Antibacterial molybdenum disulfide with silver phosphate coating on titanium fixator in rabbit models of rib fracture infection

Introduction

The prevalence of trauma and fractures has escalated due to the rise in traffic, natural disasters, and industrial accidents (1). Blunt chest wall trauma is a significant contributor to trauma admissions, representing >15% of the cases (2,3). Rib fractures are the most commonly encountered injuries after thoracic trauma, representing about 10–39% of such cases and accounting for approximately 10–15% of all trauma-related hospital admissions (4-7).

Surgical stabilization of rib fractures (SSRF) has resulted in favorable outcomes (8,9), but infection still occurs in about 0.5% of cases (10). Fracture-related infection (FRI) is a significant complication in trauma surgery and is often initiated by exogenous factors stemming from the trauma itself, including surgical intervention and potential bacterial contamination during the fracture management process (11). These infections impede wound healing and soft tissue coverage. The complexity of FRI’s etiology is underscored by the role of bacterial colonization and fracture instability. Complications arise from biofilms on foreign bodies, intracellular infections, and bacteria colonization of ectopic sites (12,13). FRIs impact patient health and represent significant socioeconomic burdens (14-16). Despite prophylactic efforts, FRIs affect 1–30% of patients with orthopedic trauma, with rates of 1–2% for closed fractures and up to 30% for open fractures (17-19). Staphylococcus aureus is the most prevalent pathogen in FRIs, being responsible for 30–42% of the cases (20).

Titanium (Ti) alloy metal implants are commonly used as rib fixators. Although infections of Ti fixators are relatively rare, at about 3%, they pose significant clinical challenges, often necessitating prolonged antibiotic use, debridement, or implant removal (21), impacting recovery from fracture and long-term functional outcomes (22-24). Therefore, the prevention of bacterial infections is critical for the long-term outcomes of patients undergoing SSRF (25).

Antibacterial coatings on implants decrease the occurrence of surgical site infections (26,27). A molybdenum disulfide with silver phosphate [MoS₂(S)-Ag₃PO₄] coating has been recently developed for Ti implants, showing good biocompatibility and, compared with their naked Ti implants, demonstrated enhanced bactericidal efficacy under 808 nm laser irradiation in vitro (28). Under 808 nm near-infrared light, MoS2 generates heat that disrupts bacterial cell membranes, while Ag₃PO₄ nanoparticles produce reactive oxygen species (ROS), further boosting antibacterial activity. Hence, the available studies examined the anti-biofilm and anti-infection properties of MoS₂(S)-Ag₃PO₄ coating for implants (26-31); although the successful management or prevention of infections will promote rapid bone healing, whether the MoS₂(S)-Ag₃PO₄ coating affects osteogenesis and fracture healing remains unknown. This issue is critical for bone healing, as it helps avoid delays in repair, prevents infection, and accelerates the recovery process, leading to better patient outcomes.

Rabbits are ideal models for infections due to their immunogenetic proximity to humans (32,33), but the literature is scant on rabbit models for rib fracture infections (RFIs), and the influence of such infections on fracture healing and local tissue environments remains under-explored. Establishing a stable rabbit model is thus pivotal for examining the role of infections in rib fracture prognosis and will pave the way for future preventative research in this field.

Therefore, this study aimed to determine the most optimal MoS₂(S)-Ag₃PO₄ coating for fracture healing in a novel rabbit RFI model. The hypothesis of this study is that MoS₂(S)-Ag₃PO₄ coating on titanium implants will effectively reduce bacterial infection and inflammation, while promoting osteogenesis and enhancing fracture healing in a rabbit RFI model. The results could help determine the clinical potential of MoS₂(S)-Ag₃PO₄ coating in surgical applications. The new model could also provide some standardization to research on the subject. We present this article in accordance with the ARRIVE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2024-2214/rc).

Methods

Rabbit RFI model

Thirty-two adult male New Zealand white rabbits (weighing 2,000–2,500 g) were purchased from the China Institute for Food and Drug Control. The experiments were approved by the Animal Use Ethics Committee of Tianjin University Tianjin Hospital (approval #2020-064), in compliance with Tianjin University Tianjin Hospital guidelines for the care and use of animals.

