Alternative dental impression fillers made of nanorod glutinous rice flour particles through precipitation

In this work, nanorod particles were synthesized from a locally available source, glutinous rice flour, using sodium hydroxide (NaOH) through a simple precipitation process. The synthesized nanofillers were then presented as an alternative organic filler for dental impression application to support the making of a diagnostic and working model. Dynamic Light Scattering, Scanning Electron Microscope, Fourier Transform Infrared Spectroscopy, x-ray Diffraction, Energy Dispersive Spectroscopy, Thermogravimetric Analysis, and Differential Scanning Colorimeter were used to characterize the fillers. The particle size measurement, morphology interaction, and composition of glutinous rice flour nanorod particles were also investigated. The cell viability using 3T3L1 cells was assessed to determine the safety of nanorod particles using the MTT assay and trypan blue solution. All treated samples exhibit a change in particle morphology from polyhedral to rod. Additionally, a decrease in crystallinity, dehydration, and gelatinization temperature was observed. The functional group interacting with sodium hydroxide also changes slightly after size reduction. The samples treated with 3000 centrifugation speed without surfactant addition showed changes from the control sample’s 3931.71 nm to the smallest average width particle size of 73.26 nm with an average length of 865.15 nm. All of the treated samples with NaOH and NaOH-surfactant additions met the non-cytotoxicity acceptance criteria in the range of 73.54%–99.58%, according to the cell viability results. The incorporation of 15 wt% of the synthesized nanorod fillers resulted in a 20 μm continuous line as the impression materials specimen, yielding a satisfactory detail reproduction test result. In conclusion, nanorod particles with biocompatible properties have been successfully manufactured and can potentially be used in the future as an alternative dental impression filler materials.


Introduction
It is common practice to disperse fillers in a matrix to enhance a material's characteristics. Dental impression materials are one of the uses of fillers in dentistry. To create the model, dental impression materials that serve as a negative representation of the anatomical form and structure of oral tissues are required. This model will be a true-to-scale representation of the soft tissues and extra-and intra-oral structures, including teeth, that will be utilized as a diagnostic tool and construction tool for prosthetic devices that should be accurate for patients. There are elastic and inelastic types of impression material that can first be molded against the tissues before setting [1,2].
The hydrocolloid and elastomer are forms of elastic impression materials that can be easily stretched and revert to their original shape if unstressed. Dental elastomers come in a variety of viscosities, ranging from extralow, low, medium, and heavy to putty, depending on the amount of filler in the mixture. Various fillers, including borax, silica, diatomaceous Earth, wax, rubber, clay, and certain inert materials are added to regulate the consistency, strength, and rigidity of dental impression materials, including hydrocolloid types. These consistencies are useful in creating an accurate reproduction because they allow a heavy body to keep the impression on a tray, while a light body could replicate the gingival crevices and interproximal region [1][2][3]. However, various type of consistencies that are typically found in elastomers are relatively expensive compared to dental alginate, which is one type of irreversible hydrocolloid impression materials. When compared to other elastomeric impression materials, these alginate impressions do not exhibit dimensional stability and cannot accurately recreate finer details or surface details. The influence of the filler size and composition, which typically contain about 7.94-14.59 μm filler size [2,4,5], may be responsible.
Nanoparticle fillers are widely used in biology, the chemical industry, and medicine, and they offer potential benefits for dentists in accurately restoring patients' teeth by improving flow and detail when creating dental impressions of narrow or thin sections in oral structures [6][7][8]. As previously mentioned, inorganic fillers are the most commonly used reinforcement for dental impression materials. However, it is important to carefully consider the possible harmful consequences of alginates, such as the inhalation of aerosols, which can lead to silicosis with prolonged exposure and pose a risk to dentists or dental assistants [4,[9][10][11]. Silica nanoparticles, which are less than 3 μm in width and over 20 μm in length, are particularly concerning for inducing cytotoxicity [4,11]. While manufacturers have created 'dust-free' alginate by adding glycol additives to prevent aerosol generation [3], it is important to be aware that improper handling of the impression materials could still cause silicosis.
As an alternative to inorganic particles, organic fillers derived from glutinous rice flour are being considered to prevent lung disease. In our previous work [12], we were able to reduce the size of glutinous rice flour particles to about 259.5 nm. However, further reduction to nano-sized particles is still required. One way to achieve this is through precipitation, which involves adding a solution dropwise to a non-solvent [13]. Sodium hydroxide is commonly used to dissolve starch as part of the starch modification process. It reacts with the hydroxyl groups in the starch molecule and become an alkoxide [14][15][16]. Therefore, it is expected that the addition of sodium hydroxide will aid in the precipitation during the production of these glutinous rice flour nanoparticles. Since small particles are prone to agglomeration, alternatives to using a surfactant to maintain the size of the particles are being considered. Previous studies by Chin et al and Xiao et al have shown the advantages of smaller nanoparticles in rice starch and sago [14,17].
Therefore, the aim of this research is to use a simple and secure precipitation method to produce organic nanoparticle filler from glutinous rice flour in order to produce the desired particle sizes for accurate dental impressions. Dynamic Light Scattering (DLS), Scanning Electron Microscope (SEM), Fourier Transform Infrared Spectroscopy (FTIR), x-ray Diffraction (XRD), Energy Dispersive Spectroscopy (EDS), Thermogravimetric Analysis (TGA), and Differential Scanning Colorimeter (DSC) were then used to characterize the fillers that were produced. This work also looked into morphology interaction models, investigated biocompatibility properties through cell viability, and evaluated the detail reproduction using the alternative fillers as the impression materials.

