1 Volume 24 2025 e254367 Original Research Braz J Oral Sci. 2025;24:e254367http://dx.doi.org/10.20396/bjos.v24i00.8674367 1 Department of Restorative Dentistry, National Dental Centre of Singapore, Singapore. 2 Department of Dentistry, Ng Teng Fong General Hospital, Singapore. 3 National Dental Research Institute Singapore (NDRIS), Duke-NUS Medical School, Singapore. 4 Faculty of Dentistry, National University of Singapore, Singapore. Corresponding author: Rui Ling Kong Department of Restorative Dentistry, National Dental Centre Singapore, Singapore Singapore 168938 Phone: +65 6324 8802 Email: kong.rui.ling@ndcs.com.sg Editor: Dr. Altair A. Del Bel Cury Received: August 24, 2023 Accepted: January 7, 2025 In-vitro antibacterial activity of contemporary bioactive dental restorative materials Rui Ling Kong1*, Adrian Ujin Yap2 , Chaminda Jayampath Seneviratne3 , Keson Beng Choon Tan4 , Christina Poh Choo Sim1 Aim: Secondary caries can lead to a failure of composite and glass ionomer cement (GIC) restorations. Bioactive dental restoratives have been introduced to address this problem. However, the evidence supporting the antibacterial efficacy of these materials remains limited. The objective of this study was to compare the antibacterial efficacy of contemporary bioactive and conventional restoratives against S. mutans. Methods: A conventional composite (Filtek Z350 - FT), a high viscosity GIC (Equia Forte - EQ) and three bioactive restoratives comprising a giomer (Beautifil II - BF), an alkasite (Cention N - CN), and a zirconia reinforced GIC (Zirconomer - ZC) were evaluated. Biofilm quantification was performed using crystal violet (CV) assays and colony forming units (CFU) counts to determine the biofilm biomass and S. mutans viability respectively. The surface roughness of the specimens was measured. Results: FT had the lowest biofilm biomass (OD570nm) and BF had the lowest S. mutans viability (LogCFU/ml). The highest surface roughness was observed for EQ while the lowest surface roughness was observed for FT. Surface roughness after exposure to bacteria suspension was significantly higher in ZC compared to FT and BF. Conclusion: GIC-based materials displayed significantly greater biofilm biomass, S. mutans viability, and surface roughness compared to resin-based ones. Surface roughness (immediately after fabrication) showed a strong correlation to S. mutans viability. Further studies are required to verify if surface roughness is the dominant factor affecting the antibacterial efficacy of dental restoratives. Keywords: Bioactive glass. Glass ionomer cements. Composite resins. Anti-bacterial agents. https://orcid.org/0000-0003-0361-6209 https://orcid.org/0000-0003-0816-538X https://orcid.org/0000-0002-5530-6838 2 Kong et al. Braz J Oral Sci. 2025;24:e254367 Introduction Composite resins have been the material of choice for dental restorations due to good aesthetics, reparability and their ability to bond to tooth structure. Despite these qualities, composite resins often fail clinically due to bulk fractures and sec- ondary caries arising from microleakage at the tooth-restoration interface1. Another group of commonly used tooth-colored restorative materials is glass ionomer cement (GIC). Unlike composite resins, these bioactive materials release fluorides and enhance remineralization while minimizing demineralization of the tooth struc- ture and impeding bacterial growth. However, clinical surveys still report secondary caries as a common reason for the failure of GIC restorations, suggesting that the level of fluoride released from GICs is insufficient to achieve bacteriostatic and bac- tericidal effects2. To overcome the limitations posed by conventional dental restorative materials, ion-releasing materials (IRMs) have been introduced. These bioactive materials form a calcium phosphate-rich layer at the tooth-restoration interface and reduce micro- leakage arising from bacterial invasion at the margins. The purported antibacterial mechanisms of these materials are mainly that of (i) elevating pH levels within the biofilm, (ii) raising osmotic pressure, and (iii) inactivating bacteria by damaging their cell walls. The first proposed mechanism occurs through the dissolution of bioactive glass in IRMs, which releases alkali ions such as sodium, calcium and phosphate, resulting in an increased pH. This alteration in the pH gradient influences the cytoplas- mic membrane permeability of bacteria, and inhibits the enzymatic activity of enolase, ATPase, and sugar transport, resulting in bacteriostatic and even bactericidal effects3. Secondly, this release of ions also causes perturbations of the membrane potential of