Firstly, this part aims to determine optimal bacterial concentrations for RFI model. Following a one-week acclimation period at the animal breeding center, twenty rabbits were randomly divided into four groups of five animals each. Rabbits were anesthetized with sodium pentobarbital (30 mg/kg) administered via intravenous injection through the marginal ear vein. Rib fractures were induced using tissue scissors to cut off the ribs. The eighth rib was chosen for its width and to avoid upper extremity scapula interference, with a large intercostal space minimizing pleura damage risk. Before closing the wound, each animal received 20 µL of culture medium at the rib fracture site, with varying S. aureus concentrations: 0, 1×10⁵, 1×10⁶, and 1×10⁷ colony-forming units (CFU)/mL. The group with 0 CFU/mL was designated as the negative control (NC) group, the group with 1×10⁵ CFU/mL as the 1×10⁵ CFU/mL group, the group with 1×10⁶ CFU/mL as the 1×10⁶ CFU/mL group, and the group with 1×10⁷ CFU/mL as the 1×10⁷ CFU/mL group. Strict hemostasis was applied to the surgical wound, followed by layered sutures on the wound.

Using the data from the rib measurements, a specialized rib fracture fixator and corresponding forceps were designed and manufactured specifically for the rabbits. The fractures were fixed using the designed fixators.

Secondly, this part aims to examine MoS₂(S)-Ag₃PO₄ coatings using a RFI model. Twelve adult male New Zealand white rabbits (2,500–3,000 g) were divided into a NC group with only a Ti fixator and an experimental group with a Ti fixator plus MoS₂-Ag₃PO₄ coating. For all rabbits, 20 µL of Luria-Bertani (LB) solution containing S. aureus at 1×10⁶ CFU/mL was applied to the rib fracture fixator. The operation procedure was the same as that used to establish the infection models of rib fracture in rabbits, except that the rib fracture fixator was placed at the fracture ends of the rib to reposition and stabilize the fracture. The fixators were placed with the fracture line at their center. Clamping pliers were used to press the wrapping jaws of the fixators in place. The operation site was then irradiated with 808-nm near-infrared light (0.4 W/cm²) for 15 minutes (Figure 1).

Figure 1 Research process. Created with BioRender. NIR, near infrared.

Specimen preparation

All rabbits from each group were sacrificed after 4 weeks by air embolism. A piece of 2×2×0.5 cm of soft tissue around the fracture was obtained. The specimen was placed in 10% neutral formalin solution and incubated at room temperature overnight or 24 h. The fixed specimens were rinsed with water for about 5 min. The specimens were placed in 80% ethanol for 1.5 h, 90% ethanol for 2 h, 95% ethanol for 1.5 h, 95% ethanol again for 2 h, and 100% ethanol for 2 h. The dehydrated tissue was sequentially placed in 50% ethanol + 50% xylene for 0.5 h, xylene for 0.5 h, and xylene again for 0.5 h. The specimens were embedded in paraffin.

Histopathological evaluation

After euthanasia on day 28, the major organs (heart, liver, spleen, lung, and kidney) of the rabbits from each group were collected for histological analysis. For hematoxylin-eosin (HE) staining, the specimens were cut into 4-µm sections. The sections were incubated at 70 ℃ for 1 h. The sections were rehydrated in xylene dewaxing solution, xylene dewaxing solution again, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol, for 10 min for each reagent. The sections were stained with the hematoxylin solution for 5 min and placed in 5% acetic acid for 1 min. The sections were rinsed with running water for 5 min and stained with the eosin staining solution for 1 min. The sections were dehydrated using the reverse sequence of solutions used for rehydration, with 2 min for each solution. The sections were fixed with neutral resin and observed using a light microscope.

Safranin-O staining

For the safranin-O staining, the specimens were cut into 4-µm sections. The sections were incubated at 70 ℃ for 1 h. The sections were rehydrated in xylene dewaxing solution, xylene dewaxing solution again, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol, for 10 min for each reagent. The sections were stained with safranin-O dye solution for 1 h, and the excess was washed with distilled water. The sections were incubated in ethanol at 50%, 70%, and 80%, each for 10 s. The sections were stained with the solid green staining solution for 60 s. Absolute ethanol was used for dehydration, three times, for 60 s each time. The sections were incubated in xylene for 5 min and mounted with medium gum. The sections were observed using a light microscope.

Bacterial culture

After euthanasia, the Ti and Ti-MoS₂-Ag₃PO₄ fixators were removed and placed separately into glass bottles containing 2 mL of pre-prepared sterile LB liquid medium and incubated at 37 ℃ and 200 rpm on a shaking incubator for 4 h. Then the culture medium was taken to measure the absorbance at 600 nm. Next, 20 µL of the culture medium was added to the LB solid medium. After incubating for 20 h at 37 ℃, the colony formation was observed and recorded, and the antibacterial efficiency was calculated.

