Edited by: Mark Smeltzer, University of Arkansas for Medical Sciences, United States
Reviewed by: Devendra Dusane, Nationwide Children’s Hospital, United States; Lichong Xu, Pennsylvania State University, United States
†These authors have contributed equally to this work
This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Recent advances in 3D printing have led to a rise in the use of 3D printed materials in prosthetics and external medical devices. These devices, while inexpensive, have not been adequately studied for their ability to resist biofouling and biofilm buildup. Bacterial biofilms are a major cause of biofouling in the medical field and, therefore, hospital-acquired, and medical device infections. These surface-attached bacteria are highly recalcitrant to conventional antimicrobial agents and result in chronic infections. During the COVID-19 pandemic, the U.S. Food and Drug Administration and medical officials have considered 3D printed medical devices as alternatives to conventional devices, due to manufacturing shortages. This abundant use of 3D printed devices in the medical fields warrants studies to assess the ability of different microorganisms to attach and colonize to such surfaces. In this study, we describe methods to determine bacterial biofouling and biofilm formation on 3D printed materials. We explored the biofilm-forming ability of multiple opportunistic pathogens commonly found on the human body including
Biofouling is the process of microorganisms attaching to solid inanimate surfaces as biofilms. It is estimated that biofouling costs billions of dollars per year and is a problem within many fields of science and industry, including the medical fields (
The major cause of biofouling in the medical setting is bacterial cell attachment (
Use of 3D-printed prosthetics is increasing because of their relative cost-effectiveness compared to that of conventional prosthetic devices (
Because 3D-printed materials are being used more frequently as medical materials, there is an overwhelming need to develop antifouling 3D-printing materials. There are many materials already on the market that utilize the simple method of impregnating the polymer filament with nanoparticles, fibers, or metal flakes (
The resolution on 3D printers is improving rapidly and high-resolution printers are affordable even as desktop devices. The resolution at which a typical extruder printer operates is 200 μm between layers and all polymers lead to an inherent surface roughness upon printing. These rough surfaces can provide an ideal environment for the initial attachment of bacteria and subsequent formation of biofilms (
In this study, we worked with several common
PLA polymers (
3D polymers used in this study.
Brass 3D Printer Filament | Yoyi 3D | 33- 40% metal powder and 67-60% PLA | BRS |
Copper 3D Printer Filament | Yoyi 3D | 33% metal powder and 67% PLA | CU |
Aluminum 3D Printer Filament | Yoyi 3D | 33% metal powder and 67% PLA | AL |
3D Printer Filament Frosted Bronze | AMOLEN | 20% metal powder and 80% PLA | BRZ |
Carbon Fiber Black | 3D Solutech | 70% PLA and 30% carbon fiber. | CF |
3D PLA Wood color | AmazonBasic | 70% polymer and 30% wood. | WD |
Black Soft PLA | MatterHackers | SoftPLA | |
Silver Metal 3D Printer PLA Filament | 3D Solutech | Silver Dye (no metal infill) | PLAS |
Purement | Purement BnK Chemical Company | Copper | Pur |
PLActive | Copper3D | Copper | PLAc |
Antibacterial PLA | XYZ Printing | Silver ions | XYZ |
The Dataphysics (Charlotte, NC, United States) Optical Contact Angle OCA 15EC measuring system was used with MilliQ® water to measure surface wettability. The surface angle of 1 μL drop was recorded in 4 different locations along the 3D printed slides and controls.
