Safety of 3D-Printed Acrylic Resins for Prosthodontic Appliances: A Comprehensive Cytotoxicity Review

Abstract

Additive manufacturing resins used in dental prosthetics may retain uncured monomers post-polymerization, posing potential long-term patient exposure risks. Understanding the biological safety of these materials is crucial, particularly for 3D-printed acrylic-based prosthodontic devices such as occlusal nightguards, complete and partial dentures, and temporary fixed prostheses. This paper reviews the literature evaluating the cytotoxicity of such materials. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, we conducted a scoping review using the MESH keywords related to population (P), intervention (I), comparison (C), and outcome (O) across databases, including OVID Medline, EMBASE, and SCOPUS. Our search, limited to peer-reviewed English language articles from 2015 to 2023, resulted in 22 papers. These studies, utilizing digital light processing (DLP) or stereolithography (SLA) printing methods, varied in examining different 3D-printed materials, as well as washing and post-curing protocols. The primary experimental cells used were human gingival fibroblasts (HGF) and mouse fibroblasts (L929). There are no statistical differences in biocompatibility regarding different commercially available resins, washing solutions, or methods. Improvements in cell viability were related to an increase in washing time, as well as post-curing time. After the polishing procedure, 3D resin-based printed occlusal devices perform similarly to milled and conventionally processed ones. Our findings underline the importance of appropriate washing and post-curing protocols in minimizing the cytotoxic risks associated with these 3D-printed resin-based devices.

1. Introduction

The use of additive manufacturing (AM), or 3D printing, has expanded significantly within dentistry, particularly in the fabrication of prosthodontic devices such as occlusal nightguards, complete and partial dentures, and temporary fixed prostheses. This innovative technology offers the advantage of rapid workflow, customization to patient-specific needs, and the potential for cost reduction (Figure 1). In restorative dentistry, materials used in additive manufacturing are called printable biomaterials and are available as acrylates, resin-based composites, and, more recently, ceramic-infiltrated composites [1].

The additive methods most frequently applied in dentistry are stereolithography (SLA) and digital light processing (DLP) technology. Each process uses light to activate the polymerization of the printable resin. While SLA uses UV light, DLP uses a high-power LED to start the chain reaction of the monomers [2].

The introduction of 3D-printed materials into clinical practice raises concerns about the biological safety of these materials, especially regarding the release of residual monomers from the polymerized products [2]. Residual monomers, primarily from acrylic-based resins, can be cytotoxic. Since prosthodontic devices are intended for long-term use, often in direct contact with sensitive oral tissues, releasing these monomers poses a health concern [3].

Consequently, evaluating the cytotoxic effects of these materials is essential to ensure the safety and efficacy of 3D-printed dental prostheses. This review aims to systematically evaluate the cytotoxicity of 3D-printed materials used in dental prosthetics, focusing on identifying and synthesizing existing research findings while highlighting areas where further study is needed. This paper seeks to contribute to the safe and effective integration of 3D printing technologies in dental care by comprehensively analyzing the cytotoxic risks and mitigation strategies associated with these innovative materials.

As with directly photocured resin-based composites, 3D printable resins are not fully converted to the polymeric chain after exposure to light. This deficiency in the degree of conversion must be overcome by a post-curing process, usually, a light oven that uses extra-time light exposure and heat to improve the degree of conversion of these materials (Figure 2). However, complete conversion is rarely achieved, and the free unreacted monomers represent a well-known risk to biological properties [4].

The extensive range of available 3D printing polymer materials from different manufacturers, each using a slightly different manufacturing and curing protocol, might lead to different outcomes regarding these materials’ mechanical and biological properties [5]. Three-dimensional printing materials can be indicated for surgical guides or custom trays to be used quickly (Figure 3). Other appliances, such as interocclusal nightguard devices and complete and partial dentures, may be produced using these materials, and long-term use occurs [1]. While mechanical properties have been widely investigated and documented [6], and data from these experiments are more easily translated to clinical practice, the biological behavior of 3D printing materials has a complex methodology, which may confuse the dentist about their biological safety [7,8].

Some published papers present data on the biocompatibility of printable resins for temporary and permanent prosthetic devices. Different methodologies can assess the biological response, such as inflammatory response, oxidative stress, biofilm interaction, cytotoxicity, genotoxicity, cell adhesion, cell proliferation, and cell viability [7,8]. Considering the amount of data produced, the complexity of methodology and data interpretation, and the need for further understanding by the general dentist regarding the cytotoxicity outcomes of printable materials, this paper aims to review the literature on the cytotoxicity evaluation of 3D-printed materials used in long-term acrylic-based prosthodontic devices, such as occlusal nightguards, complete and partial dentures, and temporary fixed prostheses.

