Enhancing Presurgical Infant Orthopedic Appliances: Characterization, Mechanics, and Biofilm Inhibition of a Novel Chlorhexidine-Halloysite Nanotube-Modified PMMA

Objectives This in vitro study aimed to develop a novel nanocomposite acrylic resin with inherent antimicrobial properties. This study evaluated its effectiveness against microbial biofilm formation, while also assessing its physical and mechanical properties. Methods Polymethylmethacrylate (PMMA) was modified with four different concentrations of chlorhexidine halloysite nanotubes (CHX-HNTs): 1%, 1.5%, 3%, and 4.5 wt.% by weight, along with a control group (0 wt.% CHX-HNTs). The biofilm inhibition ability of the modified CHX-HNTs acrylic against Candida albicans, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus agalactiae was assessed using microtiter biofilm test. In addition, ten samples from each group were then tested for flexural strength, surface roughness, and hardness. Statistical analysis was performed using one-way ANOVA and Tukey's test for comparison (P < 0.05). Results CHX-HNTs effectively reduced the adhesion of Candida albicans and bacteria to the PMMA in a dose-dependent manner. The higher the concentration of CHX-HNTs, the greater the reduction in microbial adhesion, with the highest concentration (4.5 wt.%) showing the most significant effect with inhibition rates ≥98%. The addition of CHX-HNTs at any tested concentration (1%, 1.5%, 3%, and 4.5 wt.%) did not cause any statistically significant difference in the flexural strength, surface roughness, or hardness of the PMMA compared to the control group. Conclusions The novel integration of CHX-HNT fillers shows promising results as an effective biofilm inhibitor on acrylic appliances. This new approach has the potential to successfully control infectious diseases without negatively affecting the mechanical properties of the acrylic resin. Clinical Relevance. The integration of CHX-HNTs into presurgical infant orthopedic appliances should be thoroughly assessed as a promising preventive measure to mitigate microbial infections. This evaluation holds significant potential for controlling infectious diseases among infants with cleft lip and palate, thereby offering a valuable contribution to their overall well-being.


Introduction
Cleft lip and palate (CLP) present a signifcant surgical challenge due to facial asymmetry, tissue absence, and the size of the gap.Since the 1950s, orthodontists and surgeons have collaborated on developing presurgical infant orthopedic (PSIO) to align the soft tissue and osseous structures before surgery [1,2].PSIO procedures, particularly nasoalveolar molding (NAM), have become widely adopted worldwide over the past 40 years [3][4][5][6].In the United States, 71% of centers use some form of PSIO device before cleft lip surgery, with NAM being the most common approach (55%) [6].
While polymethyl methacrylate (PMMA) is widely used in dentures and orthodontic appliances due to its afordability and aesthetics, its porous surface and the fuctuating oral environment promote bacterial and fungal growth, leading to plaque buildup [7][8][9][10][11][12].Tese concerns are even more signifcant for serious pathogens such as methicillinresistant Staphylococcus aureus, which have been found on such appliances and can potentially lead to a local or systemic infection [13,14].Oral microorganisms derived from removable acrylic appliances have been implicated in bacterial endocarditis, pneumonia, chronic obstructive pulmonary disease, and gastrointestinal infections [15].Maintaining proper hygiene with these removable appliances is especially difcult for children and disabled patients [16].Terefore, integrating antimicrobial properties directly into PSIO appliances presents a promising solution to minimize plaque formation and improve overall oral hygiene.
Nanotechnology has emerged as a powerful tool for developing materials with superior biological and mechanical properties [17,18].Halloysite nanotubes (HNTs), a natural nanomaterial similar to kaolin and derived from weathered alumina silicate clays, have gained particular interest for their drug delivery capabilities [19].Studies have demonstrated successful encapsulation of various compounds such as doxycycline, chlorhexidine (CHX), and tetracycline within HNTs, leading to a more sustained release compared to free forms or those incorporated in alternative delivery systems [19][20][21][22][23].
CHX, a broad-spectrum antimicrobial agent with low cytotoxicity, ofers promise for incorporation into acrylic resins.However, the amount of CHX included is critical as excessive levels can compromise the resin's mechanical properties and potentially exhibit cytotoxicity [24,25].An ideal scenario would involve a controlled release system for CHX within the resin, enabling localized action at the application site.Tis approach could minimize the total CHX needed, mitigating cytotoxicity risks while maintaining antimicrobial efcacy and preserving the resin's integrity.Tis is particularly relevant for applications such as PSIO appliances, where acrylic surfaces can harbor microbes and pose a challenge for immunocompromised patients susceptible to infections.Te development of controlled release systems for antimicrobials within the oral cavity holds signifcant value for improving patient outcomes.To address this challenge, this study pioneers the development of PMMA-based PSIO appliances with integrated antimicrobial properties.
Terefore, the aim of this study was to synthesize a CHX-HNT-modifed acrylic resin with inherent antimicrobial properties and subsequently evaluate its efectiveness in inhibiting microbial bioflm formation using a crystal violet (CV) assay while also assessing its mechanical properties.