Materials
Glutinous rice flour was purchased from a particular store (PT Sungai Budi 2016, RosebrandTM, Jakarta, Indonesia). Analytical-grade Tween80 (Merck), sodium hydroxide (NaOH), ethanol, and bi-distilled water were used as received. In 100 μl of culture medium in microplates, 10,000 cells of the 3T3L1 fibroblasts cell line (ATCC CL-173) were seeded per well (tissue culture grade, 96 wells, flat bottom). Analytical grade balanced salt solution, trypan blue solution, MTT reagent, and dimethyl sulfoxide (DMSO) were also used in the study.
Food grade bovine gelatine, xanthan gum, and sodium tripolyphosphate were purchased from local store (Kimiamart and Dunia Bahan Kue, Bandung, Indonesia). Plaster of paris was used as dental gypsum without further modification. Dental alginate (Cavex, Holland) was used as the control sample for dental impression materials, and the mold was prepared in accordance with BS EN ISO 21563: 2013 for the detail reproduction test.

Preparation of glutinous rice flour/NaOH solution
Bi-distilled water and 0.5 M NaOH were combined to make solution at room temperature while being magnetically stirred. Glutinous rice flour (0.1 gr ) was then mixed with the 0.5 M sodium hydroxide solution and stirred for an hour at a temperature of 62°C to create the glutinous rice flour solution.