Western blotting

The samples were homogenized (Huio Testing Equipment Co., Shanghai, China) and lysed in radio immunoprecipitation assay (RIPA) buffer supplemented with 2 mM phenyl methane sulfonyl fluoride (PMSF). A bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL, USA) was used to determine the protein concentration. Equal amounts of protein (30 µg) were separated using 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Lake Placid, NY, USA). The membranes were blocked with 5% bovine serum albumin (BSA) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and incubated overnight at 4 ℃ with diluted primary antibodies: mouse anti-β-actin (1:3,000; Sigma-Aldrich, St. Louis, MO, USA), rabbit anti interleukin (IL)-6 (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), and mouse anti-tumor necrosis factor (TNF)-α (1:1,000; Bioss Antibodies, Inc., Woburn, MA, USA). The membranes were exposed to enzyme-labeled secondary antibodies (1:1,000 dilution; Thermo Fisher Scientific) for 1 h at room temperature. Finally, the bands were visualized using Pierce Enhanced Chemiluminescence (ECL) substrate (Pierce Chemical, Dallas, TX, USA).

Hemolysis safety evaluation

Blood samples from the 12 rabbits with fixators were collected. Then, 1 mL of blood was diluted with 1 mL of phosphate-buffered saline (PBS) and centrifuged at 2,000 rpm for 15 min to obtain the red blood cells (RBCs). Different MoS₂-Ag₃PO₄ coatings were incubated with diluted RBCs for 4 h at 37 ℃ (n=3). The optical density (OD) was measured at 570 nm. The hemolysis positive control was 1% TritonX-100.

Blood test

After implanting the Ti and Ti-MoS₂-Ag₃PO₄ fixators in rabbits, RBC, platelets (PLT), neutrophils (NEUT), lymphocytes (LYPH), hemoglobin (HGB), and white blood cells (WBC) were measured on days 14 and 28 after implantation using hypocardial blood collection.

In vivo antibacterial test

Tissue specimens from the fracture sites of the rabbits were collected on days 2 and 28 for HE staining (as described above) and Gram staining. For Gram staining, rehydrated sections were incubated in crystal violet for 1 min, rinsed twice with distilled water, each time for 1 min, and excess water was removed using absorbent paper. Lugol’s iodine solution was added dropwise to the slide to cover the tissue and incubated for 1 min. The solution was rinsed twice with distilled water for 1 min each time, and excess water was removed using absorbent paper. A few drops of 95% alcohol were added to the tissue for 20 s for decolorization and then washed with distilled water, and excess water was removed using absorbent paper. Saffron solution was used for counterstaining for 1 min, washed with distilled water, and excess water was removed using absorbent paper. The sections were examined using a light microscope.

Statistical analysis

Statistical analysis was performed using Statistical Product Service Solutions (SPSS) 21.0 (International Business Machines Corporation (IBM Corp., Armonk, NY, USA). The data were described as means ± standard deviations and analyzed using one-way analysis of variance (ANOVA) and the Fisher’s least significant difference (LSD) post hoc test. P values <0.05 were considered statistically significant.

Results

Standard rabbit RFI model

The average thickness of the eighth rib was 1.82±0.21 mm, and the width was 2.99±0.26 mm (Table 1).

Two rabbits in the 1×10⁷ CFU/mL group died from pneumothorax caused by pleural rupture and were excluded. The changes in weight are shown in Figure S1A; the weight was stable in the NC, 1×10⁵ CFU/mL, and 1×10⁶ CFU/mL groups, but two out of three surviving rabbits in the 1×10⁷ CFU/mL group experienced weight loss. Tremors were also observed in two animals in the 1×10⁷ CFU/mL group (Table 2). As shown in Figure S1B, there were no toxic or inflammatory effects on the major organs. As shown in Figure S2 and Table 3, the blood parameters remained within the normal range for all rabbits.

Effects of bacterial load on wound healing and inflammation

The incision and rib fracture are shown in Figure 2A. In the NC group, the incision healed well, but the 1×10⁵, 1×10⁶, and 1×10⁷ CFU/mL groups showed different degrees of pus coating and abscesses. The bacterial culture of the intercostal tissues was positive (Figure 2B-2D) (1×10⁵ group vs. the NC group, P=0.006; 1×10⁶ group vs. the NC group, P=0.003; 1×10⁷ group vs. the NC group, P=0.002), as confirmed by Gram staining (Figure 2E).

