Development of poly(methyl methacrylate)/poly(lactic acid) blend as sustainable biomaterial for dental applications

Poly(lactic acid) (PLA) is gaining popularity in manufacturing due to environmental concerns. When comparing to poly(methyl methacrylate) (PMMA), PLA exhibits low melting and glass transition temperature (Tg). To enhance the properties of these polymers, a PMMA/PLA blend has been introduced. This study aimed to investigate the optimal ratio of PMMA/PLA blends for potential dental applications based on their mechanical properties, physical properties, and biocompatibility. The PMMA/PLA blends were manufactured by melting and mixing using twin screw extruder and prepared into thermoplastic polymer beads. The specimens of neat PMMA (M100), three different ratios of PMMA/PLA blends (M75, M50, and M25), and neat PLA (M0) were fabricated with injection molding technique. The neat polymers and polymer blends were investigated in terms of flexural properties, Tg, miscibility, residual monomer, water sorption, water solubility, degradation, and biocompatibility. The data was statistically analyzed. The results indicated that Tg of PMMA/PLA blends was increased with increasing PMMA content. PMMA/PLA blends were miscible in all composition ratios. The flexural properties of polymer blends were superior to those of neat PMMA and neat PLA. The biocompatibility was not different among different composition ratios. Additionally, the other parameters of PMMA/PLA blends were improved as the PMMA ratio decreased. Thus, the optimum ratio of PMMA/PLA blends have the potential to serve as novel sustainable biomaterial for extensive dental applications.

From the graph in Fig. 4, M100 showed the highest water sorption and water solubility while M0 showed the lowest water sorption and water solubility (p < 0.05).The water sorption and water solubility of the polymer blends were decreased when the amount of PLA reduced, and they are also different to each other (p < 0.05).

Degradation
The weight changes of different polymer blend ratios after immersion in distilled water and 0.3% citric acid from day 0 till day 63 were displayed in Fig. 5.
The weight of the specimens significantly increased over the first week after immersion, regardless of solution.Then, the weight started to rise steadily until it reached a plateau on day 35.The percentage of weight change of specimen after immersion in solutions for 63 days (w w ) was analyzed.Two-way ANOVA was performed to analyze the effect of 'blend ration' and 'types of solution' .The results showed that blend ration was significantly affected after immersion for 63 days (p < 0.05).However, the type of solution showed no significant difference (p > 0.05).The M100 showed the highest weight of wet polymer (w w ), followed by M75, M50, M25, and M0.The interaction between two factors was not observed (p > 0.05).Thus, one-way ANOVA and Tukey HSD were used to analyze the difference between groups.The results showed in Table 3.
Analysis of the percentage of weight change of dried specimen after immersion in solutions (w d ) exhibited normal distribution for all groups.Then, two-way ANOVA was performed.The result showed that solution did not affect the w d (p > 0.05), but the blend ration had an effect on the dried weight (p < 0.05).The interaction between two factors was not found (p > 0.05).By analyzing with one-way ANOVA and Tukey HSD, the results showed that even though the weight loss of M100 and M75 was not significantly different, their weight loss was significantly higher than the others (p < 0.05).While the M0 showed the lowest weight loss (p < 0.05) shown in Fig. 6.

Color stability
The results showed that the color change of M0 was much greater than that of other groups in both solutions (p < 0.05).However, there were no significant difference in color change between M100, M75, M50, and M25 (p > 0.05) (Fig. 7).Two-way ANOVA showed significantly difference in both 'blend ratio' and 'solutions' (p < 0.05) but no interaction between these factors (p > 0.05).However, coffee caused more significantly change in color than tea.Thus, the data was separately analyzed depending on solutions by one-way ANOVA with Tukey HSD.

Biocompatibility
The cell viability (%) of each group was calculated and compared to the control group.The results demonstrated that the elution in every test group was not toxic (% cell viability > 70), according to ISO 10993 19 .The cell count of each group was calculated by the standard equation [y = 0.028x + 0.0288].The R 2 was reported at approximately 0.96 which showed a strong correlation between OD and cell count.Then, the cell amount was compared between   www.nature.com/scientificreports/groups to define the statistical difference.The data was analyzed by one-way ANOVA and revealed no significant difference between groups (p > 0.05) Fig. 8.