2. Materials and Methods

A systematically conducted scoping review was utilized to summarize the currently available research on the cytotoxicity of 3D printable acrylic resins. The workflow followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement to identify, screen, elect, and include scientific papers in this review. MESH keywords were inserted into the databases to perform the search. The keywords selection obeyed the P (population), I (intervention), C (comparison), and O (outcome) question system to associate them with the advanced search option of each database portal.

Table 1. MESH Keyword.

PICO	Keywords
Population	3D print$4 OR 3-D print$4 OR 3-dimensional print$4 OR three-dimensional print$4
Intervention	“dental night guard *” OR “occlusal splint *” OR “night guard *” OR bruxism OR prosthodontic * OR “dental restoration *” OR denture * OR “artificial tooth” OR “artificial teeth”
Comparison	“three dimensional print *” OR “three-dimensional print *”
Outcome	Biocompatib * OR cytotoxic * OR genotoxic *

* This is a key used by the librarian to decrease the chance of losing an important paper during the search.

describes the PICO keywords association.

The scoping review search was conducted at the OVID Medline, EMBASE, and SCOPUS databases to compile papers evaluating topics related to the cytotoxicity of 3D printable resins. English-language peer-reviewed journal papers were selected from 2015 to 2023. Papers, such as dentures and occlusal nightguards, were included when investigating 3D-printed acrylic resin designs for long-term clinical use. Papers were excluded if they did not focus on 3D-printed acrylic materials used for prosthetic devices, if they studied temporary rehabilitation materials, if they investigated experimental non-commercially available materials, resin-based composite materials, ceramic and metallic materials, materials designed for biodegradable scaffolds or implant dentistry. These papers were compiled into Covidence for further review and selection, and a librarian removed duplicate entries.

Two independent reviewers used the Covidence software (www.covidence.org accessed on 14 July 2023) to screen the titles and abstracts to filter out papers relevant to the topic, applying inclusion and exclusion criteria. To ensure consistency, both reviewers evaluated all article abstracts and voted “yes”, “no”, or “maybe” for the relevance of the articles, with ties broken by a third investigator. The reviewers performed another screen, now evaluating the full text, and finalized the selection of these papers, resulting in 24 papers included in this scoping review. Figure 4 states the PRISMA workflow from identification to the inclusion of papers.

Both reviewers extracted data from the selected papers, considering the following criteria: author, year of paper publication, 3D-printed materials evaluated, 3D-printer device, post-processing method performed, variables tested in the experiment, cytotoxicity assay and cell lineage used, the mains results, and conclusion. All data were extracted independently, and the results from each reviewer were discussed with a third researcher when needed and merged into

Table 2. Extracting datasheet.

