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Research Article
This study evaluates biofilm formation on 3D-printed polylactic acid (PLA) and polyethylene terephthalate glycol (PETG) using static, semi-static, and dynamic in vitro models. The methodology supports safer medical device development and advances biofilm research in 3D-printed biomaterials.
Three-dimensional (3D) printing has accelerated the development of customized medical devices, but biofilm formation on printed materials remains a major threat to implant safety and performance. Because material chemistry and print-dependent surface features can influence bacterial attachment and antibiotic tolerance, standardized in vitro approaches that enable meaningful comparisons across commonly used 3D-printing polymers are needed. Here, we compare biofilm development on polylactic acid (PLA) and polyethylene terephthalate glycol (PETG) using a multi-model in vitro evaluation framework that captures complementary aspects of biofilm biology, including early adhesion, maturation, and antimicrobial tolerance, under both static and flow conditions. S. aureus biofilms were established and assessed using quantitative (viable bacterial load and minimal biofilm eradication concentration [MBEC]) and qualitative (scanning electron microscopy) endpoints. Both PLA and PETG supported biofilm formation across models; however, PLA tended to show higher early adhesion and greater biofilm density. In static assays, PLA demonstrated higher CFU values than PETG, whereas vancomycin MBEC values were similar between materials. Assay-dependent differences in MBEC were observed across platforms, underscoring how model structure can influence apparent antimicrobial susceptibility. Under dynamic flow, biofilm burden increased relative to static conditions, with minimal material-dependent differences. Collectively, these results highlight the susceptibility of both polymers to biofilm formation and demonstrate the value of a multi-model framework for evaluating material-associated biofilm behavior and benchmarking antimicrobial performance on 3D-printed device-relevant substrates.
Recent advances in 3D printing have led to the growing adoption of 3D-printed medical devices due to their cost-effectiveness, high precision, complex structural design, efficient material utilization, and patient-specific design1,2. These advancements are driving significant clinical progress in the development of customized orthopedic3,4,5,6, craniofacial7,8, dental1,9
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All hazardous chemicals (e.g., glutaraldehyde, sodium cacodylate buffer, Hexamethyldisilazane [HMDS], and ethanol) were handled in accordance with institutional Environmental Health and Safety (EHS) policies. Preparation and use of volatile/toxic reagents were performed in a certified chemical fume hood with appropriate personal protective equipment (lab coat, eye protection, and chemically resistant gloves), and flammable solvents were kept away from ignition sources. Chemical wastes were segregated and collected in properly labeled containers (including dedicated arsenic-containing waste for cacodylate and solvent waste for HMDS/ethanol) for disposal through the ins....
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Disc model of static biofilm
This assay is used to determine the MBEC on individual specimens. Progressive two-fold dilutions of vancomycin demonstrated baseline biofilm formation of 7.48 × 105 CFU/mL [IQR 6.34–8.72] for PLA and 3.60 × 105 CFU/mL [IQR 3.08–5.52] for PETG. Although both materials showed biofilm densities within the 105 range, a statistically significant difference in biofilm production was observed between them (p = 0.015). When exposed to vancomycin, .......
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This study proposes a comprehensive in vitro evaluation of biofilm formation on 3D-printed medical-grade polymers, specifically PLA and PETG, using four complementary experimental platforms: static disc assays, 24-well plate models, the CBD, and a dynamic flow system based on an MRD. By integrating these models, the study aims to characterize how distinct printing materials behave when exposed to S. aureus biofilm induction, and to determine the MBEC of vancomycin across different test conditions. This .......
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Authors have no conflicts of interest. ChatGPT was used for grammar review.