Discussion
The properties of the polymer blend are dependent on the miscibility of the blends.The miscible blend could either obtain the benefits of each polymer or improve the properties of polymer blends, while the immiscible blend can cause the poor properties 20 .Thus, the miscibility of the blend is one of the important factors.The    www.nature.com/scientificreports/miscibility could be investigated by many methods such as scanning electron microscope (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD).The DSC and SEM were methods used in this study to investigate miscibility 21 .
The DSC showed single T g value in all PMMA/PLA blend ratio.The T g of PMMA/PLA blends were within the T g range between neat PLA and PMMA.The T g of polymer blends increased when the PMMA ratio increased.This is also found in the widening of T g .The endothermic peak of melting was only observed in M0 (neat PLA) at approximately 150 °C.This could be implied that neat PLA had crystalline phase of the structure.The endothermic peak in polymer blend groups could not be detected.This could be that the polymer chain arrangement in PLA was disturbed by amorphous PMMA leading to no PLA crystalline occurring.This phenomenon agreed with the previous study by Zhang et al., who observed the endothermic peak of melting temperature of PMMA/ PLA blend when the composition of PLA was more than 90% 22 .Gonzalez-Garzon et al. also found that the crystallization of PLA in the polymer blend was restricted when the composition of PMMA was more than 30% 14 .
No evidence of phase separation was found in the fracture surface under SEM investigation, which also indicated homogeneity in all polymer blend ratios.This suggested that all PMMA/PLA blends used in this study were miscible blends.The miscibility of the polymer blend could be affected by other factors such as blending method.The polymer could be blended by either melting or mixing the polymer beads with screw extruder, called "melt-blending".Another method is to dissolve the polymer beads with solvent, called "solution casting" 16 .Samuel et al., found that PLLA/PMMA blend with melt-blending followed by injection molding was miscible in all composition by shear force during blending by twin-screw.This could support the result of miscibility this study 16 .
To prepare the specimens, the injection molding technique using polymer beads was chosen.The suitable melting temperature and time in order to sufficiently melt and to be injected into the mold were selected.If the temperature was too low, the melted polymer might not fill the mold and caused defects of the specimen.In contrast, the high temperature could cause thermal degradation which lead to color changing, pore formation or decrease the mechanical properties 23,24 .Our study found that increasing the melting temperature could lead to a decrease in the flexural properties.The common failure of M0 was 'Tear' which was different from other groups.This failure might be caused from the testing temperature being close to the T g of the PLA.This could make polymer more rubber-like and ductile 25 .But when M0 was melted at 250 °C, the failure mode of all test specimen was 'Fracture' and occurred at lower flexural properties than the others.This might be resulted from the thermal degradation of the polymers when fabricating at high temperature causing polymer more brittle 26 .
The flexural properties indicate the ability of material to resist fracture from the masticatory forces.In this study, the flexural strength of PMMA was higher than PLA but not different from the other polymer blends.In contrast to flexural strength, PLA showed higher flexural modulus than PMMA but was not different from the other polymer blends.Thus, mixing PMMA to PLA could gain the benefit of both polymers.The PMMA/PLA blend showed both comparable flexural strength to PMMA and comparable flexural modulus to PLA.
The previous studies showed that the 3D-printed PLA by FDM has lower flexural strength than PMMA 13,27 .Additionally, the flexural strength of the milled PLA was reported to be lower 28 than that of milled PMMA was reported by the other studies 29,30 .This could be caused from the milled PLA block prepared by printing method, which printing orientation would affect the strength of material 31 .Unlike, the milled block or disc fabricated by other techniques instead of 3D printing could provide the optimum properties of the specimens 32,33 .Thus, the polymer beads fabricated in this study could be used as high strength raw materials.
Residual MMA monomer in polymerized acrylic resin could affect the mechanical and physical properties of denture base.It has shown that residual monomer acts as a plasticizer which can reduce flexural strength, increase water sorption and solubility, decrease color stability, and be toxic to human cells 2,34,35 .The conventional heat-polymerized PMMA showed residual monomer about 1.16-3.00mg% 36,37 .When comparing to M100 (neat PMMA) the residual monomer was much lower than those studies.This may be due to the different types of raw material between polymer beads and powder-liquid polymer.The thermoplastic polymer bead used in this study was nearly completely polymerized.In contrast to the fabrication by melting and injecting thermoplastic polymer bead into specimen mold, fabrication by conventional heat polymerized PMMA using powder and liquid could produce more residual monomer after polymerization.
The amount of residual monomer observed in this study significantly decreased when the PMMA was blended with PLA.The detected residual monomer dramatically decreased from M100 to M75, and then gradually decreased from M75 to M50 and M25 accordingly.The decreasing of residual monomer in PMMA/PLA blend was as expected due to the PMMA component being reduced when PLA was blended.The previous study reported that the residual monomer could be affected by heat and time 38 .The residual monomer was obviously decreased from neat PMMA beads (M100) and PMMA/PLA blends (M75, M50, and M25).This could result from heat during fabrication of the PMMA/PLA blends which caused the residual monomer to reduce during polymerization.As previously described, the blend process was operated at 210 °C, and fabrication was performed between 210-270 °C which were above the boiling point of MMA (about 101 °C).This may cause either monomer evaporation or continuing polymerization.Even though lactic acid is used as component of solution to produce artificial caries in vitro, the residual lactic acid monomer was not investigated in this study 39 .The reason was that the concentration of lactic acid must be high enough to decrease the pH to critical value and the previous study reported that the leaching lactic acid from PLA was very low (about 0.24 µg/cm 2 in 30 min) 40 .Thus, our finding could imply that blending the PLA to PMMA could significantly reduce the residual MMA monomer.