Author/Year	Materials/n	Printer	Processing Method	Variables	Bio Method/Cell	Main Results/Conclusion
Aati S, 2022 [9]	PMMA-based 3D-print (Dentca Denture Base II) heat-cured (Vertex Rapid Simplified) n = ?	DLP Kulzer 3D Printer System	Cleaned sonicated with isopropanol. Soaked in a glycerol bath and post-cured. Curing unit at 200 W wavelength 390–540 nm. Finished with ascending grit size: 600, 800, and 1200. Polishing with 1, 0.25, and 0.05 µm paste.	Post-curing for 0, 5, 10, or 20 min	(APH) assay extract method: 24 and 72 h; direct contact method: 24 h Human periodontal ligament fibroblasts (HPLFs)	Extracts of uncured resin reduced cell viability by 50% (p > 0.001). Direct contact: uncured resins reduced cell viability by approximately 60–65% (p < 0.0001). This effect was significantly (p < 0.0001) reduced when resins were cured for at least 5 min. An increase in post-curing time enhanced cell viability and biocompatibility.
Alamo A, 2022 [10]	Prizma 3D Smart Print Bio; Cosmos DLP Temp n = 6	DLP FlashForge Hunter	Specimens were immersed in isopropyl alcohol for 10 min under agitation; finished using 600- and 1200-grit abrasive paper; post-polymerization in an ultraviolet (UV) light chamber (90 W, 405 nm).	Post-curing for 1, 10, or 20 min	Cell metabolism (Alamar Blue; n = 6); cell viability (Live/Dead assay; n = 2)/1, 3, and 7 days. Immortalized normal oral keratinocytes (NOK-Si; ID:CVCL_BW57) 3D culture of human gingival fibroblasts (HGF)	Severe reduction in metabolism (>70%) and viability of keratinocytes occurred for post-curing 1 min. 10 and 20 min promoted a mild–moderate cytotoxic effect. The 3DP resins submitted to post-polymerization for 20 min showed a pattern similar to that of resin composite. The biological compatibility of 3D printing resins for interim restorations can be negatively influenced by inadequate post-polymerization processing.
Atria PJ, 2022 [11]	Temporary Crown and Bridge (Formlabs); Crowntec C&B Nextdent, Permanent Bridge resin n = 30	SLA-printer Formlabs A2; DLP Nextdent 5100	Formlabs: 3 min washing machine (99% IPA). Post-curing time was set to 60 °C for 20 min; Crowntec: alcohol-soaked (96%) cloth. Final curing UV-light box 320–500 nm, twice, for 180 s each. Nextdent: specimens cleaned in 91%IPA (ultrasonic unit) for 5 min, post-curing for 30 min.	Three commercially available resins and the experimental resin	Cell viability PrestoBlue assay; /24, 48, 72 h, 5 days, and 7 days human periodontal ligament fibroblasts (hPDLF)	There was no significant difference in cell proliferation of hPDLF cells in contact with different 3D-printed materials. Evaluation of cytotoxicity through the LDH assay yielded no cytotoxicity of the materials. There were no differences in cell viability, cell proliferation, and cell toxicity among different evaluated resin materials.
Bayarsaikhan E, 2022 [12]	crown and bridge resin (C&B resin, NextDent, Soesterburg, Netherlands) n = 5	DLP 3D printer	405 nm UV LED light (1.4 mW/cm3) Washed (ethanol) for 10 min. Four different UV-light polymerization chambers and a handheld curing device (5, 15, and 30 min). Post-curing chambers [LC-3D Print Box (LC), Form Cure (FC), Veltz 3D (VE), and Cure M (CM)]. Post-cured for 20, 40, and 60 s using VALO.	Post-curing for 5, 15, and 30 min four different chambers VALO	CELLOMAX™ viability kit /24 h Primary human gingival fibroblasts	The cell viability of the post-cured resin specimens ranged from 56.46% to 92.29%. The cell viability did not differ significantly with the post-curing time in the LC, FC, and VA groups, whereas it did in the CM and VE groups (p < 0.05). The cell viability varied significantly in all 30 min PCE groups and in the 20, 40, and 60 s VA groups (p < 0.05). Post-curing with different equipment showed significant changes in cell viability of the 3D-printed crown and bridge resin.
Bayarsaikhan E, 2021 [13]	PMMA resin (Denture teeth resin A2, Formlabs) n = ?	SLA 3D printer (Form 3, Formlabs)	Cleaned with 90% isopropanol for 10 min. Post-cured in a UV-light chamber. 405 nm light source heating up to 80 °C. Post-cured at temperatures of 40, 60, and 80 °C for 15, 30, 60, 90, and 120 min.	Post-cured at 40, 60, and 80 °C for 15, 30, 60, 90, and 120 min	CELLOMAX™ viability; a lactate dehydrogenase (LDH) release assay/24, 48, and 72 h Primary human gingival fibroblasts (HGFs)	There was a significant interaction effect between cultivation time, post-curing temperature, and post-curing time. Increasing the post-curing temperature and time results in significant enhancements in biocompatibility.