bacteria via rapid water efflux and a pressure drop across the cell membrane, resulting in altered cell size, cell shape, and membrane stress level4. Thirdly, “needle-like” debris found on the surface of Bioglass 45S5 has been found to be capable of destroying the cell walls of bacteria, resulting in bacterial death5. Understanding the antibacterial properties of restorative materials allows the clini- cian to make an informed decision when restoring cavities, especially since biofilm formation at the tooth-restoration interface due to bacterial adhesion is known to be the cause of recurrent caries6. Some of the contemporary IRMs claim to have greater ion release7 and superior mechanical properties compared to the traditional GICs8,9. However, the current evidence on the antibacterial properties of IRMs remains lim- ited and inconclusive due to differing testing methodologies and surface treatment of the materials in studies. Materials can also be broadly categorized into two groups, which are either resin-based (e.g. Filtek Z250, Beautifil II and Cention N) or glass-ion- omer-based (e.g. Equia forte and Zirconomer). The categorization is largely based on the presence of methacrylate monomers (which are believed to have bacterio- static properties) in the resin-based group and the absence of these monomers in the glass-ionomer-based group. Limited studies have been done to compare the antibacterial efficacy of IRMs against their conventional counterparts and among themselves. Hence, the objective of this 3 Kong et al. Braz J Oral Sci. 2025;24:e254367 study was to evaluate the antibacterial activity of IRMs. Additionally, we sought to determine the correlation between surface roughness and antibacterial activity. Methods and Materials Selection of materials and determination of sample size The technical profiles of the materials are listed in Table 1. This study used 3 types of contemporary IRMs namely: (i) a giomer with pre-reacted glass-ionomer fillers (Beautifil II [3M-ESPE, St Paul, MN, USA])(BF); (ii) an alkasite with alkaline fillers (Cention N [Ivoclar Vivadent, Liechtenstein])(CN); and (iii) a zirconia reinforced GIC (Zirconomer [Shofu Inc., Kyoto, Japan](ZC). A conventional composite, Filtek Z250 (3M-ESPE, St Paul, MN, USA)(FT) and a GIC, Equia Forte (GC America INC, Alsip, IL, USA)(EQ) were used as controls. The minimum sample size of 60 (i.e., n=6) was determined using R language program (version 4.0.2; R Foundation for Statisti- cal Computing, Vienna, Austria) based on the assumption of a standard deviation of 0.025 (derived from pilot data) to detect a difference of 0.1 using two-sided two-sample equal variance t-test, with an alpha error of 0.05 (Bonferroni adjusted), and power of 80% for 5 different materials. Table 1. Technical profiles of the materials chosen for study Manufacturer Material (Abbreviation) Type Resin (Photo-initiator) Filler Filler size (µm) Filler content % by weight/ % by volume Curing mechanism 3M-ESPE, St Paul, MN, USA Filtek Z250 (FT) Micohybrid composite Bis-GMA Bis-EMA UDMA TEGDMA (CQ) Zirconia/ Silica cluster and Silica nanoparticle Average 0.6 (0.01-3.5) 78.5/63.3 Light cured (20 sec) GC America INC, Alsip, IL, USA Equia Forte (EQ) High viscosity glass- ionomer cement (Glass hybrid) NIL Fluoroalumino silicate glass, polyacrylic acid Average 7 84/70 Light cured (20 sec) Shofu Inc., Kyoto, Japan Beautifil II (BF) Giomer Bis-GMA TEGDMA (CQ) S-PRG based on F-Br-Al-Si glass Average 0.8 (0.01-4) 83.3/68.8 Light cured (10 sec) Ivoclar Vivadent, Liechtenstein Cention N (CN) Alkasite UDMA DCP PEG-400 DMA Barium aluminum silicate glass, ytterbium trifluoride 0.1–35 78.4/57.6 Self cured Shofu Inc., Kyoto, Japan Zirconomer (ZC) Zirconia reinforced glass ionomer NIL Fluoroalumino silicate glass, zirconium oxide Average 7 (2-30) 34/64 Self cured Bis-GMA, Bisphenol-A glycidyl methacrylate; Bis-EMA, Ethoxylated bisphenol-A-glycidyl methacrylate; CQ, Camphorquinone; TEGDMA, Triethylene glycol dimethacrylate; S-PRG, Surface modified pre-reacted glass; F-Br-Al-Si, Fluoroboroaluminosiliciate; UDMA, Urethane dimethacrylate; DCP, dimethanol dimethacrylate; PEG-400 DMA, polyethylene glycol 400 dimethacrylate 4 Kong et al. Braz J Oral Sci. 2025;24:e254367 Preparation of specimens and immersion protocol There were 6 specimens of each restorative material which were prepared separately for the crystal violet (CV) assay and colony forming units (CFU) assay measure- ments. Specimens of 8 mm in diameter and 1.5 mm in thickness were fabricated using cylindrical-shaped stainless-steel molds of 12 mm in external diameter, 8mm in internal diameter and 1.5 mm in internal height. All specimens were prepared