To investigate the water sorption and water solubility, it showed that M100 had the highest values while M0 had the lowest ones.The water sorption and water solubility decreased when the ratio of PLA was increased as indicated in Fig. 4. Salih et al. reported the water sorption and water solubility of PMMA denture base at 23.2 and 1.8 µg/mm 3 , respectively, which were closely to M100 in this study 41 .When considering the polymer structure of PMMA, the functional groups of PMMA (-COCH 3 ) can make PMMA more hydrophilic due to being a polar group.PLA is strongly hydrophobic polymer even the ester group exists in backbone chain, but the side chain is methyl group 42,43 .High water sorption and solubility leads to a decrease in mechanical properties, and color stability of denture base materials.The water sorption into PLA networks can cause hydrolysis of the polymer backbone at ester site 12 .Thus, these properties of polymer should be as low as possible to maintain the properties of materials.
The mean weights of wet polymer (w w ) and weight after degradation (w d ) were not statistically different, regardless of solution.Thus, immersion in 0.3% citric acid, polymer blended was affected by water uptake and released the soluble substances as similar as in distilled water.However, the blend ration could have an influence on both weights of wet polymer and weight after degradation.This might be explained by the result of water sorption and water solubility which M0 exhibited the lowest value and M100 showing the highest weight change.
Color is another important property of materials.When exposed to staining substances found in foods and beverages, materials may lose their original color.The color change could be observed by visual perception or spectrophotometer.By using spectrophotometer, the color changed (∆E*) is calculated from different of L*, a*, and b*.The human eye could detect the difference in color when ∆E* is above 1.0 44 .However, Ruyter et al. reported that ∆E* above 3.3 was unacceptable in dentistry 45 .The coffee and tea were selected as representative of daily consumed beverages due to the staining ability of these two beverages was higher than other beverages 46,47 .In this study, color change of M0 group was the highest while all PMMA/PLA blend ratios showed no significant difference in color stability, compared to PMMA.Color stability of the denture base materials depends on many factors including surface roughness, T g , UV resistance, chemical composition, or water sorption 48,49 .According to the lower water sorption, water solubility and residual monomer of PLA than those of PMMA, the thermal characteristics and chemical resistance of material could be results in lower color stability of PLA.
Color change of materials could occur by staining at the surface or penetration of colorants into materials.As previously described, the T g of PLA was about 55 °C which is closely to the testing temperature (37 °C), while PMMA/PLA blend showed higher T g than neat PLA.When the environmental temperature is close to T g , the material appears to be softened.Consequently, the solutions can easily penetrate the bulk materials.Thus, PLA appears to be degraded by bulk erosion as a result of the inner core deteriorating more quickly than the outer region 50,51 .This suggested that PLA could be permeated by the solutions more than PMMA.As the present result, the colorants were able to enter the materials but were not easily removed by mechanical methods such as rinsing, scrubbing, or grinding.
The in vitro cytotoxicity tests are used as screening tests for biocompatibility.To investigate the cytotoxicity of dental materials, the result of MTT test showed no toxicity in all groups and no difference from each other.It has been well accepted that the cytotoxicity of the denture base materials was correlated with the amount of residual monomer.A previous study showed that PLA is biocompatible because of its degradation property.PLA can degrade into lactic acid or carbon dioxide (CO 2 ) and water.These degradation products could be metabolized intracellularly and excreted through kidney filtration in urine and breath 52 .This study confirms that the neat polymers and the polymer blends were not toxic.This may be due to the relatively low level of residual monomer.Thus, PMMA, PLA, and PMMA/PLA blends in this study could be an alternative material for dental application with good biocompatibility.
From this study, the PMMA/PLA blend polymer beads could be used as raw materials for manufacturing processes such as injection molding and digital fabrication in dentistry.PMMA/PLA beads could be either formed into disc shape or ingot for CNC fabrication.Furthermore, PMMA/PLA beads could be prepared into filament, powder-liquid, and liquid resin for polymer 3D printing.However, this blended biomaterial may need some further modification to meet the requirements of each application such as color, characteristic, translucency.Therefore, a variety of dental applications can be further developed.Additionally, the thermoplastic materials could be recycled to reduce waste from manufacturing, and the biodegradable properties of PLA still existed after blended to PMMA.Thus, these polymer blends are more environment friendly than conventional PMMA and could be introduced to more dental applications in the future to promote sustainability.
Due to the growth in digital fabrication in manufacturing process and environmental concern, the properties of materials, especially the strength of material, should be considered.The benefits of PLA are biocompatibility, biodegradable, physical and mechanical properties close to PMMA.This PMMA/PLA blend could have potential applications in the dental field, including denture base materials, temporary restorations, or possibly as a component in dental implants or bone graft substitutes.
According to the result of PMMA/PLA blends (M25, M50 and M75) in this study, the M25 is not suitable for long-term temporary or permanent restoration due to the low glass transition temperature.Even though M75 had higher T g than M50, the flexural properties, color stability and biocompatibility were not different from each other.The M50 showed less residual monomer, water sorption, water solubility, and degradation than M75.Furthermore, the melt temperature of M50 was less than that of M75 which can be benefit due to less energy required for fabrication.From our finding, M50 ratio could be a suitable PMMA/PLA blend ratio for long-term temporary and permanent restoration in dental applications.However, it is necessary to do further investigation on the different ratios between M50-M75 to optimize the properties of polymer blend in this range and find the most favorable ratio.

