Introduction

Recently, manufacturing technique is useful to create physical models for different medical and pharmacological application based on three-dimensional (3D) computed data [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19]. All these applications strongly rely on material properties such as mechanical and thermal characteristics, biocompatibility, and in the last years, the antibacterial capability became important factor because of the undesirable infections. Parallel with this demand the different sterilization methods used in case of 3D printed models were checked more rigorously [20, 21]. The most frequently used sterilization methods for poly-lactic acid (PLA), which is the favorite among the 3D printing materials, are the follows: (1) steam sterilization (at high pressure and ~ 130 °C), (2) dry sterilization (close to 200 °C), (3) sterilization with radioactive sources and (4) gas sterilization (mainly in ethylene oxide). Their application depends on the local possibilities, but each one has advantage/disadvantage (deformation, degradation, toxic residues, etc.). Of course, the sterilization method and printing temperature are greatly dependent on the thermal characteristics of the most commonly used 3D printing materials. Another important point in the fabrication of PLA-based medical device is the biocompatibility and antibacterial character [22, 23]. The 2009 WHO Guidelines for Safe Surgery summarizes all the evidences and recommendations about perioperative precautions and round of duties [22]. According to it, minimizing contamination in the operating room is essential because generally the environment of interventions can pose a risk to patients. Moreover, medical device development used outside of the operating room must also fulfill the requirements stated in the ISO 10993 series [23]. Overall, minimizing contamination risk is a basic principle in all these cases.

Silver nanoparticles are widely investigated in connection with many different aspects such as antibacterial agents [24,25,26,27], its possible antiviral effects [28], its antifungal [29] and antiparasitic activities [30], and all agree on silver ions and silver-based compounds have distinct antimicrobial effects. Adopting these findings to 3D printing is a recent concept that has a limited literature yet, and it is mostly confined on creating custom conductive complex structures [31,32,33]. A 3D printed antimicrobial medical device study has already been conducted [34], but Sandler et al. used a drug–polymer mixture containing antimicrobial active pharmaceutical ingredient (API) for that purpose. Utilizing silver nanoparticles in 3D printing process as an antimicrobial agent is a novel aspect.

In recent paper, we planned to check the thermal behavior of a novel PLA-Ag, and nanoparticles containing 3D printing materials using TG/DTA in wide temperature range (from 25 to 250 °C), comparing with the data of our previous paper [35]. Simultaneously, we checked their response on the attack of clinically most aggressive bacteria.

Materials and methods

Materials

PLA

We have tested different PLA-based plastic fibers made experimentally with twin-screw extruder by Filamania LLC/HU (Limited Liability Company, H-2310 Szigetszentmiklós, Fenyőfa str. 23., Hungary) planned for usage in additive manufacturing technology (FFF/FDM printing). The source of base material was purchased from NatureWorks LLC/US (15305 Minnetonka Boulevard Minnetonka, MN 55345 USA). The density of raw material (pellet) was between 1.23 and 1.25 g cm−3, with approx. 67% amorphous and 37% crystalline structure. The D-lactide content—according to NatureWork database—is between 1.2 and 14%; Filamania LLC/HU did not provide the exact value for the tested materials.

PLA-HDT

The base material was the same as in case of PLA; the preheat treatment procedure during the production was made by Filamania LCC/HU.

PLA-Ag

It is a PLA-based plastic 3D printing filament mixed with Ag-NPs (NP: nanoparticle). Fabrication was done similarly, as in case of PLA, by the same company. The silver content, the applied colloid and homogeneity of the distribution of the nanoparticles, is a part of the production patent.

In case of printing of all tested materials, the recommended speed of the print head in X–Y direction is 60 mm s−1 and the thread pull speed is about 15–20 mm s−1. The proposed printing temperature for PLA and its composites is 215 °C, recommended for 100–400-micron print thickness with reduced print head/extruder and tray sink speed.

Bacterial testing

The tested bacteria were: (1) Micrococcus luteus–Sarcina lutea ATCC 9341. This bacterium is common in transient skin flora of humans; it can produce carotenoid pigments, resulting in growth of yellow colonies on agar media. The Gram-positive Micrococcal and Bacillus species, e.g., (2) Bacillus subtilis ATCC 6633, have industrial uses as bioassay organism for detection of antimicrobial agents. They are occasionally isolated from human clinical specimens where they usually represent contaminants from the skin flora or from the environment [36].