Bieger V, 2023 [14]	Dental LT Clear Resin (Formlabs Inc); FREE-PRINT splint (Detax GmbH & Co KG); n = 9	SLA-printer (Form 2; Formlabs)	Rinsed (IPA 90%) for 5 min. Post-cured (2000 flashes—UV-light). Unpolished specimens were washed in the Millipore water (2 × 4 min). Polished specimens were cleaned ultrasonic bath of 96% ethanol (2 × 4 min) and washed in Millipore water (4 min).	Unpolished and polished	A WST-1 cell viability assay (Cell Proliferation Reagent WST-1; F. Hoffmann-La Roche)/24 h Human gingival fibroblast (HGF-1) cells	Overall, material (p < 0.001) and surface treatment (p < 0.001) significantly influenced the viability of HGF-1 cells. Similar behavior on conventionally processed, milled, and printed resin materials for occlusal devices when polished. On unpolished surfaces, cell viability on printed materials was reduced compared with conventionally processed and milled materials. Polishing of printed materials is essential to reduce cytotoxic effects.
Britto VT, 2022 [15]	Cosmos Temp, n = 3	DLP Bego, Varseo 3D printer,	Wash specimens using 96% ethyl alcohol sonic bath for 10 min. Post-polymerization in a lighting chamber 400–450 nm; for 13 min.	Comparing a bis-acryl resin (BA) and an acrylic resin (AR)	Sulforhodamine B; MTT/72; 24 h primary gingival fibroblasts	AR (71.94 ± 11.82%) showed a lower cell viability (p < 0.05) than 3D (92.88 ± 11.36%) and bis-acryl composite resin (90.85 ± 11.60%) for sulforhodamine B analysis. No significant difference among materials for MTT analysis (p > 0.05). 3D-printed polymer fulfilled the material’s requirements for clinical use.
Bürgers R, 2022 [16]	Med610 (Stratasys), V-Print splint (FREEPRINT) ortho 385; Dental LT Clear n = 25	Not reported	Not described	3D resins compared to conventional resins	WST-8-based-assays; 24 h explosion L929 cells	No significant differences between the 3D-printed or milled resins. No significant differences between 3D printing, milling, thermoforming, and pressing. 3D printing and milling showed no significant differences compared with conventional methods. In human gingival fibroblasts, cytotoxicity of all resins was below a critical threshold.
Chen H, 2021 [17]	AA temp; C&B MFH n = 6	DLP MiiCraft Ultra 125; Phrozen Sonic mono LCD	All specimens were washed with 95% alcohol and subjected to post-polymerization treatment by using the FormCure or PhrozenCure units for different times to enhance the polymerization.	3D printing resins or DLP 3D printers can be used in a mono-LCD 3D printer	MTT/24 h Mouse fibroblasts (L929)	Without post-polymerization, both AA and CB had viability < 70% (cytotoxic potential). Post-polymerization (FormCure—15 min; PhrozenCure—1 min), all material-printer combinations reached near 100% cell viability. Resins designed for DLP 3D printers can be used in a mono-LCD 3D printer post-polymerized.
Dai J, 2022 [18]	Freeprint Denture, Detax GmbH, Ettlingen, Germany n = 4	DLP printer UltraCraft DS	Ultrasonically cleaned in 99% isopropanol for 3 min twice and post-cured for 15 min in a light chamber (UltraCraft PCU mini, with an ultraviolet light 360 to 440 nm.	Not described	(LDH) release (LDH cytotoxicity assay)/24 h Mouse fibroblasts (L929)	L929 fibroblasts cultured in specimen extracts exhibited no compromised membrane integrity. No differences between extracts (p > 0.05). Relative LDH release of all the groups <30% of negative control. No differences between groups (p > 0.05). The coating treatment did not cause cytotoxic effects.
Guerrero-Girones J, 2022 [19]	Keysplint Soft NextDent Ortho Freeprint Splint (Detax) traditional resin Orthocryl (Dentaurum) n = 40	SLA 3D printer (Phrozen Sonic Mini 4k)	IPA 99% for three minutes. Second bath for two minutes. Left 30 min in a dark room. Post-cured using a UV-polymerization Box (405 nm, 40 mW/cm2). Orthocryl mixture at a ratio of 2.5:1 (powder: fluid) for 5 min. The final polymerization in a pressure vessel (2.2 bar) at a temperature between 40 °C and 46 °C for 20 min.	3D print resin and traditional resins used for dental splints	MTT; Cell migration assays; Cell cytoskeleton staining assays; Human gingival fibroblasts	At 1:4 dilution, no resin affected cell metabolic activity; at a 1:2 dilution, Freeprint Splint had the highest cytotoxicity at 24 and 72 h (p < 0.001), while other resins did not affect cell biocompatibility; undiluted Freeprint Splint-treated cells exhibited a significantly lower viability. The new dental resins for 3D printing and the conventional dental resins assessed in this study showed similar biocompatibility, except for Freeprint Splint, which was the most cytotoxic of the four dental resins studied on hGFs.