Polymer blend preparation
The PMMA polymer beads with average molecular weight (M w ) of 11.0 × 10 4 g/mol (Acrypet™ MD001, Diapolyacrylate Co. Ltd, Thailand), and PLA polymer beads with average molecular weight of 11.6 × 10 4 g/mol (Ingeo™ Biopolymer 3052D, NatureWorks LLC, USA) were used in this study.To prepare PMMA/PLA polymer blend, the PMMA and PLA beads were melt-blended using twin-screw extruder (Collin T-20, Maitenbeth, Germany) in different ratios at 210 °C, 60 rpm for 10 min under dried nitrogen airflow.There were 5 different ratios of www.nature.com/scientificreports/PMMA/PLA to be investigated (Table 1).The polymer blend beads of each group were loaded into stainless steel tube and fabricated into specific shapes for further investigations using dental injection unit (Myerson Flex Press, Myerson LLC, USA).The polymer beads were melted at melt temperature for 20 min, then the melted polymer was injected into the mold with the 8 bar of pressure and was held the pressure for 10 min.

Flexural properties testing
To find the most favorable melt temperature for these thermoplastic polymer groups, the flexural properties of 5 groups of PMMA/PLA blend ratio were investigated in coordination with 4 different melt temperatures obtained from the preliminary study and were defined as T1, T2, T3, and T4 (Table 1).The lowest temperature at which a specimen could be manufactured without having any flaws, such as a weld line or an incomplete injection, was determined as the lowest melt temperature.The highest melt temperature was determined by the temperature at which the color of specimen did not change, signifying that the specimen had not undergone thermal degradation.Ten bar-shape specimens of each group sized 10.0 × 64.0 × 3.3 (± 0.2) mm were fabricated and polished using silicon carbide paper up to P1200.All specimens were kept in 37 °C water in incubator for 48 ± 2 h prior to flexural properties testing.The specimens were tested using the universal testing machine Shimadzu EZ-S (Shimadzu, Kyoto, Japan) with load cell 500 N at crosshead speed 5 mm/min in 37 °C water.The maximum flexural strength and flexural modulus were recorded.The failure mode of each specimen was classified as either fracture (F) or tear (T).