A Mitutoyo (Kanagawa, Japan) JS210 contact profilometer was used to analyze surface properties of the materials using standard parameters like JIS, VDA, ISO-1997 and ANSI. Standard linear (2D) parameters (Ra, Rq, Rz, Rp, Rv, Rsk, Rku, Rc, RSm, RDq, Rmr, Rdc, Rt, Rk, Rpk, Rvk) (
A Leica (Leica United States) DVM6 digital microscope and MountainsMap® ver.7.4 (Digital Surf SARL, France) were used to collect and analyze 3D images. Standard surface 3D parameters S (Sq, Ssk, Sku, Sp, Sv, Sz, Sa, Sz, Smr, Smc, Sxp, Sdc) (
Printed out 3D rings were sterilized for 30 min in 70% ethanol followed by washing twice in sterile water or autoclaving in MilliQ® water (15 min) or just a dry cycle (15 min- sterilization/10min-drying). Rings were placed in 24-well plates (
Overnight cultures (25 mL) of bacterial strains in LB Miller medium were transferred into 50-mL conical Falcon tubes (
Printed pins were sterilized (autoclaved 15 min/10 min) and placed into 500 μL of 100x dilutions of bacterial overnight cultures in 48-well plates (Corning United States). Biofilms were grown for 3 days at 37°C (50 rpm) in Forma Model 3,950 incubator (> 90% humidity). Pins were submerged in the culture entire time and no change in liquid level was observed. After 3 days pins were removed and bacterial cell densities (OD600) were measured in the medium. Pins were washed twice in PBS and placed into 700 μL BacTiter-GloTM Reagent (Promega) (
A Leica DVM6 Digital Microscope was used to collect pictures of 3D-printed slides with and without bacterial biofilms. Biofilms were grown on 3D printed slides in 50 mL Falcon tubes as described in the Bacterial Adhesion section. After 3-day incubation at 37°C (50 rpm) biofilm-containing slides were washed two times with 1 × PBS solution. The entire slide was then fixed in 3% glutaraldehyde solution overnight at 4°C. After the fixation step, the slide was washed two times with distilled water and dried in a laminar flow hood for a period of 24 h. 3D projections were made using the MountainsMap® ver.7.4.
All experiments were conducted at least in triplicates. A standard unpaired t-test was used in the case of simple two-group comparisons. Two-way analysis of variance (ANOVA) was performed on the biofilm data. MountainsMap® ver.7.4. statistics module was used to analyze pictures.
Eight different PLA 3D materials (
Contact angle/wettability of water droplets on 3D printouts. AL, BRS, BRZ, CF, CU, PLAS, SoftPLA, WD (
To characterize the surfaces of the printed slides, we first used a Mitutoyo JS210 contact profilometer. The standard unit is equipped with a 5-μm radius stylus tip, which contacts the surface with 4-mN measuring force and measures 39 different parameters. For each printout, we conducted two kinds of measurements: perpendicular (
The surface profiles of 3D printed slides perpendicular (black, bottom) and parallel (red, top) the layers (representative of 4 measurements); Selected R parameters: Ra
Analyzing the cross-section profiles (
A Leica DVM6 3D digital microscope was utilized for surface profiling of 3D printed slides. Original pictures (
Digital microphotography of 3D printouts
Analyzing parameters calculated by the MountainsMap® software, we noticed that all R parameters showed lower values compared with those obtained with the contact profilometer (
In addition to linear (2D) R parameters, optical profilometry allows for calculation of areal (3D) parameters (
To analyze the effect of the 3D polymers on microorganisms, we used 3 different bacterial species:
Effect of polymers on bacterial growth
Bacterial cell density (OD600) measured after 24 h of growth at 37°C.
AL | 0.82 ± 0.14* | 0.98 ± 0.07* | 1.31 ± 0.18 |
BRS | −0.00925 ± 0.047* | 0.05 ± 0.04* | −0.02 ± 0.086* |
BRZ | 0.86 ± 0.14* | 0.76 ± 0.075* | 1.37 ± 0.18 |
CF | 0.66 ± 0.067* | 0.96 ± 0.19* | 1.46 ± 0.13 |
CU | 0.67 ± 0.18* | 0.88 ± 0.1* | 1.39 ± 0.11 |
PLAS | 0.81 ± 0.14* | 1.09 ± 0.16 | 1.43 ± 0.14 |
SoftPLA | 0.75 ± 0.24* | 0.99 ± 0.2 | 1.30 ± 0.15 |
WD | 0.64 ± 0.18* | 1.15 ± 0.17 | 1.42 ± 0.15 |
Control | 1.13 ± 0.14 | 1.26 ± 0.08 | 1.35 ± 0.22 |
Bacterial attachment is the first step in biofilm formation (
Printed 3D slides were submerged in bacterial cultures of
Effect of polymers on bacterial attachment after 2-h attachment
To test biofilm using a semi-high-throughput method, the 3D pins were designed to fit a standard 48-well plate (see
For all 3 tested bacterial species, the least amount of biofilm formation with the lowest corresponding cell density was found in the case of the BRS polymer (
Microscopic observation and analysis are the most common techniques for visualizing biofilms (
Pictures of representative biofilms formed on 3D printed materials.