Synthesis of nanoparticles precipitation-based
An aliquot of glutinous rice flour/NaOH solution (1 ml) was added dropwise into 20 ml of ethanol and stirred for 30 min to perform the instant precipitation method [12,17]. The mixture was then subjected to a 30-minute centrifugation. Following that, the samples were divided into sample A, centrifuged at 3000 rpm, and sample B, centrifuged at 6000 rpm. All samples were completely washed with ethanol three times and dried in a 37°C incubator to remove any residual ethanol. Similar procedures were carried out for sample C (centrifuged at 3000 rpm) and sample D (centrifuged at 6000 rpm), except that a surfactant was added. Initially, 1 ml of 4% Tween80 surfactant was added, followed by 1 ml of the glutinous rice flour/NaOH solution for instant precipitation into 20 ml of ethanol, and stirred for 30 min. The colloidal phase's particle size was determined using the Horiba SZ-100 Nano Particle Analyzer (Horiba, Japan) in Dynamic Light Scattering characterization.
The morphological structure of nanorod particles has been studied using a Scanning Electron Microscope (JEOL-IT300). The average particle width distribution was also analyzed. The sample suspension was poured onto a cover glass and allowed to dry in a 37°C incubator. Prior to SEM measurement, a thin layer of gold was applied to the sample. For x-ray Diffraction, a copper tube with a wavelength of 1.5406 Å was used in the x-ray diffractometer (RigakuSmartLab, Japan), operating at 40 kV and 30 mA. The relative crystallinity (RC%) of the control and treated samples was determined using XRD. The calculation of RC% followed the method described by Ahmad [13], where RC% is the crystalline area divided by the sum of the crystalline and amorphous areas, multiplied by 100.
To identify the functional group of the materials, Fourier Transform Infra-red Spectrometer (Prestige 21 Shimadzu, Japan) was employed. The FTIR spectra were produced by measuring 40 times at a resolution of 4 cm −1 at a wavenumber of 4000-400 cm −1 .
The EDS was conducted using SEM (JEOL-IT300) to examine the strong signal of the peak at 1 keV for the absorption and to verify the production of filler through elemental mapping.

Thermal properties
The TGA, thermogravimetric measurement was performed using Hitachi STA7300, Japan. According to the previous work, the TGA measurement was carried out under a nitrogen atmosphere with a gas flow of 20 ml min −1 . Approximately 3 mg of samples were loaded into an aluminum pan for the analysis. The heating process was carried out from 45°C-450°C with a 10°C min −1 rate [12,18]. The degree of gelatinization was assessed using the DSC measurement conducted with DSC 214, Polyma Merck Netzsch. Prior to examination, the dry basis samples (approximately 3-8 mg) were sealed hermetically in an aluminium pan. The samples were then heated at a rate of 10°C min −1 in the temperature range of 25°C to 120°C. The empty aluminium pan served as the reference for the analysis.

Cell viability
Direct contact and cell counting were used to determine the viability cell number. A balanced salt solution was used to create the cell suspension, and then 0.5 ml of 0.25% Trypan Blue Solution (w/v) was added to a test tube. The 2.5 ml cell suspension and 0.5 ml balanced salt solution were added and well mixed. A small amount of the mixture was then employed to improve Neubauer chambers. The cells were then counted in the 1 mm center square and the four 1 mm corner squares, which were stained blue to indicate non-viable cells. The following formula was used to determine the cell viability. Cell viability (%) is equal to the total number number of viable cells (unstained) divided by the total of cells (stained and unstained cells) multiplied by 100 [19].
For the 3T3L1 cells, they were seeded in 96-well plates at a density of 10,000 cells per well for 24 h. Following that, aliquots of 10 μl of MTT solution (0.5 mg ml −1 ) were added and incubated at 37°C for another 4 h before aspirating the medium and kit. Meanwhile, 100 μl DMSO aliquots were added to each well. The dye was dissolved by shaking it until the complete dissolution of formazan crystals. The absorbance was measured spectrophotometrically at 550 nm using a microplate reader. The darker the solution, the greater the number of viable, metabolically active cells [20,21].

Detail reproduction of the impression materials
The replication of sample A method was conducted to prepare glutinous rice flour nanorod fillers for the evaluation of detail reproduction. The nanofillers were incorporated into a mixture of glutinous rice flour and bovine gelatine at three different concentrations: 5 wt% (group 1), 15 wt% (group 2), and 25 wt% (group 3).
Dental alginate and a mixture of glutinous rice flour and bovine gelatine without nanofillers were used as control samples. Another ingredients, such as ±0.3-0.5 wt% xanthan gum, ±2-2.5 wt% STPP, and ±16 wt% gypsum were also used in glutinous rice flour and bovine gelatine mixtures. The preparation process involved adding the fillers to a powder composition in beaker glass, followed by the addition of a bovine gelatine solution. The mixture was then mixed with a spatula for 30 seconds in all groups, except for dental alginate, which was mixed in a rubber bowl using a spatula in the usual manner.
To simulate the oral temperature, the impression in the mold was immersed in a water bath at a temperature of 35°C until the setting time was completed. The detail reproduction test was conducted using a mold that contained three lines measuring 20 μm, 50 μm, and 75 μm, following the guidelines outlined in the BS EN ISO 21563:2013 standard [22]. After the impression was made, the lines were examined with a stereomicroscope (10x) (Nikon SMZ800). The detail reproduction of a 20 μm line, which was continuously reproduced in two of three specimen, was considered satisfactory.