Materials and Reagents.
Self-polymerizing PMMA resin (Orthocryl, Dentraum) was used to fabricate acrylic specimens for this study.Te specimens were further modifed by incorporating chlorhexidine digluconate solution and halloysite nanotubes (Sigma-Aldrich, Germany) to create the nanocomposite material.

Overview.
Te complete timeline of this experimental in vitro study was 12 weeks.Acrylic resin samples were divided into fve groups: one control group with no modifcations, and four groups containing increasing concentrations of chlorhexidine (1%, 1.5%, 3.5%, and 4.5 wt.%).Te researchers assessed the efcacy of these modifed resins in inhibiting bioflm formation by Candida albicans (C.albicans), Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae), and Streptococcus agalactiae (S. agalactiae) using a microtiter plate method.In addition, the mechanical properties of the resins, including fexural strength, surface roughness, and hardness, were evaluated.

Synthesis and Characterization of Chlorhexidine-Halloysite Nanotubes.
For the synthesis of chlorhexidinehalloysite nanotubes, 20% chlorhexidine digluconate solution (CHX) was diluted to 10% by dissolving it in doubledistilled water (2.5 ml CHX/2.5 ml H 2 O).Tis solution was then stored in darkness at room temperature.Following a published method [19,26], 1.25 g of HNTs were mixed with 5 ml of 10% CHX solution.Te mixture was vortexed for 20 seconds and then sonicated for 2 hours to ensure the CHX was evenly distributed throughout the HNTs (encapsulation).Te CHX-HNTs solution was then dried in a vacuum oven for an hour.Finally, the mixture was centrifuged twice at 3500 rpm at ambient temperature, with vortexing in between.Te resulting CHX-HNT nanocomposite was dried in an incubator and stored for further testing.

Characterization Methods.
Field emission scanning electron microscopy (FE-SEM) (Inspect F50, FEI, Holland) operated at an accelerating voltage of 2 kV was used to observe the morphological topography of the HNTs, and to ascertain the level of CHX-HNT dispersion within the PMMA.In order to determine the elemental composition of the samples more accurately, energy-disperse x-ray spectroscopy (EDS) spectra and elemental mappings of the prepared samples were performed.Raman spectroscopy was performed on a Raman spectrometer (532 nm preconfgured Raman spectrometer system) with an excitation wavelength of 532 nm to identify molecules and evaluate functional groups with intramolecular bonds of CHX and HNT before and after loading.Finally, X-ray difraction (XRD, DX2700BH, Haoyuan Instrument Co., Ltd., Dandong, China) with Cu K radiation at 40 kV, 30 mA, a 2 range of 20-100 °, and a step size of 0.02 °was used to acquire the difraction patterns of HNTs, CHX, and CHX/HNTs samples.Tis analysis aided by X'Pert Highscore Plus software allowed researchers to confrm the successful loading of CHX onto the HNTs by identifying their characteristic crystalline patterns.