In this work, we noticed that some of the tested polymers showed bacteriostatic effect on microbes. Recently, 3 antimicrobial PLA polymers have been released to the market: Purement Antimicrobial PLA, Copper 3D PLActive Antibacterial Nanocomposite, and YXZPrinting Antibacterial PLA (
In growth curve experiments, we noticed no difference between the PLActive polymer and normal PLAS (
Next, we compared biofilm formation on the antimicrobial polymers and our previously tested ones (
We showed that the BRS, BRZ, and CU polymers were the most inhibitory for
The smallest differences in biofilm formation on 3D polymers were observed in the case of
To conclude, none of the commercial antimicrobial polymers were better than our metal-filled polymers BRS, BRZ, and CU and the differences between the remaining polymers were not really striking.
Since its development in the early 1980s, 3D printing technology has become ever more popular in many fields of science and industry (
Several of our tested polymers have been filled with metals/alloys such as copper, brass, bronze, and aluminum. Copper, brass, and bronze have a long history of being used as antimicrobial agents (
Optical profilometry is a rapid, non-destructive, and non-contact surface metrology technique. An optical profiler is a type of microscope in which light from a lamp is split into two paths by a beam splitter and each light beam is used in forming topographic information (
Three common human-associated bacterial species (
In the case of
While analyzing biofilm formation on 3D printouts, we realized that 3 principal parameters (antibacterial properties, surface structure, and hydrophobicity of the polymers) play important roles. All 3 (or 4) metal-filled polymers with their antimicrobial properties showed the least amount of biofilm formed for all 3 bacteria species (
In the biofilm image analyses, it was not surprising that we observed that most of the biofilms filled the grooves between printed layers. In a several cases, the biofilms formed bridges between higher layers.
The antimicrobial properties of the polymers play a crucial role in reducing biofilm formation. The first antimicrobial PLA polymer with different concentrations of silver nanoparticles was developed in 2013; however, the fibers (not printouts) were tested only against planktonic cells (
To the best of our knowledge, this is the first report describing biofilm formation on 3D printed surfaces, with as many as 8 standard and 3 antimicrobial PLA polymers, as well as 3 of the most prevalent human colonizers:
The original contributions presented in the study are included in the article/
DH and JK designed the study, performed the experiments and analyses, wrote the original draft, and edited the manuscript. PP performed experiments, analyzed data, and edited the manuscript. GE supported the project and edited the entire manuscript. H-FJ supported the project. All authors read and approved the final version of the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Miller Microscopes (Leica), Steve Bucks (Hirox-USA), and Mark LaMarre (Keyence) for supplying 3D digital microscopes and Digital Surf for supplying a demo version of the MountainMap® software. We also thank Jocelyn Hammond for editing, proofreading, and comments on the manuscript.
The Supplementary Material for this article can be found online at:
Designed 3D slides, rings, and pins
Schematic representation of adsorption experiment. 1: 3D slide placed in overnight culture of bacteria; 2: 2 h incubation at 37°C, 50 rpm; 3&4: washing in sterile PBS; 5: Shaking 3 min 1000 rpm on Mini-G, SPEX-Sample-Prep; 6: Dilutions were plated by drip titration method.
Selected Mitutoyo R parameters on cross-sections of printouts ranked ascendant (average data from 4 measurements). Ra - Arithmetic mean deviation of the roughness profile; Rq - Root-mean-square (RMS) deviation of the roughness profile; Rz - Maximum Height of roughness profile; Rp - Maximum peak height of the roughness profile; Rv - Maximum valley depth of the roughness profile.
RSm (mean peak width) parameters on cross-sections of printouts (average data from 4 measurements).
Selected R parameters on cross-sections of printouts (average data from 9 measurements). Ra - Arithmetic mean deviation of the roughness profile; Rq - Root-mean-square (RMS) deviation of the roughness profile; Rz - Maximum Height of roughness profile; Rp - Maximum peak height of the roughness profile; Rv - Maximum valley depth of the roughness profile; Rc - Mean height of the roughness profile elements.
Pictures of representative biofilms formed by
3D projections of selected printouts obtained with Keyence VHX-7000 Laser Scanning Confocal Microscope. From left top: CU, SoftPLA, WD, CF, BRS, BRZ.
3D projections of selected printouts obtained with Hirox KH8700 microscope. From left top: CU, WD, SoftPLA, PLAS, BRS. Bottom:
Most commonly used surface parameters.
Characteristics of the 3D printouts generated by MountainMap® software.