Size and morphological determination
The particle size reduction of native glutinous rice flour after treatment with NaOH or NaOH with surfactants is shown in table 1.
The measurement displays an intensity weighted mean size from the peaks cumulant analysis as z-average. Each polydispersity index (PdI) provides information about the homogeneity of the samples. Although sample B has the lowest mean and z-average value, sample A exhibits the lowest PdI value (0.180) and smaller particle size compared to samples C and D. As shown in figure 1, Sample C has two distribution mean peaks.
The treatment of the samples resulted in a reduction in size according to the DLS results, although they did not reach the nano-sized particle range. Thus, SEM characterization was performed to confirm these findings. Figure 2 shows SEM images of the morphological changes from polyhedral native glutinous rice flour as control sample transformed into rod-shaped particles in the treated samples. The SEM images of the treated samples  revealed the presence of smaller particles, indicating optimization through the precipitation method, with rod particles exhibiting a nano-sized width. Among the treated samples, sample A exhibited the smallest particle width (73.26 nm) and length (865.15 nm) of the treated samples. In contrast, sample B displayed wider particles (195.96 nm) with visible agglomeration indicated by the orange arrow. Sample C had longer rod particles (1102.09 nm length), consistent with the DLS mean particle size measurement represented by the green rectangle. On the other hand, sample D exhibited longer rod particles (1835.75 nm), but the DLS mean particle size measurement indicated shorter length particles compared to sample C. This discrepancy in length measurement between SEM and DLS might be attributed to the presence of a bent particle, as indicated by the purple circle and the yellow arrow.
Furthermore, the zeta potentials of treated samples A-D were found to be −8.5 mV, −32.3 mV, −28.8 mV, and −35.6 mV, respectively. These zeta potential values provide insights into the long-term stability of nanoparticles. The prediction of incipient instability in the nanoparticles was confirmed by the smallest zeta potential observed in sample A (−8.5 mV). Sample B and D, on the other hand, exhibited zeta potentials greater than −30 mV, indicating moderate stability. The presence of Van der Waals force attraction, which leads to larger particles, may explain this moderate stability [13].

XRD and FTIR analysis
The diffractogram in figure 3 shows the relative crystallinity analysis, which was determined by XRD. The measurement was made by comparing the nearly identical diffraction peaks of treated particles to control samples, which had the nearly identical structure of rice flour with diffraction peaks at 2theta 15.8°, 17.1°, 18.3°, and 23.1° [12,18]. The treatment with NaOH detected the change of glutinous rice flour crystalline conformation, which promoted not only main peaks of the glutinous rice flour but also identified additional peak formation originating from sodium hydrogen carbonate. These additional peaks could be detected overlapping at 2theta 18.3°and also strong peak at 2theta 27.7°, 29°, and 34.6° [23]. In comparison to the control sample (21.51%), the relative crystallinity of samples A-D decreased to 10.03%, 7.52%, 9.89%, and 9.24%, respectively.
The XRD diffractogram reveals that additional peaks of NaHCO 3 appeared as a crystalline phase with much higher crystallinity compared to glutinous rice flour. The glutinous rice flour only showed by the small in XRD due to its more amorphous phase. Nevertheless, the FTIR spectra can clearly confirm the presence of glutinous rice flour from its characteristics peaks as depicted in figure 4.
Since sodium hydroxide was used in the precipitation method, the functional group depicted in the FTIR spectra has absorption peak changes at 3285 cm −1 and 2850 cm −1 of -O-Na+, which is a typical combination of sodium hydroxide and C6 unit of glutinous rice flour [24]. The special spectra also shown the peak at ∼1450 cm −1 (pointed by red arrows) as the absorption peak of sodium content [25] that remained in all treated samples, which are different from control sample. The rest of the functional groups, however, are almost identical to the native glutinous rice flour, shown by peaks at ∼3400 cm −1 for OH-and ∼1630 cm −1 for -CHO; as assigned to crystal water in the flour [26]. The treated sample demonstrated a change in hydrogen bonding as sodium present to bind the polymers. The study also compared the FTIR peaks of glutinous rice flour as the control sample with sodium hydroxide. Figure 4(b) shows the functional group changes in 800 cm −1 of NaOH, followed by the strong prediction of the -O-Na+ combination of the treated samples.