Hwangbo NK, 2021 [20]	NextDent C&B; Formlabs Denture Teeth resin n = 15	DLP 3D printer (NextDent) SLA 3D printer (Form 3, Formlabs)	Washed with a 90% IPA or TPM solution for 3, 5, 10, 15, 30, 60, or 90 min. A non-washing group was the control group. The specimen was then post-cured for 30 min at 60 °C in a UV-light polymerization chamber using a 405 nm light source.	Washing solutions (IPA or TPM); Washing times	CELLOMAX™ viability assay; lactate dehydrogenase (LDH);/24, 48, and 72 h Primary human gingival fibroblasts (HGFs)	The cell viability varied significantly with the washing time (F = 216.669, p < 0.001) and material used (F = 79.899, p < 0.001), but not with the washing solution used (F = 1.298, p = 0.255). Cell viability increased with the higher washing time. Cytotoxicity decreased with an increase in washing time. Biocompatibility increased with the washing time for both solutions.
Jin G, 2022 [21]	Temporary crown and bridge resin (Nextdent C&B resin, 3D Systems n = ?	DLP printer (Nextdent 5100 3D printer)	Washing by a rotary washer (3, 6, 10, 20, and 60 min); soaking in a tank and washing by an ultrasonic bath (3, 6, 10, 20, and 60 min); specimens manually dunked and rinsed for 10 s without a post-washing process, with the residual liquid resin left on the surface (control group).	Different washing protocols	Cell viability assay: CELLOMAX viability kit; cell cytotoxicity assay: lactate dehydrogenase (LDH) release assay/24 h; 24 and 48 h. Primary human gingival fibroblasts (HGFs)	Cell viability rises with the prolonged post-washing time. Cytotoxicity decreases with the prolonged post-washing time. Ultrasonic bath provided more powerful detoxification effects to the Nextdent C&B resin than the rotary washer. The ultrasonic bath had a superior washing outcome in eluting residual monomers compared with using the rotary washer. Prolonged post-washing can efficiently reduce the quantity of the residual monomers
Kim JH, 2022 [22]	C&B MFH (NextDent) n = 3	DLP 3D printer (ND5100; NextDent)	The printed specimens were washed (10 min) in a washer with IPA 99%. Specimens were kept in aluminum zipper bags to shield them from light.	Post-polymerization devices: D1; LC; FO; ME and MP	Water-soluble tetrazolium (WST)-1 assay/24 h L929 mouse fibroblast cells	No post-polymerization group showed a cell viability of 16.41%, whereas all other experimental groups showed results from 66.79% to 96.53%. D1 group had the highest level of cell viability (96.53%), MP group showed the lowest level (66.79%). Light intensity and time management improve the post-cure process.
Ping L, 2021 [23]	V-Print Denture & Solflex 650, n = 15	Not reported	Rinsed in an ultrasonic bath 96% ethanol post-cured with different post-polymerization devices: Otoflash G171 (OF), Labolight DUO (LL), PCU LED (PCU), and LC-3DPrintbox (PB).	Different post-curing devices	Cell membrane damage: live/dead staining; cell morphologies: fluorescence microscope; metabolic activity: CCK-8 assay;/24 h; mouse fibroblasts (L929)	L929 fibroblasts cultured in the extracts of 3D-printed specimens displayed metabolic activities > 70% of the negative control. No differences in cell metabolic activities between different 3D-printed surfaces. Cytotoxic effect of the 3D-printed material can be decreased by any different post-curing methods.
Nam NE, 2023 [24]	NextDent C&B (NextDent) n = 5	DLP 3D printer (NextDent 5100)	Specimen surface was washed (IPA 90%—10 min). Post-curing for 30 min (UV—220 µW/cm2—60 °C). The completed specimens were kept in darkness and divided into groups based on their heat-treatment method.	Storage at room temp. (25 °C); RT water; 80 °C and 100 °C water; autoclaving	Cell viability: CELLOMAX™ viability kit; cytotoxicity: (LDH) release assay; Primary human gingival fibroblasts	RT = cell malformation was observed. Water storage = better cytotoxicity; Autoclave = not significant; Greater time of immersion = better cell viability. Cell viability increases and cytotoxicity decreases by immersing a printed resin in 100 °C water for 1 or 5 min.
