Testing survival of cells found in oral environment on Liqcreate Bio-Med Clear 3D-print resin
Liqcreate collaborated with Fontys University of Applied Sciences to investigate cell adhesion and cell survival rate on Bio-Med Clear, a 3D-printable photopolymer resin developed and manufactured by Liqcreate. Previous studies have demonstrated that various bacterial species and human cell lines are capable of adhering effectively to this material with a high cell survival rate.
The current study aims to evaluate whether the micro-organisms Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans can continue to adhere to and remain viable on the Bio-Med Clear resin over an extended period of up to three weeks. These particular microorganisms were selected due to their frequent presence in the human oral environment, making them relevant for assessing the potential clinical applications of the material.
Image: successful growth of Candida albicans yeast on Sabouraud agar in the presence of Liqcreate Bio-Med Clear, biocompatible 3D-printed resin parts.
Tested bacteria and yeast on Liqcreate Bio-Med Clear
A wide variety of bacterial species can be used to study adhesion capacity. The most critical factor in evaluating this property is the surface characteristics of the bacterium, particularly the composition of the cell wall and the associated surface proteins. For this experiment, three micro-organisms were selected, each exhibiting distinct characteristics in terms of Gram staining, oxidase, catalase, and coagulase reactions. These tests were chosen because they represent fundamental and widely used methods for differentiating between microbial species. As such, a well-defined selection of three taxonomically diverse micro-organisms was established.
Additionally, these specific micro-organisms were selected due to their known presence in the human oral microbiota. This relevance to the oral environment supports the potential future use of Liqcreate materials for dental applications.
Staphylococcus aureus
Staphylococcus aureus is a Gram-positive coccus that typically occurs in clusters. It is part of the commensal flora in approximately 30% of the population, residing on mucosal surfaces, the throat, and the skin. S. aureus exhibits strong adhesive properties, attributed to its hydrophobic cell surface, which facilitates adherence to synthetic materials such as plastics. Furthermore, S. aureus produces a biofilm composed of extracellular polysaccharide matrices. Under unfavorable environmental conditions, it can secrete toxins that may lead to infections, particularly in individuals with compromised immune systems.
Image: Gram staining of staphylococcus aureus.
Image: Gram staining of Pseudomonas aeruginosa.
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative rod-shaped bacterium that is strictly aerobic and classified as a non-fermenter, meaning it does not metabolize glucose. It is capable of surviving in nutrient-poor environments and can persist under unhygienic conditions, provided there is sufficient moisture. Like S. aureus, P. aeruginosa produces a biofilm and possesses a hydrophobic cell surface, which enhances its ability to adhere to plastic materials. In addition, it utilizes type IV pili to facilitate surface attachment. P. aeruginosa can also produce toxins, posing a risk of infection in immunocompromised individuals.
Candida albicans
Candida albicans is a Gram-positive diploid fungus with a unique ability to undergo phenotypic switching between unicellular yeast and multicellular hyphal forms. This transition can occur spontaneously and repeatedly. Each morphological form exhibits different affinities for host tissues and distinct patterns of antigen expression. In its yeast form, C. albicans engages in hydrophobic interactions with hydrophobic surfaces and exhibits relatively weak biofilm formation. In contrast, the hyphal form is associated with stronger biofilm development and increased expression of genes regulating adhesion proteins. C. albicans can also secrete toxins and form spores under stressful conditions, enabling it to cause opportunistic infections in immunocompromised hosts.
Image: Gram staining of candida albicans.
In conclusion, all tested micro-organisms were able to survive on the 3D-printed Bio-Med Clear resin material over a three-week period, supporting its potential application in biomedical and oral use where microbial interaction is a relevant factor.
Researchers from the University of Applied Science, Eindhoven, Netherlands
viability and adhesion capacity of cells on 3D-printed Bio-Med Clear resin
The study commenced by assessing the viability and adhesion capacity of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans over a period of five days. These micro-organisms were incubated in 8 mL of BHI alongside a 3D-printed polymer, without any addition of fresh growth medium. This preliminary step aimed to determine whether the organisms could remain viable under nutrient-limited conditions and whether they could successfully adhere to the 3D-printed material. Following successful validation of this approach, a modified protocol was implemented.
In the second phase, the micro-organisms were incubated in a reduced volume of 1.5 mL BHI, again in the presence of the 3D-printed resin, for five days. This method served a dual purpose: to assess microbial survival in a more restricted environment and to test an alternative technique for detaching adhered organisms from the polymer surface. After confirmation of this method’s effectiveness, a long-term experimental design was employed.
For the long-term test, the micro-organisms were cultured for a duration of three weeks in 1.5 mL of BHI with the 3D-printed polymer. To evaluate the impact of nutrient availability, parallel sample sets were maintained—one with periodic replenishment of the growth medium and one without. This distinction allowed for observation of microbial survival and adhesion under both sustained and nutrient-depleted conditions.
The results demonstrated that all tested micro-organisms retained the ability to adhere to the 3D-printed polymer after three weeks. In the non-replenished samples, P. aeruginosa exhibited strong adhesion at week two, followed by a slight decline by week three. S. aureus showed relatively stable adhesion across the three-week period, correlating with the bacterial concentration in the surrounding medium. C. albicans displayed a progressive increase in adhesion capacity over the same duration.
In the medium-replenished condition, P. aeruginosa again showed peak adhesion at week two, followed by a decrease in week three. S. aureus exhibited reduced adhesion, likely due to contamination that negatively impacted its growth and surface attachment. C. albicans continued to show increasing adhesion over time, although the increase was less pronounced than in the non-replenished condition.
Conclusion
The objective of this study was to investigate whether the micro-organisms Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans retained their adhesion capacity on the 3D-printed polymer resin Bio-Med Clear and whether they could survive over a period of three weeks without the addition of fresh nutrients. The results demonstrated that all three micro-organisms were capable of adhering to the Bio-Med Clear 3D-printed resin over an extended period and could survive in the absence of nutrient replenishment.
Pseudomonas aeruginosa exhibited increased adhesion during the second week, followed by a decline in the third week. Staphylococcus aureus maintained a relatively stable level of adhesion, although a decrease was unexpectedly observed in the nutrient-supplemented samples. Candida albicans displayed a continuous increase in adhesion capacity over the three-week period, potentially influenced by environmental stress resulting from nutrient limitation. Among the tested organisms, P. aeruginosa demonstrated the highest overall adhesion to the Bio-Med Clear polymer.
In conclusion, all tested micro-organisms were able to survive on the 3D-printed Bio-Med Clear resin material over a three-week period, supporting its potential application in biomedical and dental environments where microbial interaction is a relevant factor.
Liqcreate Bio-Med Clear
Liqcreate Bio-Med Clear is a rigid clear biocompatble photopolymer resin and can be processed on most resin based 3D-printers. 3D-printed parts from this material exhibit biocompatible properties when post processed according to the processing instructions1. After washing and post-curing according to the instructions, printed parts from Liqcreate Bio-Med Clear pass the biocompatibility tests of:
| ○ Cytotoxicity | ISO 10993-5:2009 |
| ○ Sensitization | ISO 10993-10:2021 |
| ○ Irritation | ISO 10993-23:2021 |
Printed parts from Bio-Med Clear can be disinfected with commonly used disinfectants and sterilized by steam sterilization using an autoclave.
Key benefits |
3D-Printer compatibility |
| · Biocompatible | · Asiga series |
| · Steam sterilization possible | · Phrozen series |
| · High accuracy | · Elegoo & Anycubic series |
| · Dimensional stable | · And many more |