Element analysis
EDS confirm the presence of C and Na that could support the FTIR spectra with element analysis as shown in figure 5. Table 2 shows the percentage of atom element pointed at nanorod particle of all treated samples composed of C, carbon, and O, oxygen atoms dominantly originating from the glutinous rice flour and Na, natrium atom might resulted from the precipitation method that used sodium hydroxide. Previous study by Chin, et al in 2014 reported alkalization between starch and sodium hydroxide that transformed the molecule into sodium starch alkoxide (Starch-O-Na) [14]. This finding supports our discovery that the percentage of Na atoms is lower compared to the atoms C and O, which can form bonds on the outside surface of the molecule. This observation is consistent with predicting the potential structure of the resulting rod particles [27], as illustrated in figure 6.

Thermal analysis
TG, Thermogravimetric; DTG, Derivative Thermogravimetric; and DSC were used to analyze the thermal properties of polyhedral control samples and nanorod particles from treated samples. The thermogravimetric analysis of the control sample reveals only one main loss step, as shown in figure 7. However, all treated samples that were precipitated with sodium hydroxide show stages of decomposition. The maximum DTG peaks represent the maximum rate of mass loss [28].
The thermograms of the gelatinization of these native and nanorod particles of glutinous rice flour are based on DSC characterization from figure 8, which shows peak temperature results. The first peak temperature after treatment was lower than the control and possessed a second peak temperature from the alkali used in the precipitation method [29].

Cell viability analysis
The cytotoxicity of treated samples was evaluated using the Trypan blue solution and the MTT assay, as depicted in figure 9, with ANOVA statistical analysis of mean and SD, standard deviation. The Trypan blue test results in significant mean results (p< 0.05), and the trend shows that 48 h of exposure time is higher than 24 h.
According to Jiang et al in 2017, all samples in this study are considered to be non-cytotoxic [30], but sample A has the highest cell viability percentage of MTT Assay, with 82.30% in 24 h and 99.58% in 48 h of exposure. Based on the morphology visualization of the staining cells, the Trypan blue solution has a higher cell viability percentage than the MTT assay. Because of their biocompatibility, these two methods produce similar results in terms of non-cytotoxicity of nanorod particles synthesized from glutinous rice flour, which could be used as an alternative to dental impression fillers.

Detail reproduction analysis
The reproduced 20 μm, 50 μm, and 75 μm lines of the native and nanorod particles fillers of glutinous rice flour in impression materials are based on detail reproduction test were shown in figure 10, which compared also with the dental alginates line results. The pictures were taken from the microscope, which were from the camera embedded in microscope directly and also from the phone camera. The satisfactory result of the detail reproduction of a 20 μm line (the center among three lines) that reproduced continuously in all specimens are in group 2, which incorporated 15 wt% nanorod fillers as the alternative fillers in the impression materials.