Oh R, 2023 [25]	Next Dent C&B, Next Dent BV, Netherlands n = 5	DLP method (ASIGA MAX UV)	Washed for 5, 10, 15, 30, and 60 min at various temperatures with 95% ethyl alcohol, temperature modification (N/T, 18–20 °C), 30 °C, 40 °C, and 50 °C.	Washing and time	CELLOMAXTM viability/24 h Primary human gingival fibroblasts (HGFs)	Temperature-treated groups had significantly improved cell viability. The washing solution temperature and time significantly affected biological properties. Washing of 3D-printed samples at 30 ◦C for 30 min presented a significantly improved cell viability.
Srinivasan M, 2021 [26]	AvaDent Denture base puck, Avadent Extreme CAD CAM shaded NextDent Base, NextDent C&B, n = 9	DLP Rapid Shape D30	Rinsed twice (3 min) in a 96% ethanol solution in an ultrasonic bath to remove excess material. Placed in an ultraviolet light box for 10 min for additional polymerization, a wavelength of blue UV-A 315 to 400 nm, and an output of 43.2 kJ.	Milled or; 3D-printed	Resazurin assays/4, 7, 14, and 21 days human epithelial cells (A-431); human gingival cells (HGF-1)	Epithelial cells (A-431) and gingival cells (HGF-1) grew gradually around a 3- to 4-fold increase from day 4 to day 21, (same trend as the control group). No statistical difference among different resin groups (MB, MT, PT, and PB1) from day 4 to day 21 for either A-431 or HGF-1. CAD-CAM milled and complete denture resins had similar biocompatibility.
Wuersching SN, 2022 [7]	Tetric EvoCeram (TEC); Tetric CAD (TC); VarseoSmile (VSC); NextDent (ND); Protemp 4 (PT); Telio CAD (TEL); VarseoSmile Temp (VST); Temp PRINT (TP) P Pro Crown & Bridge (P)	DLP printer P30 (RapidShape); DLP printer DLP NextDent 5100 (NextDent); DLP printer Varseo XS (BEGO).	Managing different materials following the manufacturer’s instructions.	Different materials	Cell viability: RealTimeGlo® MT Cell Viability Assay; Induction of apoptosis: e RealTimeGlo™ Annexin V Apoptosis and Necrosis Assay/over 72 h; 24 h; 24 h; 24 h. human gingival fibroblasts (hGF-1)	Among the resins for permanent FDP, TEC, VSC, and ND were severely toxic. Four of the five resins intended for temporary FDP, namely, PT, VST, TP, and P, were also severely toxic. TC and TEL were the only resins that exhibited slight toxicity to hGF-1. In the presence of most resins, RLUs after 24 h were slightly higher compared to the basal level with DMEM. However, when incubated with TEC or PT, the RLUs were significantly lower than the negative control. TC and TEL showed more favorable cytotoxicity results. We suggest that further post-processing steps, such as additional light curing and washing, may improve the biocompatibility of printable materials.
Wullf J, 2022 [27]	Luxaprint OrthoPlus, DMG; V-Print Splint, Voco n = 25	DLP (P30 printer, Straumann)	Alignment: Printing was performed under 90° (A1), 45° (A2), or 0° (A3) alignment to the building platform in a standard of 100 µm layers. Post-processing: Specimens were either automatically washed or manually cleaned with Isopropanol. Post-polymerization was performed with LED or Xenon light.	Printer alignment; wash system; post cure; materials	Crystal violet assay The murine RAW264.7 mouse macrophage cell line	Materials influenced cell survival in most cases (p ≤ 0.029). The wash system showed differences for most combinations (p ≤ 0.032). The type of post-cure provided significant differences for most systems (p ≤ 0.022). The results for the criterion alignment were not consistent. The individual parameters of material selection as well as the postprocessing (post-polymerization, washing procedure) affect in vitro cytotoxicity. Alignment during manufacturing does not have any effect on in vitro cytotoxicity.
Xu Y, 2021 [28]	Dental LT Clear Resin, Formlabs; Orthocryl Clear, Dentaurum GmbH & Co. KG, Ispringen, Germany (control); n = 4	SLA 3D printer (Form 3B,)	Ultrasonically rinsed with IPA for 5 min, 12 min, 20 min, 30 min, 1 h, and 12 h, post-curing at 80 °C for 20 min.	Washing time	Cell morphology; cell viability (live/dead fluorescence staining) Direct contact test: Metabolic activity (CCK-8 assay); L929 mouse fibroblasts	Cell viability of groups from 5 min to 12 h did not differ from control. All post-processed samples with different post-rinsing times showed no cytotoxic effect (L929 fibroblasts). The removal of cytotoxic methacrylate monomers by post-rinsing could be achieved in 5 min. Extending the post-rinsing time did not improve the cytocompatibility.