according to manufacturers’ instructions. The lateral walls of the stainless-steel molds were smeared with a thin layer of Vaseline using a microbrush to prevent material adhesion. The various materials were packed into a stainless-steel mold, covered entirely with Mylar strips and compressed between two glass slides using finger pressure to extrude excess material. The specimens were cured with two overlapping irradiations from the top and bottom surfaces using an LED curing light unit (Radii-cal, SDI, Australia). This was done with an 8 mm curing tip diameter with output irradiance of 1200 mW/cm (20s) and 460nm wavelength following the man- ufacturers’ curing times, where applicable. The light curing unit was maintained at full charge before use, and the light intensity and wavelength were monitored after every 5 specimens using a radiometer (LED Radiometer, Kerr, Orange, CA, USA). No finishing or polishing was performed, to maintain a standardized surface smooth- ness for all the specimens. Additionally, the smooth surface achieved with the Mylar strip is often considered to be a final finish10. The specimens of each group were then immersed in 20ml of distilled water in a sterile Falcon tube for seven days in complete darkness at 37°C and 100% humidity11. Sterilization It was important to ensure that the specimens were sterile to avoid contamination by other microorganisms that could interfere with the biofilm culture and ultimately affect the results obtained. Sterilization of the specimens was done through UV irra- diation. The UV lamp built within the biosafety cabinet was used at an ambient tem- perature. The UV lamp emitted UV light with a wavelength of 254 nm at an intensity of 10W. Specimens were irradiated for 15 minutes on each side12-14. Biofilm formation The S. mutans biofilm formation protocol was adapted from Ho et al.15 (2017). S. mutans strain ATCC 35668 was stored at 37°C and 5% CO2 in aerobic conditions and reactivated onto Mitis Salivarius (MS) agar. Single colonies from the MS agar were inoculated in sterile brain heart infusion broth with 2% sucrose supplementa- tion at 37°C at 200rpm for 24 hours. Bacterial cultures were adjusted to the inocu- lum of 108 CFU/ml (equivalent to an optical density(OD)600 of 0.5). The specimens from the different experimental groups were then inoculated with 1.5 mL of the inoculation medium (sterile brain heart infusion broth with 2% sucrose supplemen- tation) in a 24-well plate platform and incubated at 5% CO2 and 37 °C for 24 hours at 200rpm to allow for biofilm formation16. A duration of 24 hours was chosen to allow for S. mutans biofilm formation to reach the stationary phase of the S. mutans growth curve17. Additionally, this is a commonly used duration in many in-vitro bio- film studies as there is no need for a change in the medium, allowing undisrupted 5 Kong et al. Braz J Oral Sci. 2025;24:e254367 bacteria growth18. After 24 hours of exposure to S. mutans suspension, the bacteria suspensions were aspirated from each well and biofilms were washed gently with Phosphate Buffered Saline (PBS) to remove the non-adherent cells on one side. The samples were then carefully flipped and transferred to a new well for gentle washing with PBS again. Biofilm quantification To provide a complete assessment of the antibacterial effect of the materials tested against S. mutans, biofilms were quantified by 2 methods: CV assay and CFU counts. CV assay was used to determine the biofilm biomass by measuring the optical den- sity of the content using a microtiter plate spectrophotometer (µQuant Microplate Spectrophotometer, Biotek, Winooski, VT, USA) at 570 nm. CFU counting method was used to determine S. mutans viability, whereby the bacterial colonies were counted and converted to the corresponding log CFU values. Surface roughness measurements Surface roughness measurements were determined with a profilometer (Model MR200, Mitech, China) at 2 time points; immediately after fabrication and after expo- sure to S. mutans suspension for 24 hours. Mean surface roughness (Ra) was mea- sured from 3 different points on each specimen before and after 24 hours of exposure to bacteria suspension in the CFU count experiment. An average of 3 readings was recorded for each specimen, and the median was taken from the 6 specimens per group. Change in surface roughness was defined as the difference in surface rough- ness measured immediately after specimens were fabricated and after 24 hours of exposure to S. mutans suspension. Statistical analysis All statistical analyses were conducted using the R language program (version 4.0.2; R Foundation for Statistical Computing, Vienna, Austria). Statistical