Discussion
Organic nanoparticles, which are typically derived from polymer materials, are widely used in biomedicine due to their biodegradability and non-toxicity. Glutinous rice flour, as an organic polymer source, could be useful in this matter [31,32]. Nanoprecipitation is a technique for preparing small particles that has the advantages of obtaining nanoparticles by gradually adding polymer solution to a non-solvent with a simpler, less toxic material use, and low cost [6,33]. A general definition of a nanomaterial is one that is smaller than 100 nm in one dimension and could be categorized as nanorod particles are those with aspect ratios greater than one but less than 20 [34,35]. All of our treated samples (Samples A-D in figure 2) led in nano-sized and rod morphology of the glutinous rice flour particles that were evenly distributed throughout all samples.
The particle's rod morphology could be explained by the disruption of glutinous rice flour structure aided by sodium hydroxide dissolution. The sodium hydroxide reacts with hydroxyl groups to form sodium starch alkoxide after breaking the intermolecular and intramolecular hydrogen bonds between water and glutinous rice flour molecules. This treatment is also referred to alkaline modified starch [14,15,36]. The resulting nanorod particles are based on amylopectin, which has a length and branch framework that allows for double helix conformations [37,38], and are shielded on the outside by sodium starch alkoxide binding. Figure 6 depicts a potential illustration of the proposed morphology, which is supported by the EDS element atom analysis in      figure 4, in which sodium ions substituted the attached hydrogen at those secondary alcoholic hydroxyl groups in C2 and C3 positions [36].
Tween80 was used as a surfactant on samples C and D, resulting in larger particles than samples treated without surfactant. This surfactant is more hydrophilic than Span80 or hexadecyl (cetyl) trimethylammonium bromide (CTAB), which interacts with the molecules more strongly [39], but it also causes the amylopectin molecules to bind with the surfactant first before precipitating, resulting in large aggregates. Aside from that,   despite being nano-sized width samples, the use of surfactant addition in samples C and D still has wider dimension than sample A, which is likely due to the use of a high concentration of the surfactant, which is 4% [17,40].
Sample A has a smaller average width but a longer z-average length than sample B, which has a higher centrifugation speed. On the contrary, with the addition of surfactant, the width of sample C becomes wider than that of sample D, while the average length of sample D becomes longer than that of sample C. This finding is similar to that of Gavory et al (2010), who discovered that the average particle diameter of Polylactic Acid (PLA) with stabilizer decreased systematically from 3000 to 5500 rpm. As a result, the speed of centrifugation extracted aggregation. Meanwhile, the centrifugation speed was used to increase the average particle diameter of PLA without a stabilizer [41].
The XRD results show that the crystallinity of glutinous rice flour changes as it transitions to a more amorphous phase. The determination of crystallinity of native glutinous rice flour is tricky because of the small crystal size and the role of hydration [37]. As a control sample, the crystallinity of native glutinous rice flour is 21.51%, decreasing to 10.03%, 7.52%, 9.89%, and 9.24% for treated samples A-D, respectively. Sample B also shows the decrease in intensity and an increase in the half peak width that indicated the formation of a more amorphous structure than samples A, C, and D. The decrease in relative crystallinity can be attributed to an increase in amorphous region of the glutinous rice flour after size reduction that could contributed from the destroyed of crystal structure by NaOH treatment, which makes sample B has the lowest relative crystallinity than other samples. Although the crystallinity of all treatment samples has changed, the resulting nanorod particles may be useful in improving detail reproduction of 1-25 μm and dimensional accuracy in dental impressions, which are typically available in 7.94-13.16 μm filler on the commercial market [5,42]. The potential advantages of nano-sized and rod particles are that they can not only flow to a narrow area such as the subgingival part of oral structures, but their morphology can also reinforce the impression so that it has a good tear strength when the clinician takes the impression from the patient's mouth for diagnostic treatment.