Preparation of Acrylic Resin Nanocomposite Samples.
Te experiment utilized a self-polymerizing acrylic resin for both control and experimental specimens.Te acrylic resin was prepared according to the manufacturer's instructions, mixing the liquid and powder in a specifc ratio (2.5 g of polymer to 1 ml of monomer).For the experimental group, CHX-HNT fller was added at various percentages into the 2 International Journal of Biomaterials acrylic powder before mixing it with the liquid monomer.
Te mixture was then vortexed to ensure even distribution of the nanotubes.After thorough mixing to a doughy consistency, the resin was shaped in molds and cured using hydrofask at 2.2 bar with 40 °C water for 15 minutes to remove porosity and accelerate curing.Any excess material was removed with a standard tungsten carbide bur, and both surfaces of the samples were polished with sandpaper.Finally, the dimensions of each specimen were precisely measured with a micrometer, and all samples were sterilized using ultraviolet light for 30 minutes.Bioflm inhibition on the tested microbes was evaluated using a CV assay on a sterile 96-well microtiter plate, as described previously [27].In brief, three to fve microbial colonies were suspended in 4 ml of 0.85% (w/v) NaCl solution and compared with a 0.5 McFarland scale (equivalent to 1.5 × 10 8 CFU•ml −1 ).A colony of each isolate was inoculated into tubes containing 2 ml of brain heart infusion broth (BHIB) and incubated at 37 °C for 24 hours.Ten acrylic discs from each test group, measuring 0.5 mm thick and 6 mm in diameter, were immersed in wells of a 96-well microtiter plates containing a microbial suspension.Ten microtiter plates were sealed and incubated at 37 °C to create the microbial bioflms.After 24 h, the planktonic microorganisms with weak attachments were removed by rinsing for 1 minute with sterile saline (PBS; pH 7.4).Each specimen was stained with 0.1% (w/v) crystal violet for 20 minutes at room temperature before being rinsed twice with distilled water.For 20 minutes, the adhering bioflm was dissolved in 200 μL of 95% (v/v) ethanol, as seen in Figure 1.Te dye associated with the bioflm was solubilized in 33% (v/v) acetic acid, and the optical density of each group was measured with a spectrophotometer (Apel PD-303, Japan).Te bacterial inhibition percentage was calculated according to the following equation [28]: Percentage inhibition � 100 − OD620 of cells treated with CHX − HNTs OD620 of non − treated control cells   × 100  . (1)

Measurement of Physical and Mechanical Properties
2.7.1.Flexural Strength.Te fexural strength test was done according to ASTM D790-86.Rectangular acrylic bars measured (65 mm × 10 mm × 2.5 mm) in length, width, and thickness, respectively, were prepared for both the control and experimental acrylic specimens.All specimens were stored in deionized water at 37 °C for 48 hours prior to investigation in order to reduce the free residual monomer, and then removed and air dried.A Tinius Olsen 330-012 3-point fex testing machine with a displacement rate of 1.0 mm/min, and a span of 40 mm was used to perform the test.Five samples from each group were used to evaluate fexural strength using the following equation and expressed in MPa: where P is the load at fracture (N), I is the span length, b is the specimen's width (mm), and d is the specimen's height (thickness mm), while P/Y is the slope of the linear part of the stress-strain curve within the elastic portion.

Surface Roughness.
Te surface topography was verifed by using atomic force microscopy (AFM workshop/TT-2, USA) in tapping mode to reduce potentially damaging forces caused by contacts between the tip and the specimen.All samples were scanned with a 10 μm × 10 μm feld of view to ensure even surface coverage.First, acrylic specimens were stored in deionized water at 37 °C for 48 hours before being tested.Te specimens were placed on the device's stable stage, and the location of the tested area was selected.Te analyzer then traversed along each tested area at six diferent points.Topographical analysis was performed by importing the resulting AFM data fles into the software to calculate the average roughness (Ra) and compare it among specimen groups.All of the roughness data presented in this study is an average of fve scans.

Surface Hardness.
Following a standardized protocol [29], the surface hardness of the specimens was measured.First, the samples submerged in deionized water at 37 °C for 48 hours.Afterward, a durometer hardness tester (TR 220, International Journal of Biomaterials China, Shore D hardness) was used to determine the surface hardness.Tis involved pressing an indenter frmly and swiftly onto the sample surface and recording the highest reading.Tis measurement was repeated fve times on diferent areas of each specimen, and the fnal value reported was the average of these fve readings.