Blend miscibility testing
The miscibility was observed in 2 aspects including thermodynamic properties and topographic investigation.First, the thermodynamic properties of polymer beads were investigated by Differential Scanning Calorimetry (DSC).The polymer beads of each group were fragmented into 10 mg and analyze by DSC 204 F1 Phoenix (NETZSCH-Gerätebau GmbH, Selb, Germany).The samples were processed under nitrogen gas with heat rate at 10 °C/min from 0 − 220 °C to erase thermal history and cool down rate at 10 °C/min to 0 °C.The cooling and second heating scans were used to evaluate the material's ability to crystallize and the glass transition temperatures.The melting temperature of each group was also determined.
The other miscibility aspect was investigated by Scanning electron microscope (SEM).The polymer bead of each group was immersed in liquid nitrogen and later broken into pieces.The fracture surfaces of each specimen group were gold-coated and observed under SEM with JSM-IT300 InTouchScope (Joel Ltd., Tokyo, Japan) at 5000 × magnification.

Residual methyl methacrylate (MMA) monomer testing
Six specimens of each group were fabricated into disc shaped size 50 mm in diameter and 2 mm thickness.The residual monomer test was conducted in accordance with ISO 20795-1.To serve as the m sample for calculation, the samples from each group were given a 650 mg weighting.The residual MMA monomer was analyzed by high-performance liquid chromatography (HPLC) system with 5 μm particle size, 250 mm length and 4.6 mm internal diameter column.The amount of residual MMA monomer (m MMA ) was obtained from the standard calibration curve.Therefore, the residual monomer (%mg) was calculated from equation.

Water sorption and water solubility testing
Five specimens of each group were fabricated as 50 mm diameter, 0.5 mm thick disc and polished using the same previous procedure.The specimens were kept in desiccator containing freshly dried silica gel in 37 °C incubator and frequently weighted until the mass remained constant, at which point "m 1 " was recorded.The volume of specimen (V) was calculated using the mean of three measurements of diameter and the mean of five measurements of thickness.The test method complied with ISO 20795-1, so the specimens were immersed in 37 °C water for 168 h.The specimens were dry towel cleaned and weighted as "m 2 ".The specimens were reconditioned to constant mass in the desiccator and recorded as "m 3 ".The water sorption (w sp ) and water solubility (w sl ) were calculated by following equation:

Degradation test
To investigate the degradation of polymer blends, six disc-shaped specimens of each group sized 50 mm diameter and 2.0 mm thick were prepared following the same procedure.The specimens were also kept in desiccator and the specimens were weighed until their mass remained constant, at which point "d 0 " was recorded.The specimens were randomly divided into 2 groups and immersed in sealed container with either distilled water or 0.3% citric acid at 37 °C incubator for 63 days.The solutions were changed every week and the specimens were rinsed with distilled water before immersing in the fresh solutions.The specimens were weighted every day from day 1 till day 8.Then, the specimens were weighted at day 10, 12, 14, 21, 28, 35, 42, 49, 56, and 63.The specimens were taken

Figure 5 .
Figure 5. Graphs of weight change (w w ) after immersion from day 0 till day 63 in distilled water and 0.3% citric acid solutions.

Figure 6 .
Figure 6.Mean and SD of dried weight (w d , %) in each group.

Figure 7 .
Figure 7. Mean and SD of color change (∆E*) of each group.

Table 2 .
Mean (SD) of flexural properties (MPa) of polymer blend groups.*The same superscript letter means there is no significant difference between groups in each row.F means fracture and T means tear in mode of failure.

Table 3 .
Mean (SD) of weight of wet polymer (w w , %) of each group (M0 − M100).*The same superscript letter means no significant difference.