The diffractogram from XRD result reveals additional peaks besides the main peaks from native glutinous rice flour, which have strong peak at 2theta between 25-45°. The result also show peak at 2theta 18.3°was identified higher than the control sample. This could indicate an overlapping peak that possibly as a result of starch and sodium interaction and also additional peaks originating from sodium hydrogen carbonate [23]. Since the main peaks in XRD diffractogram considered low compared to the additional peaks, which indicated the amorphous results, then our study confirmed the starch and sodium combination using FTIR characterization. The spectra shows absorption peak changes at 3285 cm −1 and 2850 cm −1 for the combination and also at 1450 cm −1 that indicated sodium content [24,25]. The EDS result also could possibly support the XRD diffractogram's higher diffraction peak around 18.3°. Although it is not good for organic materials, but crystalline structure could be collected well using an XRD, while EDS shall detect the elemental composition or atomic concentrations within a compound across a surface and at a specific point. The higher percentage of C atom element indicate the assurance of the source [43], which mainly provided by glutinous rice flour and Na would only has a role as to support the bonds of the molecule resulted, which make sense in providing the morphology of nanorod particles.
The thermogravimetric analysis of TG/DTG found nearly identical temperatures on samples A-D. Despite this, it did not have the same temperature as the control sample because it did not include the sodium hydroxide addition. The first loss associated with gelatinization began at approximately 80°C-90°C is due to the departure of physisorbed water that generate penetrated water into the crystalline areas and dropping the required energy. Then, it was followed by the second loss at approximately 230°C-250°C, which was acting as the main decomposition of glutinous rice flour as the organic matter. The thermal stability of nanorod glutinous rice particles as biopolymers from chemical treatment with an alkali-like sodium hydroxide decreased with increasing hydrolysis [44]. Through DSC characterization, the gelatinization endotherm peak temperature is determined at Tp1, and the melting endotherm peak temperature is determined at Tp2. The sodium hydroxide was considered to promote molecule realignment; it could be referred to as a plasticizer. It could also solubilize glutinous rice flour in the amorphous region of gelatinization, promoting hydrogen bond scission in the crystalline region. This exceptional ability also aids in the mobility of molecules during gelatinization [29].
According to the DSC curve, the gelatinization temperature of the treated sample becomes broader, shallower, asymmetric, and shifts to a lower temperature than that of the control sample. The sample with the lowest gelatinization peak temperature was sample A, in which sodium hydroxide stimulated gelatinization by breaking intermolecular hydrogen bonds in the crystalline region, causing new realignment without being interrupted by any other additives, such as surfactant in samples C and D. Meanwhile, sample A's gelatinization and melting peak temperatures are still lower than sample B's due to a smaller width size, which is attributed to a faster mechanism for water [12,18,45], and sodium penetration to make the alignment. Thermal properties can be used to calculate the time required to manipulate the dental impression reinforced with this nanorod particle filler.
The cytotoxicity test assessed biocompatibility for medical devices that used the useful and relatively simple cell culture technique. This test will be used to determine whether the materials will support as a dental impression filler [46,47]. The test was conducted through direct contact and was left for 24 and 48 h exposure. Because the cell viability test using Trypan blue solution was performed qualitatively by cell counting, this study performed two cell viability tests to ensure the results were consistent. Furthermore, the MTT assay was carried out as an absorption using a spectrophotometer.
When compared to the cell control, the 24 h exposure time resulted in a lower percentage of viable cells. Sample D, on the other hand, has the lowest (73.54%) on both viability tests, while sample A (82.30%) has the highest MTT assay result and sample C (85.33%) has the highest Trypan blue viability test result. The lower cell cytotoxicity observed in sample D is most likely due to a surfactant (Tween80) that does not bind properly and produces larger particles than sample C. This finding is corroborated by Pithon et al study in 2010, which discovered Tween80's high toxicity to biological membranes. The surfactant component shall stimulate protein secretion in order to alter the cell surface wall [48]. Although sample C has the highest viability test result, it was only counted using a qualitative method in the Trypan blue viability test. In contrast, the surfactant in this sample most likely binds to the molecule properly. Surprisingly, sample A had the highest percentage of viable cells in the MTT assay after 48 h of exposure (99.58%), nearly matching the viability of the cell control (100%). Biocompatibility testing using cell viability revealed that all materials tested met the non-cytotoxicity acceptance criteria. Nonetheless, sample A may be the safest dental impression alternative fillers.