.

3. Results

Twenty-one papers ranging from 2021 to 2023 were finally selected and had their information and data extracted. The main characteristics of each study, as well as the outcomes related to our research question, are presented in Table 2. The study assessed various 3D-printed dental resins and their biocompatibility, focusing also on the effects of post-processing methods on cell viability. The results evidenced that the post-curing impacts the biocompatibility of such materials. For instance, uncured resins consistently demonstrated high cytotoxicity, with cell viability reduced by 50–65% depending on the method of testing and resin type [9]. Post-curing for at least 5 min notably improved cell viability, with further increases in post-curing time enhancing biocompatibility [9]. However, varying post-curing times and methods showed differential effects across different studies. For example, resins post-cured for 20 min in UV light chambers exhibited varying levels of cytotoxicity, with some showing improved biocompatibility compared to others [10].

Additionally, the type of post-processing equipment and protocols influenced the cytotoxicity outcomes. Different UV light chambers and washing protocols resulted in variations in cell viability, indicating that optimal post-processing conditions are critical for reducing cytotoxic effects [12,21]. Materials with extended post-curing times and more thorough washing procedures generally demonstrated lower cytotoxicity, although certain resins like Freeprint Splint showed higher toxicity even with extensive post-processing [19].

4. Discussion

Overall, the studies reviewed here indicated that acrylic resins used in prosthodontic appliances can exhibit varying levels of cytotoxicity, with significant differences observed across different resin formulations. Residual monomers, such as methyl methacrylate (MMA), are a primary source of cytotoxicity, as these compounds are released over time and negatively affect cellular viability [29]. Moreover, we observed that the cytotoxic response seems to be influenced by factors such as the curing process, resin composition, and storage conditions.

Cytotoxicity analysis of dental materials is of utmost importance as it ensures the safety and biocompatibility of these materials when in contact with human tissues. By evaluating the potentially toxic effects of dental materials on cells, such as pulp cells or gingival fibroblasts, cytotoxicity analysis provides valuable insights into their potential adverse effects on oral tissues. This analysis aids in identifying any potential risks or side effects associated with dental materials, allowing dentists and dental professionals to make informed decisions when selecting suitable materials for various dental procedures. Ultimately, cytotoxicity analysis plays a crucial role in promoting patient safety and optimizing the long-term success of dental treatments.

According to International Standard Organization (ISO) 10993-5 [30], the in vitro cytotoxicity test plays a vital role in assessing the biocompatibility of biomedical materials. It serves as the first step in determining the safety of these materials before further tests, including those involving laboratory animals. In vitro techniques offer numerous advantages compared to in vivo methods. These include controlling experimental variables, obtaining relevant data more efficiently, and often having shorter testing periods. Although there is a challenge in extrapolating in vitro data to clinical applications of biomaterials, this can be addressed using suitable reference materials currently utilized in clinical settings. Overall, by establishing the non-toxicity of a material through in vitro cytotoxicity analysis, researchers can proceed with confidence in studying its overall biocompatibility, which will include other materials’ biological properties.

ISO 10993-5 also establishes methodologies and cell types for cytotoxicity analysis. In this review, we observed that most of the studies used different methods and cell types compared to the suggested by ISO. According to ISO 10993-5, if viability drops below 70%, the material is considered to have cytotoxic potential. ISO suggests the MTT test, which uses the reagent (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) to indicate cell metabolism. Many studies included in this review applied analyses based on this cell behavior, which differs from cell viability. Despite being widely applied, the MTT assay has some disadvantages compared to viability tests such as the sulforhodamine B (SRB) assay, which are also found in the studies along with this review. The SRB method calculates cell enumeration based on protein content, minimizing the interference of endogenous and exogenous substances, which strongly influence the MTT outcomes. For this reason, despite not being the suggested ISO method, the SRB test has presented superior predictive power than the MTT assay and is encouraged to be performed.

Microscopy analyses with Live/Dead kit were performed in some of the selected studies. This methodology is a valuable tool for assessing the cytotoxicity of materials due to its ability to visualize the cell damage directly. One major advantage of live/dead microscopy is its real-time assessment, enabling the observation of cellular responses immediately after exposure to a material. This provides valuable information regarding the speed and extent of cell death or damage. However, despite the name, live/dead kits do not necessarily stain live and dead cells.

In contrast, they stain cells with or without membrane damage. Another drawback is that live/dead microscopy can only depict the current status of cells and does not provide information about the underlying mechanisms or long-term effects of cellular damage. For these reasons, it is recommended that other complementary tests are performed to gain a more comprehensive understanding of cytotoxic effects.