Characterization by EDX Spectroscopy.
In order to determine the elemental composition of the samples accurately, the EDS spectra and elemental mappings of the Te molecular formula of pure chlorhexidine gluconate is C 34 H 54 Cl l2 N 10 O 14 [30].From Figure 3, it can be seen that carbon, oxygen, nitrogen, and chlorine are the main elements detected for the CHX sample, which are the main elements in the chlorhexidine gluconate molecular structure.It should be noted that hydrogen cannot be detected with the EDS analysis because it has a very low atomic number and does not emit X-rays with sufcient energy to be detected by the EDS detector.Te EDS detector is designed to detect X-rays emitted by elements with higher atomic numbers, typically ranging from boron to uranium.In addition, hydrogen has a very low electron density, making it difcult to generate X-rays through interactions with the electron beam used in the EDS analysis.Furthermore, silicon, sodium, calcium, and magnesium elements were also detected (with a total atomic percentage less than 4%) as impurities.Elemental mapping (Figure 3(b)) shows that the elements are well distributed throughout the surface.
Te main elements found in the HNT sample were Si, O, and Al, indicating the presence of halloysite nanotubes with a chemical structure of Al 2 Si 2 O 5 (OH) 4 .Te theoretical atomic percentages for oxygen, silicon, and aluminum atoms (ignoring the hydrogen atom) are about 70%, 15%, and 15%, which are close to those derived from experimental data (about 72%, 14%, and 14% for O, Si, and Al, respectively) as shown in Figure 4(a).
Te analysis of the CHX-HNT nanocomposite confrms its successful synthesis.Tis is because the presence of elements from both original materials (Al, O, and Si from HNTs and C, N, and Cl from CHX) is detected.Furthermore, impurities such as magnesium, sodium, and calcium found in the initial CHX sample are absent in the fnal nanocomposite, indicating their removal during synthesis.Interestingly, the data reported in Figure 5(a) suggest the nanotubes (HNTs) are the dominant phase (10 times more than CHX), acting as a scafold for the CHX molecules.Tis is further supported by the mapping results (Figure 5(b)), where some areas lack carbon, chlorine, and nitrogen, implying these regions are the HNT nanotubes decorated by the CHX molecules on the outer surface.

Raman Spectroscopy.
Raman spectroscopy is used in chemistry to identify molecules and study chemical bonding and intramolecular bonds [31,32].Figure 6 shows the Raman spectra of CHX, HNT, and CHX-HNT samples in the Raman shift range of 0-1800 cm −1 .
In the Raman spectrum of CHX, a broad band can be seen in the Raman shift range of 800-1600 cm −1 with  XRD is a well-established technique for determining the arrangement of atoms within a crystalline material and can reveal any structural modifcations that occur during the formation of the nanocomposite (Figure 7).It can be observed that the XRD pattern of the CHX sample has a predominantly amorphous profle with a broad peak in the 2θ range of 10 °-50 °, as was seen previously for this structure [37,38].For the XRD pattern of the HNT International Journal of Biomaterials sample, by coinciding the peaks with the reference diffraction patterns using the difractor software, it was found that the meta-halloysite phase with the chemical formula of Al 2 Si 2 O 5 (OH) 4 and the JCPDS No. 00-029-1487 reference code was the main crystalline structure in the sample.According to the reference card, the crystalline phase has a hexagonal crystal structure with a space group of P. In this pattern, the difraction planes are (001), (100), (002), (110), (003), (210), and (300) appeared at 2θ � 11.6 °, 19.9 °, 24.2 °, 35.9 °, 38.2 °, 55.2 °, and 62.4 °, respectively.Te peak at 2θ � 26.5 °can be attributed to the (011) plane of quartz crystalline phase, which usually present as an impurity in halloysite nanotubes [39].Te crystallite size of the sample was calculated using the Scherrer equation [40]: where D is the mean dimension of the homogeneous crystallites along an axis perpendicular to the hkl system considered, β is the width of the difraction profle (the full width at half maximum, FWHM), K is the Scherrer constant approximation close to unity, and λ is the corresponding Xray wavelength (units are 2θ scale in radians).According to the equation, the crystallite size for the HNTsample based on the sharpest peak (at 2θ � 19.9 °) was about 11 nm.Te XRD analysis of the CHX-HNT nanocomposite indicated the preservation of the HNT's crystalline structure, evidenced by the presence of difraction peaks at similar positions to pristine HNTs.However, these peaks exhibited broadening, suggesting a decrease in crystallinity likely due to the incorporation of the amorphous CHX phase, as previously observed by Wu et al. [41].