The detailed reproduction test results displayed in figure 10 show that the impression using an alternative nano-sized filler based on glutinous rice flour succeeded in reproducing continuous lines without interruption in sample group 2 using 15 wt% nanofiller at all line sizes (20 μm, 50 μm, and 75 μm) compared to the control group and other sample groups. Although the line size of 20 μm was successfully reproduced in the negative control group using dental alginate impression material, porous formations were still visible, especially on line sizes of 50 μm and 75 μm. This is similar to the positive control of glutinous rice flour-based impression materials without the use of nano-sized fillers. In group 1 with 5 wt% nanofiller, the imperfect condition of 20 μm and 50 μm reproduce lines was observed, which was ensured by the use of microscope magnifications that captured from both a mobile phone camera and a camera attached to the microscope.
In general, the viscosity increases with the proportion of filler used, but it is also affected by particle size, morphology, and quantity [2][3][4]. However, with nano-sized and uniform morphology of the filler particles, a higher percentage can affect viscosity changes. As seen in group 3 with a higher percentage of nanofiller (25 wt%), all sizes of lines are reproduced, but the details are still more visible in sample group 2. This condition could be the result of a decrease in viscosity caused by molecular conformations, such as those described previously due to the high cation content of the nanofiller used. As a result, sample group 2 has the optimal amount of nanofiller percentage of glutinous rice flour-based impression material, which is 15% wt. Fillers with nano-sized and uniform morphology can support surface area increases that easily duplicate thin areas, resulting in good detail reproduction [3,[49][50][51].
For dental procedures, accurate casts and models for diagnostic and treatment purposes are required, and one of these factors is filler. The size and morphology of this filler should affect the material's properties, whereas larger ones do not contribute to reinforcement [10]. Although the time exposure for making the impression in the patient's mouth only lasts a few minutes [2], our study performed well in terms of cell viability percentage over a long period of time to give the impression times if it casts in the technical lab. As a result, the nanorod particles derived from glutinous rice flour can serve as an effective organic filler in dental impression procedures. These particle offer the advantage of determining the viscosity and strength of the dental impression filler while ensuring biocompatibility due to their non-cytotoxic nature. By utilizing these organic nanorod particles, it could enhance the performance and safety of an alternative dental impression fillers.
Dental impression materials must meet the following requirements, such as (1) flowability to adapt to the oral tissue, (2) has a certain viscosity to be applied into the impression tray, (3) non-soluble in oral fluids and may set (transforms into an elastic/rubbery state or a rigid/rigid solid in oral cavity for the appropriate time (about less than 7 min), (4) ability to withstand the distortion and tearing when removed from the mouth, (5) dimensionally stable when cast repeatedly, (6) biocompatible, and (7) effective in terms of time and cost, including costs in equipment for processing [1,2]. This research only conducting to synthesizes fillers made from glutinous rice flour proposed as alternative dental impression fillers, which is only fulfilled adequate setting and working time and also a preliminary biocompatibility test. Therefore, continuing research still need to be developed in order to be accepted as suitable alternative dental impression materials through its performance, including viscosity, dimensional stability, mechanical properties and also biocompatibility when it is used in the clinical procedure.

Conclusions
In this work, nanorods (73.26 nm width) with reduced length size particles (865.15 nm length) of organic fillers made from glutinous rice flour were successfully produced using a precipitation technique without the addition of surfactant at a centrifugation speed of 3000 rpm. Because of their biocompatibility, the resulting particle fillers allow for lower gelatinization temperatures and may have a non-cytotoxic effect on cells. Given what has been said thus far, this study suggests that 15 wt% of the resulting nanorod particles could be used as alternative fillers to improve dental impression materials, which have satisfactory created continuous 20 μm line of detail reproduction in order to create an accurate replica for diagnostic and treatment in dental procedures. The mechanical properties of the dental impression application reinforced with alternative fillers must be evaluated in future research.