The safety of 3D-printed acrylic resins highly depends on their degree of conversion. The incomplete polymerization may lead to a higher concentration of free monomers known to cause cytotoxicity [31]. Previous in vitro studies comparing heat-polymerized acrylic resins to auto-polymerized resins also found that the latter tend to exhibit higher cytotoxicity due to incomplete polymerization, leading to increased monomer release [32,33]. The duration of resin exposure to biological tissues also plays a critical role in cytotoxicity, with prolonged exposure correlating with heightened adverse effects on cell cultures [32]. To reduce the number of uncured monomers from 3D-printed devices, washing protocols are essential [20,28]. Usually, the rinsing liquid is a 90% or higher concentration of ethylic or isopropyl alcohol (IPA) [24,25], or tripropylene glycol monomethyl ether (TPM) [20,21]. There are no statistical differences in biocompatibility regarding the washing solutions [20]. The washing method was also investigated, and no differences in cytotoxicity were found between the rotatory wash and the ultrasonic bath [21]. The washing time presents conflicting data. While some studies do not present differences in washing time biocompatibility results, ranging from 5 min to 1 h [25,28], others demonstrate a significant improvement in cell viability related to an increase in washing time, reaching their best results at 20 min [20,21].

Surface treatment of resin-based 3D-printed dental devices is recommended to reduce surface roughness, which can minimize biofilm formation and enhance the longevity of prosthetic appliances. For a long time, it has been known that the surface roughness of materials plays a critical role in bacterial adhesion, and smoother surfaces exhibit a significantly lower risk of biofilm development [34]. A novel approach involves applying unpolymerized 3D printing resin as a coating agent to the device’s surface, which can be particularly advantageous when physical modifications, such as abrasive polishing, are not desired—for example, in the intaglio surface of dentures. This technique has been shown to improve surface smoothness and mechanical properties without altering the physical structure of the prosthetic. Additionally, in vitro studies using the L929 mouse fibroblast line have demonstrated that this method does not induce significant cytotoxic effects, as determined by LDL assay (ISO 10993-5 standards) [23,24].

Special attention must be paid to the post-curing treatment, which is essential to properly polymerize the coating resin [18]. On the other hand, when no coating is applied, mechanical polishing of 3D-printed devices with low-grit silicon carbide instruments is essential to reduce their cytotoxic effects. After a proper polishing procedure, 3D resin-based printed occlusal devices perform similarly regarding cytotoxic effects in human gingival fibroblast cell assays compared to milled and conventionally processed ones [14].

Post-curing protocols vary in the literature. The time of post-curing is the most studied variable, ranging from 1 min to 120 min [9,10,12,13]. An increase in post-curing time enhanced cell viability and biocompatibility in human periodontal ligament fibroblasts [9] and human gingival fibroblasts [10,13]. Data show that a post-curing time of around 20 to 30 min is necessary to reduce the cytotoxicity of 3D printable resins to clinically acceptable levels [9,10,12,13]. The temperature of the post-curing protocol ranges from 40 °C to 80 °C [13].

Data from the literature explained that the brand of the post-curing oven is not the most important issue to consider regarding the cytotoxicity of 3D print resins [23]. One may achieve excellent biocompatibility outcomes by associating high temperatures, such as 80 °C, a long post-curing period, such as 30 min, and high UV-light intensity [22]. These combined factors must be present in the post-curing process to promote a higher degree of conversion of the 3D-printed device, which is associated with better outcomes of cytotoxicity [27].

Different commercially available resins did not present statistical differences regarding cytotoxicity for human periodontal ligament fibroblasts when following the manufacturer’s instructions [11]. When compared to conventional materials, 3D-printed materials did not show significant differences to acrylic resins on human gingival fibroblast [16,19]. However, samples milled from resin blocks are less cytotoxic than printed ones [7], while conventional light-cured resin-based composites presented similar cytotoxic behavior to 3D printable resins [7,15]. After printing, washing, and post-curing a resin-based material, one may increase its biocompatibility by immersing the printed resin in 100 °C water for 1 to 5 min without affecting its mechanical or optical properties [24].

A limitation of this review is that it does not perform a statistical analysis, such as a systematic review with meta-analysis. Therefore, we cannot ensure what factors, overall, in the literature, significantly impact the outcomes. Future research could organize the extracted data to evaluate the statistical significance of different protocols on the cells’ viability.

5. Conclusions

This review highlights key practices for improving the biocompatibility of 3D-printed dental resins. Within the limitation of this review, it seems that washing devices for at least 20 min effectively reduces cytotoxicity by removing residual monomers and that the post-curing is crucial to ensure fewer possible cytotoxic effects. High-intensity UV-light post-curing for 20–30 min seems to significantly decrease cytotoxicity. Overall, these findings underline the importance of both post-curing and washing protocols in enhancing the biocompatibility of 3D-printed dental resins and emphasize the need for standardized post-processing of these materials.