signifi- cance was set at p<0.05. As the results obtained from CV assay and CFU counts did not follow a normal distribution based on QQ plots, non-parametric tests were employed to compare the median of the biofilm biomass, S. mutans viability and change in surface roughness among the groups. The Kruskal-Wallis test was employed to compare the biofilm biomass, S. mutans viability, and change in sur- face roughness (immediately after fabrication and after 24 hours of exposure to S. mutans) among the groups. Post-hoc Kruskal-Nemenyi tests were performed to compare individual materials in terms of the biofilm biomass, S. mutans viability and change in surface roughness. Univariate and multivariate linear regression were performed to test the association between surface roughness immediately after fabrication, surface roughness after 24 hours of exposure to bacteria suspension and type of material with S. mutans viability. The Kruskal-Wallis test was performed to determine the change in sur- face roughness after 24 hours of exposure to bacteria suspension among different groups. Wilcoxon signed ranked test was then performed to compare the change in surface roughness within each of the 5 groups tested. 6 Kong et al. Braz J Oral Sci. 2025;24:e254367 Results Comparison of biofilm biomass, S. mutans viability, and surface roughness before and after 24 hours exposure to bacteria suspension The median and interquartile range of each group are presented in Table 2. The biofilm biomass (p<0.001), S. mutans viability (p<0.001), surface roughness before 24 hours of exposure to bacteria suspension (p<0.001), and surface roughness after exposure to bacteria suspension (p<0.001) were significantly different between test groups. The amount of biofilm biomass measured from CV assay was significantly higher for ZC compared to FT (p<0.001) and BF (p=0.004). EQ reported significantly higher bio- film biomass compared to FT (p=0.004). S. mutans viability was significantly higher in ZC compared to FT (p=0.004) and BF (p<0.001). EQ reported significantly higher S. mutans viability compared to BF (p=0.004). Surface roughness before exposure to bacteria suspension was significantly higher in ZC compared to FT (p<0.001) and BF (p=0.001). EQ reported significantly higher sur- face roughness values compared to FT (p=0.012). Surface roughness after 24 hours of exposure to bacteria suspension was significantly higher in ZC compared to FT (p<0.001) and BF (p=0.001). Table 2. Comparison of biofilm biomass (CV assay), S. mutans viability (CFU) and surface roughness before and after 24 hours exposure to bacteria suspension. Median (IQR) p value Significant post hoc pairwise comparison FT BF CN EQ ZC Biofilm biomass (OD570nm) 0.19 (0.18, 0.20) 0.25 (0.23, 0.26) 0.47 (0.44, 0.55) 0.74 (0.73, 0.76) 0.89 (0.87, 0.92) <0.001 ZC, EQ > FT ZC > BF S. mutans viability (LogCFU/ml) 2.38 (2.35, 2.40) 2.13 (2.08, 2.18) 2.84 (2.83, 2.88) 2.98 (2.94, 3.01) 3.53 (3.50, 3.57) <0.001 ZC, EQ > BF ZC> FT Mean Surface roughness (Before) (μm) 0.076 (0.075, 0.078) 0.078 (0.077, 0.08) 0.170 (0.164, 0.172) 0.282 (0.279, 0.284) 0.381 (0.374, 0.387) <0.001 ZC, EQ >FT ZC> BF Mean Surface roughness (After) (μm) 0.079 (0.077, 0.08) 0.081 (0.079, 0.082) 0.172 (0.165, 0.175) 0.287 (0.281, 0.298) 0.388 (0.386, 0.389) <0.001 ZC> FT, BF FT, Filtek Z250; BF, Beautifil II; CN, Cention N; EQ, Equia forte; ZC, Zirconomer. Association between surface roughness and S. mutans viability On univariate analysis, a significant positive association was detected between sur- face roughness immediately after fabrication with S. mutans viability (p<0.001), and surface roughness after exposure to bacteria suspension with S. mutans viability (p<0.001). Immediately after fabrication, for every unit increase in surface roughness, CFU was found to have increased by 3.971 units (95% CI=3.488-4.453). After 24 hours 7 Kong et al. Braz J Oral Sci. 2025;24:e254367 of exposure to bacteria suspension, for every unit increase in surface roughness, CFU was found to have increased by 3.861 units (95% CI=3.372-4.349). On multivariate analysis, there were no significant associations between surface roughness immedi- ately after fabrication (p=0.212), surface roughness after exposure (p=0.897) and type of material (p=0.581) with S. mutans viability. Change in surface roughness The change in surface roughness after 24 hours of exposure to bacteria suspension was not significantly different among different groups (p=0.072). When comparing the change in surface roughness within each of the 5 groups, 3 out of the 5 materials, namely EQ (p=0.036), BF (p=0.034) and CN (p=0.036), reported significant changes in