Figure 2 Effects of bacterial load on wound healing and inflammation. (A) Surgical incision and fracture ends. (B) Bacterial culture medium after incubation on a 37 ℃ and 200 rpm constant temperature shaker for 4 h. (C) The optical density at 600 nm of different groups. (D) Bacterial colony images of different groups. (E) Gram staining of the tissue around the fracture ends. Red arrows indicate inflammatory cells. (F) HE staining of the tissue around the fracture ends. Red arrows indicate inflammatory cells. (G) Immunohistochemistry was performed to detect IL-6 and TNF-α in tissue sections around the fracture. (H) Safranin O/fast green staining results. Green indicates bone tissue; red/orange indicates cartilage. (I) The proportion of bone formation among different groups. **, P<0.01. CFU, colony-forming units; HE, hematoxylin-eosin; IL-6, interleukin 6; NC, negative control; TNF-α, tumor necrosis factor-α.

HE staining revealed marked inflammatory cell infiltration around the ribs in the infected groups but was absent in the NC group (Figure 2F). IL-6 and TNF-α expression in the infected groups (1×10⁵, 1×10⁶, and 1×10⁷ CFU/mL) was significantly higher than in the NC group (1×10⁵ group vs. the NC group, P=0.008; 1×10⁶ group vs. the NC group, P=0.006; 1×10⁷ group vs. the NC group, P=0.005) (Figure 2G, Figure S3). Safranin-O staining showed that osteogenesis was 18.6%±1.91% in the 1×10⁵ CFU/mL group, 13.82%±1.00% in the 1×10⁶ CFU/mL group, 8.53%±1.80% in the 1×10⁷ CFU/mL group, and 75.93%±2.93% in the NC group (all P<0.001) (Figure 2H,2I).

Biocompatibility evaluation of Ti, Ti-MoS₂­, and Ti-MoS₂-Ag₃PO₄ implants

As shown in Figure 3A, Ti, Ti-MoS₂, and Ti-MoS₂-Ag₃PO₄ did not exhibit significant hemolysis, with hemolysis rates well below 5%, indicating that the materials are non-hemolytic and exhibit blood compatibility. Furthermore, after implanting Ti or Ti-MoS₂-Ag₃PO₄ fixators into rabbits, the blood tests remained within the normal range after 14 and 28 days (Figure 3B). Figure 3C shows the HE staining of the major organ tissues of each group after 28 days.

Figure 3 Biocompatibility evaluation of Ti, Ti-MoS₂, and Ti-MoS₂-Ag₃PO₄ implants. (A) Hemolysis rates of Ti, Ti-MoS₂, and Ti-MoS₂-Ag₃PO₄. Inset shows the degree of hemolysis for different samples. (B) Complete blood test results of rabbits 14 and 28 days after implantation with Ti or Ti-MoS₂-Ag₃PO₄. Error bars represent mean ± standard deviation, n=3. (C) HE staining results of the major organs of rabbits after 28 days. n.s., no significance. HGB, hemoglobin; LYPH, lymphocytes; NEUT, neutrophil; PLT, blood platelet; RBC, red blood cell; Ti, titanium; Ti-MoS₂, molybdenum disulfide coated titanium; Ti-MoS₂-Ag₃PO₄, molybdenum disulfide with silver phosphate coated titanium; WBC, white blood cell.

Enhanced bacterial eradication and inflammation alleviation with Ti-MoS₂-Ag₃PO₄ photothermic coating

Figure 4A indicates dense bacterial accumulation in the Ti group, while the Ti-MoS₂-Ag₃PO₄ group showed sparse bacterial colonies, achieving a 99.43% antibacterial rate against bacterial biofilms (Figure 4B). After culturing samples from the ribs in LB medium, the medium of the Ti-MoS₂-Ag₃PO₄ group remained clear and transparent, while the medium of the Ti group became turbid (Figure 4C,4D).