We would like to thank Renee Fleeman for the invitation.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 0.9% NaCl | Fresenius Kabi Brasil, Ceara, Brazil | 17811 | |
| 3D printer with Fused Deposition Modeling technology (Bambu X1-Carbon) | Bambu Lab, Shenzhen, China | PF001-P | |
| Blood agar | NewProv, Paraná, Brazil | PA31 | |
| Calgary device for biofilm downloadable | No company | See link (without catalogue numbers) | https://www.printables.com/model/1464751-calgary-model-biofilm |
| Cell culture multiwell plate, 24 well | Greiner Bio-One, Kremsmünster Österreich, Austria | 662160 | |
| Centrifuge (5910 Ri) | Eppendorf, Hamburg, Germany | 5943000511 | |
| Centrifugue tube sterile 15 mL | Corning, Missouri, USA | 430790 | |
| Centrifugue tube sterile 50 mL | Corning, Missouri, USA | 430291 | |
| Electronic Pipette Controller (Easypet® 3) | Eppendorf, Hamburg, Germany | 4430000018 | |
| Ethanol (≥99.5%, ACS reagent) | Sigma-Aldrich, Missouri, USA | 459844 | |
| Glutaraldehyde | Merck, Darmstadt, Germany | 8143931000 | |
| Hexamethyldisilazane | Merck, Darmstadt, Germany | 440191 | |
| Incubator (502/4-C) | Fanem, São Paulo, Brazil | 502007700 | |
| Laminar Air Flow cabinet (BioSEG 9) | Veco, São Paulo, Brazil | 1EAC3770 | |
| Mechanical pipette (Eppendorf Reference® 2) | Eppendorf, Hamburg, Germany | 4924000061 | |
| Modified Robbins Device downloadable | No company | See link (without catalogue numbers) | https://www.printables.com/model/1481700-robbins-device-modified |
| Nephelometer | Alfakit, Florianópolis, Brazil | 7718 | |
| Orbital shaker (INNOVA 44R) | Eppendorf, Hamburg, Germany | M1282-0314 | |
| Parafilm M | Amcor, Illinois, USA | PM-996 | |
| Peristaltic pump (Minipuls® 3) | Gilson, Villiers-le-Bel, France | F117604 | |
| Petri dish (90 x 15 mm) | Greiner Bio-One, São Paulo, Brazil | 630164 | |
| Pipette tip 1–200μL | Corning, Missouri, USA | CLS4142 | |
| Polyethylene terephthalate glycol (PETG) | Creality, China | 44759 | |
| Polylactic acid (PLA) Ultrafuse® PLA PRO1 | BASF, Maarssen, Netherlands | PR1-7501A075 | |
| Rotary pump (Q150R ES Plus) | Quorum Technologies, Lewes, UK | 13034 | |
| Scanning electron microscope (Vega3 LMU) | Tescan, Brno, Czech Republic | See link (without catalogue numbers) | https://tescan.com/product-portfolio |
| Serological pipette 10 mL | Corning, Missouri, USA | CLS4488 | |
| Sodium cacodylate | Sigma-Aldrich, Missouri, USA | C0250 | |
| Staphylococcus aureus ATCC® 25923 strain | Laborclin, Pinhais, Brazil | 660674 | |
| Sucrose | Sigma-Aldrich, Missouri, USA | 59378 | |
| The 24-well flat-bottom downloadable | No company | See link (without catalogue numbers) | www.printables.com/model/104339-24-well-plates |
| Tryptic soy agar | Kasvi, Madrid, Spain | K25-1068 | |
| Tryptic soy broth | Sigma-Aldrich, Missouri, USA | 22092 | |
| Ultra Low Freezer ( IULT 335 D) | Indrel, Paraná, Brazil | 12038 | |
| Ultrasonic bath (Soniclean 15) | Sanders Medical, Minas Gerais, Brazil | See link (without catalogue numbers) | https://sandersdobrasil.com.br/produto/lavadora-ultrassonica-soniclean-15d/ |
| Vancomycin | ABL, São Paulo, Brazil | 15,56,20,041 | |
| Vortex (FLEXVORTEX) | Loccus, São Paulo, Brazil | 4500 |
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