surface roughness (Table 3). Table 3. Change in surface roughness within each group Group Median surface roughness (IQR) p valueSurface roughness (μm)(before) Surface roughness (μm)(after) Change in surface roughness (μm) FT 0.076 (0.075, 0.080) 0.079 (0.079, 0.081) 0.003 (0.002-0.003) 0.053 BF 0.078 (0.077, 0.080) 0.081 (0.078, 0.083) 0.003 (0.002, 0.003) 0.034 CN 0.170 (0.161, 0.175) 0.172 (0.163, 0.179) 0.003 (0.003, 0.006) 0.036 EQ 0.282 (0.274, 0.288) 0.287 (0.276, 0.309) 0.005 (0.003, 0.015) 0.036 ZC 0.381 (0.373, 0.391) 0.388 (0.383, 0.394) 0.008 (0.008, 0.012) 0.059 FT, Filtek Z250; BF, Beautifil II; CN, Cention N; EQ, Equia forte; ZC, Zirconomer. Discussion FT showed significantly lower S. mutans viability compared to EQ and ZC. This is a surprising finding since composite restorations are believed to be more prone to sec- ondary caries development. Reasons for this belief include the absence of antibac- terial properties in composites, and the promotion of growth of S. mutans and other cariogenic bacteria due to the release of methacrylate monomers, which result from incomplete polymerization and resin biodegradation of composites19. However, the leachable components in methacrylate-based dental resins have since been proven to be bacteriostatic20. Consequently, this resin-rich layer resulting from the adaptation of the Mylar strip during fabrication (which comprises poorly polymerized resin mono- mers) could have led to an increased elution of unbound monomers. This is a possi- ble explanation for the low biofilm biomass and S. mutans viability observed in FT. Similarly, BF also reported significantly lower biofilm biomass compared to ZC, while S. mutans viability from FT and BF were both significantly lower than ZC. 8 Kong et al. Braz J Oral Sci. 2025;24:e254367 The glass-ionomer-based groups exhibited lower antibacterial activity than the resin-based groups. S. mutans viability levels were found to be significantly higher in ZC compared to FT and BF. EQ also displayed significantly higher S. mutans viability than BF. The weak antibacterial activity of glass-ionomer-based groups observed in this study contrasts with other studies that report a significant anti- bacterial effect of GICs against S. mutans21,22. The antibacterial effect of GICs is believed to be due to their ability to release fluoride ions, which prevents bacterial growth. GICs have been reported to be capable of achieving a fluoride concentra- tion of 7 ppm or more after 14 days23, with in-vitro studies suggesting that only small amounts of fluoride (approximately 0.03–0.08 ppm) are required to shift the equilibrium from demineralization to remineralization24. A possible explanation for the poor performance of the glass-ionomer-based materials in this study was that the dissolution of fluoride ions from these materials during the 7-day water-aging process did not result in an ion concentration higher than the minimum inhibi- tory concentration required for S. mutans during the 24 hours biofilm formation25, although we did not measure fluoride concentrations for the specimens used. The short duration of exposure of specimens to the bacterial suspension could also have dampened the antibacterial effects of fluoride on the specimens. However, an in-vitro study suggested that even a continuous supply of fluorides at thera- peutic levels had minimal inhibitory effect on the aggregation of S. mutans26. A correlation between fluoride-releasing potential and antibacterial properties in flu- oride-releasing composites, compomer and resin-modified GICs has also not been shown27. In addition, it has been proposed that surface roughness plays a more important role in S. mutans viability compared to fluoride release in GICs28. Hence, further studies of the fluoride release capacity of GICs are needed to understand this relationship better. The glass-ionomer-based group was found to exhibit greater surface roughness than the resin-based group. FT displayed the lowest surface roughness values, fol- lowed by BF, CN, EQ and ZC. A similar study also found that FT exhibits the lowest surface roughness values of 0.260μm followed by CN (0.378μm)29. The surface roughness of these materials may be attributed to filler particle size. Multiple stud- ies have shown a positive association between surface roughness and filler parti- cle size30,31. The average filler particle size of EQ and ZC are both 7μm, which is much larger than that of FT (0.6μm) and BF (0.8μm). Hence, the study results are not unexpected. The study found a significant positive association between the amount of viable S. mutans and surface roughness on univariate analysis. This finding is congruent