Bioflm Inhibition
Test.Microbiological analysis revealed bioflm formation by all isolates tested.Table 1 presents the results of a normality test and descriptive statistics for the bioflm inhibition efcacy of CHX-HNTs.Te Shapiro-Wilk test confrmed normal distribution of all data.Notably, CHX-HNTs demonstrated signifcant antibioflm activity against all investigated strains, including C. albicans, S. aureus, S. pneumoniae, and S. agalactiae.
Te statistical analysis of data presented in Tables 2 and 3 revealed a dose-dependent inhibitory efect of CHX-HNTs on bioflm formation by all investigated pathogens.Te application of CHX-HNTs at concentrations ranging from 1 wt.% to 4.5 wt.% resulted in a statistically signifcant decrease in bioflm formation (p < 0.05), as seen in Figure 8. 4 summarizes the mechanical properties of the control and experimental groups.Te statistical analysis revealed no signifcant increase (p > 0.05) in fexural strength upon incorporation of CHX-HNTs into the acrylic resin.Interestingly, the unmodifed PMMA control exhibited the highest fexural strength among all groups, although this diference was not statistically signifcant.Tese results suggest a potential negative impact of CHX-HNT fller on fexural strength, with the group containing the highest concentration (4.5 wt.%) displaying the weakest fexural strength compared to others.

Mechanical Properties. Table
Consistent with the fexural strength results, CHX-HNT incorporation resulted in a slight, yet statistically insignifcant (p > 0.05) increase in surface roughness of the acrylic resin (Figure 9).Tis trend suggests a potential correlation between fller concentration and surface roughness.However, the hardness measurements revealed no signifcant diference between the control and experimental groups, indicating minimal impact of CHX-HNTs on this mechanical property.

Discussion
CLP patients' oral fora has an increased prevalence of potentially pathogenic microbial colonization, including Candida species, S. aureus, S. pneumonia, and S. agalactiae that can lead to serious infections and pose challenges during postsurgical healing due to the immunocompromised state of CLP patients [42].To mitigate these risks, meticulous oral hygiene practices and regular preventive dental care are crucial components of CLP treatment protocols [41,43].However, current methods for cleaning removable acrylic dentures, often relying solely on mechanical and chemical means, have proven inadequate in eliminating these contaminating microorganisms [44][45][46].Terefore, there is a growing demand for an afordable, easy-to-implement strategy for preventing the accumulation of dental plaque on these prosthetic devices [47][48][49].In an attempt to combat microbial adhesion on acrylic resin, this study implemented a novel approach by incorporating CHX-HNT nanocomposite fllers.Tis study used a novel method to modify acrylic base materials by adding CHX-HNT fllers in an attempt to potentially discourage microbial adhesion.Te frst objective of this research was to develop and characterize a CHX-HNT nanocomposite, and second, to evaluate the impact of integrating this nanocomposite at various  As an alternative to more expensive nanofllers such as carbon nanotubes (CNTs), HNTs have gained growing research interest due to their lower cost in the manufacturing of high-performance materials such as polymers [50].HNTs are characterized by a hydroxyl with a lower level of density that permits smooth difusion in a polymer matrix compared to other nanoclays.Te unique high aspect ratio tubular structure and the polarity of the tubule surface show that the HNTs are suitable candidates to secure generous dispersal in the polymer matrices [18,23,51].Due to the critical role of appropriate nanotube morphology in both functionality and successful nanocomposite synthesis [52], a comprehensive characterization of the HNTs was undertaken.Tis involved employing various structural analysis techniques such as FE-SEM, EDS, Raman spectroscopy, and XRD.Our analysis confrmed that the morphology and size of the HNTs matched those reported in previous studies [21,53,54].In addition, these techniques successfully verifed the homogenous distribution of CHX-HNTs within the acrylic matrix, with minimal instances of localized nanofller aggregation.
CHX was selected among other antimicrobial reagents as it has established itself as the leading agent for chemical plaque control.CHX exhibits ability to inhibit adherence of microorganisms to a surface thereby preventing growth and development of bioflms [55,56].Numerous studies have documented its efcacy in inhibiting microbial growth, dental plaque and gingivitis, particularly when used as a mouthwash [55][56][57].Notably, Redding et al. demonstrated that CHX exhibited the highest rate of inhibition against C. albicans bioflm formation on denture materials compared to other antifungal drugs like Amphotericin B and nystatin [55].Our investigation confrms the efectiveness of CHX-HNTs as an antiplaque agent.We observed bioflm inhibition rates exceeding 98% against C. albicans, S. pneumoniae, and S. agalactiae.Furthermore, a statistically signifcant decrease in bioflm formation was observed with increasing concentrations of the CHX-HNTs nanofller.International Journal of Biomaterials Considering the mechanical properties of the nanofllermodifed acrylic, research fndings revealed that while the incorporation of CHX-HNTs in acrylic resin provided satisfactory inhibition of microbial adhesion, it had no signifcant efect on the materials' properties (fexural strength, roughness, and hardness).A slight, dose-dependent decrease in fexural strength and a corresponding increase in surface roughness were observed.Tis can be attributed to the inherent hydrophilicity and high surface area of halloysite nanotubes, which may promote fller agglomeration at higher  International Journal of Biomaterials concentrations [22,23].Tese agglomerates can act as stress concentrators within the composite, compromising strength and limiting elastic modulus enhancement in dental resins [64,65].Te observed increase in surface roughness is likely due to the presence of nanoparticles on the acrylic specimen surface [66].Similar results were reported by Feitosa et al. who explored CHX-HNT applications in dental adhesives [67].Teir study demonstrated that CHX-loaded nanocomposite adhesives exhibited sufcient antibacterial activity while maintaining essential mechanical properties, including the degree of conversion, microhardness, water sorption, and biocompatibility.However, it is important to note that Barot et al. observed contrasting results [22].Teir study found that incorporating CHX-HNTs into dental resin composites actually enhanced both mechanical properties and antibacterial activity.Tis diference highlights the potential infuence of the specifc material being integrated with CHX-HNTs.
Te incorporation of HNTs into PMMA resin can improve hardness by causing the HNT fller that is not strongly adhered to the resin to disperse or dissociate under load, creating frictional force that allows stress distribution across the matrix cracks and thereby increasing the material's resistance to indentation [66][67][68][69].Te FTIR study results confrmed the absence of a chemical interaction between the acrylic resin and the CHX/HNTs fller, validating the hardness results found because the presence of the CHX/HNTs caused no statistically signifcant change in the acrylic resin property.
4.1.Limitations.Tis is an in vitro experimental study.Tus, the complexity of the bioflm community, the lack of knowledge regarding the identity and abundance of each bioflm resident, and the lack of salivary and host factors hinder our ability to precisely replicate the intraoral scenario.Te authors acknowledge that not all processes during bioflm formation can be adequately simulated with a laboratory approach; thus, the results of this study should lead to further in situ and in vivo studies to validate their use.