The Gram-positive (3) Staphylococcus aureus ATCC 25923, the Gram-negative (4) Escherichia coli ATCC 25922 and (5) Pseudomonas aeruginosa ATCC 15442, originally were isolated from clinical samples as they are human pathogenic bacteria. They can cause, e.g., wound, respiratory and urinary tract infections [37].

In our experiment, we used BBL™ Mueller–Hinton agar media (Becton, Dickinson and Co. France). The surfaces of media were continuously inoculated with test bacteria; the test disks containing presumably antimicrobial materials were placed on the inoculated surface of the media. After 24 h incubation at 37 °C in a thermostat, the disks were removed and discarded.

TG/DTA measurements

We have used a SSC 5200 SII TG/DTA equipment (made by Seiko, Japan) to study the melting (fusion) properties of the different plastic fibers. The temperature and enthalpy calibration were made by Indium (Alfa Aesar PURATRONIC, Johnson Matthey Company, Ward Hill, MA, USA) using its thermal parameters from Thermal Applications Note TA Instruments (TN-11, Ref. [38]): the heat of fusion was set as 28.57 J g−1, and the melting temperature was taken to be 156.5985 °C [39]. Open aluminum sample pans of 5 mm diameter were used. The heating rates during the measurements were 10–20–30–40 K min−1 from room temperature up to 250 °C, and we applied N2 gas with 50 mL min−1 flow rate. Under the cooling process of melted samples, we did not use external cooler. The average sample mass was 9 ± 1 mg (rounded to integer value).

Results

The heating test of base PLA, as well as PLA-HDT and PLA-Ag plastic fibers between room temperature and 250 °C, can be seen in Figs. 1–3 (heating rates of 10 and 40 °C min−1 are only in all plot, as an average of three trials). The thermal parameters determined from the experimental curves are presented in Tables 1 and 2.

Fig. 1
figure 1

Heating and (spontaneous) cooling curves (inserts) for PLA base fiber: a with 10 °C min−1 and b with 40 °C min−1 heating rate. Solid lines: DTA average curves, dashed lines TG curves. Here and in all further figure too, the endotherm process is downward

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Fig. 2
figure 2

The melting (fusion) and cooling curves of PLA-HDT (symbols as in Fig. 1). In case of 40 °C min−1 heating rate, there was no crystallization in the spontaneous cooling phase

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Fig. 3
figure 3

The TA curves of PLA-Ag nanocomposite fibers (symbols as in Fig. 1)

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Table 1 Characteristic temperature values in heating/cooling cycle of different PLA fiber samples (Ton starting, Tend final temperature of process, Tg glass transition temperature, Thc crystallization at heating, Tcc crystallization at cooling and Tm melting (fusion) temperatures)
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Table 2 Enthalpy values in heating/cooling cycle of different PLA samples [exo (crystallization) negative and endo (melting or fusion) with positive sign]
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The DTA curves of PLA fiber are demonstrated in Fig. 1a, b. It shows the well-known parts of the polymer heating process: in case of Fig. 1a, a glass transition around 60, crystallization ~ 118, the fusion (melting) at ~ 151 °C. During cooling, a crystallization ~ 61 °C (see Table 1) was observed. Applying 40 °C min−1 heating rate, the Tg increased (~ 65 °C), and instead of a definite crystallization peak it could be seen an exo-like range (105–140 °C). During cooling, a double-crystallization peak was produced: a surprising ~ 94 °C, and a shoulder around the heating Tg range. The observed heating/cooling rate dependence is in a good correlation with data published by Ádám et al. [40].

The transition enthalpies varied strongly in the function of heating rate: both crystallization and melting ones were higher at 10 than at 40 °C min−1 rate during heating. It reversed at cooling crystallization. In the investigated temperature range, a remarkable mass loosening cannot waited according to the literature, but a shoulder can be seen at 10 °C min−1 rate after the end of glass transition, while at 40 °C min−1 rate at the beginning of an “exo-like” jump. After the melting, the mass change was negligible (max. 0.5 µg).

The PLA-HDT fibers (see Fig. 2a, b) showed clearly the effect of preheat treatment. The temperature range of glass transition and the Tg as well as the Tm increased compared to PLA, and all parameters were higher at 40 °C min−1 rate. At this heating rate, a definite “baseline” was not achievable after glass transition. The crystallization transition was wider with smaller peak at 40 °C min−1 rate. During cooling only at 10 °C min−1 rate exhibited crystallization, which also had a shoulder, and its lower compound (see Fig. 2a) was only in the range of Tg. The transition enthalpies were higher in both heating rates than in case PLA. The mass losses were also significantly higher than in PLA.