with reports that rougher surfaces promoted dental plaque formation32. It has been demonstrated that surface roughness influences the early attachment of S. mutans to the surfaces of nanofilled and nanohybrid resin-based composites surface33. This is important, as the proliferation of the initial adhering microorganisms accounts for a major part of microorganism mass increase during plaque formation34. Surface roughness of 0.2μm has long been considered the critical surface roughness for bacterial adhesion and retention on dental materials35, with values surpassing 0.2μm resulting in a higher volume of bacterial attachment, and a higher caries risk. 9 Kong et al. Braz J Oral Sci. 2025;24:e254367 Out of all the materials in this study, only EQ and ZC were found to have surface roughness values greater than 0.2μm. This may explain why the amount of biofilm biomass and viable S. mutans measured were significantly higher in these two mate- rials. A significant association was also found between surface roughness (before 24 hours of exposure to S. mutans suspension) and S. mutans viability in our study, which supports the view that surface roughness is a crucial factor in influencing bac- terial adhesion on dental materials. After 24 hours of exposure to S. mutans culture, a change in surface roughness was observed in all groups. However, this change in surface roughness was only signifi- cant in BF, CN and EQ, with the change in surface roughness in FT and ZC approach- ing statistical significance. This increase in surface roughness may be due to biodeg- radation of the material from the acids produced by cariogenic bacteria in the biofilm layer, which lowers the pH of the environment, and degrades the monomers in the resin matrix36. This leads to an increase in the surface roughness of the dental mate- rial, thereby encouraging further bacterial accumulation. Several limitations of our study are worth highlighting. Firstly, we used a monospe- cies S. mutans biofilm, which cannot replicate the in-vivo biofilm model which can investigate the complex interactions between multiple species of bacteria. However, the monospecies biofilm model can still help to investigate how a chosen cariogenic microorganism would respond in an in-vitro setup and can improve our understand- ing as to how it may play a role in complex biofilm models. Secondly, a 2-dimen- sional contact profilometer was used in this study. However, contact profilometry may underestimate the surface roughness of dental composites37, and there is a possibility that the surface roughness values reported in this study may be higher if atomic force microscopy was used. Thirdly, confocal laser microscopy was not performed to confirm the proportion of live and dead bacteria on the dental materi- als. Fourthly, we did not measure the fluoride concentration of the specimens used, which may have a biological effect on inhibiting bacterial growth and biofilm forma- tion. Lastly, although materials are usually polished clinically, we opted for a Mylar finish in this study to minimize the confounders affecting the surface roughness of the materials studied. In conclusion, we found that glass-ionomer-based groups displayed greater biofilm biomass, S. mutans viability and surface roughness compared to the resin-based groups. A significant positive association was detected between S. mutans viability and surface roughness. Future in-vitro studies are required to investigate other fac- tors apart from surface roughness that could affect bacterial adhesion in IRMs, such as the extent of residual monomers and fluoride ion release. Acknowledgements None. Conflicts of interest None. 10 Kong et al. Braz J Oral Sci. 2025;24:e254367 Data Availability Datasets related to this article will be available upon request to the corresponding author. Author Contribution Rui Ling Kong: Study conception and design, Data collection, Analysis and interpre- tation of results, manuscript drafting and preparation. Adrian Ujin Yap: Study con- ception and design, Analysis and interpretation of results, manuscript drafting and preparation. Chaminda Jayampath Seneviratne: Study conception and design, Data collection, Analysis and interpretation of results, manuscript drafting and prepara- tion. Keson Beng Choon Tan: Analysis and interpretation of results, manuscript drafting and preparation. Christina Poh Choo Sim: Analysis and interpretation of results, manuscript drafting and preparation. All authors actively participated in the discussion of the manuscript’s findings, and have revised and approved the final version of the manuscript. References 1. 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