimina et al., 2020).

Group 5(C-30CHX) containing 30% ES filler same as group 3(C-30) has a much higher contact angle (22.29° vs. 64.6°) which could be explained by two factors. One is CHX being less hydrophilic than CaCO₃ and the other is the inherent adsorbent property of CaCO₃ in the form of ES powder. ES powder is an excellent adsorbent material and CHX may have formed a surface coating over the ES powder particles, thus increasing the value of Contact Angles in groups 4 and 5. Thus, incorporation of different concentration of ES powder results in different types of GTR membranes with varying levels of wettability and contact angle of the membranes (Khan et al., 2022c).