PLA-Ag nanocomposite (see Fig. 3a, b) showed similar TA characteristics than PLA-HDT during heating. Its thermal parameters were greater than at PLA, exhibiting stronger internal structure caused by Ag nanoparticles. The half width of the transitions was higher, compared with PLA, showing the decreased cooperativity among the structural compounds. The increased enthalpies are reflecting also on the more stable structure. The mass loss was also higher than in PLA in case of both heating rate.

The bacterial test of 3D printed specimens (disks with 15 mm diameter and 5.7 mm height), after their dezincification, provided an unexpected result (see Fig. 4). It was surprising that under the test disks (after its removal) the media were clear, without any colonies. This situation—free of colonies—remained for weeks independently from the type of species (see Fig. 5). The best picture (see Fig. 4) developed during M. luteus test strain: the yellow colonies and clear zones were impressive. In P. aeruginosa test strain, the clear zones were greenish discolored, as this bacterium produces two water-soluble pigments—pyocyanin and fluorescein—and these pigments diffused into the clear zones, and it is difficult to make a good-quality photograph demonstrating that these spots are free of colonies. Sometimes at the site of disk containing test material (No. 4), a few contaminating colonies were found (very probably the reason is the bad removing of the test sample by forceps, pushing some colony from the base into the original place of the sample).

Fig. 4
figure 4

PLA-based sample disks in different bacterium milieu: Symbols are: bacterial beds BS, Bacillus subtilis; SA, Staphylococcus aureus; EC, Escherichia coli; PS, Pseudomonas aeruginosa; SL, Sarcina lutea. Printed samples: 1—PLA “Model” (20% CaCO3), 2—PLA, 3—PLA-Ag, 4—PLA “Gypsum” (50% CaCO3), 5—PLA-HDT. Photographs were made 5 days after the removal of disks (row a, except of PLA), 8 days of removal (row b) and at 11th day (row c)

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Fig. 5
figure 5

PLA samples in SL—Sarcina lutea bed. Upper left PLA-Ag, upper right PLA-HDT and bottom PLA disks: a in 5 days, b after 10 days, c after 15 days and d after 19 days of removing the plastic disks. The two neighbor Petri-disks differ from each other only in the percentage of print completeness

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Discussion

There is an increased interest in medical/pharmacological practice for using the different biodegradable polymers. This way they are frequent objects of different TA analysis. They are appropriate to use (fibers or granules as basic materials) for the microencapsulation of drugs having short biological half-life but frequent administration. It is important the long-term stability of controlled-release dosage forms too [7, 41,42,43,44]. The indiscriminate use of antifungal agents has led to the advancement of microorganisms tolerant to the various drugs used in the market [45]. An antibacterial and antioxidant activity of a PLA Nano-silver composite was recently reported [46]. According to our results, the PLA-Ag composite can be used widely in different surgical intervention using 3D printed products, because it has better thermal parameters than PLA (higher Tg, Thc, Tm and Tcc as well as calorimetric enthalpy values, see Tables 1, 2). The bacterial test result was very surprising for us because the usual halo (“paraselene”) around the printed sample did not appear (see Fig. 4). Instead of it—after removal of sample disks and waiting relative long time—bacteria did not enter into the original place of sample disks from the surrounding (see Figs. 4, 5). Some entrance appeared, but it was the consequence of wrong removal of disks, and under the PLA-CaCO3 composite, a netlike structure appeared, probably as the consequence of looser structure filling.

Conclusions

Technological developments made a revolution and play a central role in the improvements of medical methods. The most commonly used PLA is well-characterized material in science, but the thermal and mechanical properties of the composites based on its application have been not well described yet. Our present results have showed that the investigated PLA-HDT and PLA-Ag can be potentially used in most biomedical applications. These results also highlighted to the careful but essential sterilization processes during development and application of surgical implants and tools. Because of the unmissable sterilization process, the thermal structural stability of the material remains a key point. It means that disinfection at high temperature should be avoided. Thus, HDT-PLA and PLA-Ag can be promising materials as a base material for composites suitable for heat-based sterilization methods. The strange antibacterial effect of the tested samples is also interesting; this way the infections after implantation of printed elements could be minimalized. The experimental setting used in our present study has several advantages to assess the quality and safety of 3D printed materials, and thus, it deserves further attention in order to determine the detailed protocol and processes necessary for possible future therapeutic use.