Strength of the Study.
Tis study set out to strengthen the early oral health preventative and maintenance programs in CLP patients.Our research will add to the existing body of knowledge by being the frst to construct a CLP infant orthopedic appliance with an antimicrobial property by successfully incorporating chlorhexidine-halloysite nanotubes into a PMMA resin.Results of this study showed that incorporation of CHX-HNT into PMMA-based resins allowed for its subsequent release from the matrices, which enabled the resins to inhibit C. albicans and bacterial bioflm without afecting the appliance's mechanical properties, demonstrating promising antimicrobial activity.

. Conclusions
Te CHX-HNT-modifed acrylic resin exhibited remarkable efcacy in inhibiting bioflm formation while maintaining satisfactory mechanical properties.Tese fndings suggest its promise as a novel preventive strategy for managing infectious complications in cleft lip and palate infants, potentially improving disease control in this patient population.

3. 1 . 1 .
Field Emission Scanning Electron Microscope.FE-SEM images at diferent magnifcation powers of HNTs before CHX loading shown in Figure2confrmed the tubular geometry of HNTs with a relative heterogeneous particle size distribution having a mean diameter of approximately 70 nm and 400 nm in length.While FE-SEM images of CHX-HNT nanocomposites after acrylic incorporation confrmed the fair dispersion with few aggregations of the nanofller within the PMMA acrylic matrix.

Figure 4 :
Figure 4: (a) EDS and (b) elemental mapping results for the HNT sample.

Figure 8 :
Figure 8: Efect of diferent percentages of CHX-HNT incorporation of acrylic samples on microbial bioflm formation.

Table 1 :
Descriptive statistics of the bioflm inhibition ability of the CHX-HNTs.

Table 2 :
ANOVA test of the bioflm inhibition test.

Table 3 :
Inferential statistics of the bioflm inhibition test using Tukey's test.

Table 4 :
Descriptive and inferential statistics of mechanical properties of diferent CHX-HNT acrylic samples.