Figure 4 Photodynamic antibacterial activity of samples in rabbits (at the site of rib fracture). (A) Bacterial plates of Ti and Ti-MoS₂-Ag₃PO₄. (B) Antibacterial efficiency statistical chart. (C) Optical images of Ti and Ti-MoS₂-Ag₃PO₄ after incubation in LB medium at 37 ℃ for 4 h. (D) Optical density values of LB medium for Ti and Ti-MoS₂-Ag₃PO₄. Error bars represent mean ± standard deviation, n=3. ****, P<0.0001. 1D, 1 day; LB, Luria-Bertani culture; Ti, titanium; Ti-MoS₂-Ag₃PO₄, molybdenum disulfide with silver phosphate coated titanium.

Figure 5A shows the HE staining results of the rib fracture site at 2 and 28 days. Two days post-operation, many inflammatory cells were observed in the Ti group (indicated by the black arrows). The Ti-MoS₂-Ag₃PO₄ group showed almost no presence of inflammatory cells. Even after 28 days, the Ti group still had significant inflammatory cells, indicating ongoing inflammation at the site of the rib fracture. Meanwhile, the Ti-MoS₂-Ag₃PO₄ group showed no inflammatory cells. The number of bacteria in the Gram staining images (indicated by red arrows in Figure 5B) also supports the above results.

Figure 5 Enhanced inflammation alleviation and antibacterial efficacy with Ti-MoS₂-Ag₃PO₄ coating. (A) HE staining of tissues surrounding the implant site within rabbits. Black arrowheads indicate inflammatory cells. (B) Gram staining of tissues surrounding the implant site in rabbits. Red arrows indicate Staphylococcus aureus. HE, hematoxylin-eosin; Ti, titanium; Ti-MoS₂-Ag₃PO₄, molybdenum disulfide with silver phosphate coated titanium.

Discussion

The primary purpose of this study was to examine the effect of a titanium rib fixator MoS₂(S)-Ag₃PO₄ coating on S. aureus infection in the rabbit model. The study demonstrated that MoS₂(S)-Ag₃PO₄-coated titanium implants effectively manage infection and enhance fracture healing in a rabbit RFI model. Compared to controls, the coated implants showed significantly lower bacterial colonization and reduced inflammatory markers (IL-6 and TNF-α) while promoting osteogenesis, achieving a remarkable 99.43% antibacterial rate. Silver-based implant coatings are known to have infection-control properties (26,27). In the present study, after phototherapy, S. aureus was almost completely eradicated in the Ti-MoS₂(S)-Ag₃PO₄ group. After that, the small amount of Ag⁺ being released from the coating would synergize with the host’s immune system to suppress or kill the remaining bacteria, significantly reducing the number of viable bacteria over time and promoting healing. These results are supported by some previous studies that showed high bactericidal efficacy after 808 nm laser irradiation in vitro (25,28).

The present study also suggested that there were no significant damages or organ toxicity, strongly suggesting the excellent in vivo safety of Ti-MoS₂-Ag₃PO₄, as supported by the in vitro data (25,28). The Ti-MoS₂-Ag₃PO₄ group showed no inflammatory cell infiltration around the implant, suggesting that the micro-release of Ag⁺ from the coating alleviated tissue inflammation (probably through the bactericidal effect), and normal tissues and cells were gradually recovering from the bacterial infection damage. Under 808 nm near-infrared irradiation, the Ti-MoS₂-Ag₃PO₄ group did not exhibit significant bacterial infection issues.

The establishment of stable rabbit RFI model is another main research content of this study. The present study showed that the rabbit model of RFI was stable, without toxic and inflammatory effects on the major organs. Still, two rabbits died of pneumothorax and pleural effusion in the 1×10⁷ CFU/mL group, and the high bacterial load could be responsible. IL-6 and TNF-α play a critical role in the initiation and progression of inflammation and serve as important indicators for evaluating inflammation (34,35). The results showed high IL-6 and TNF-α levels in the soft tissues surrounding the rib fractures in the infected groups compared with the NC group. Safranin-O staining showed that osteogenesis was significantly lower in the infected groups (8.53–18.6%) compared with the NC group (75.93%), indicating that the bacterial infection inhibited osteogenesis, as previously reported (36,37). The spectrum of animal models for rib fracture research spans mice, rabbits, dogs, and pigs (38-45). Rabbits are ideal due to their immunogenetic proximity to humans (32), showing similar susceptibilities to infections and analogous disease mechanisms. Unlike rodents, rabbits typically do not spontaneously eliminate bacterial infections, and infections caused by S. aureus often result in chronic infection scenarios similar to what is observed in humans, enhancing the viability of the infection models (32,33). On the other hand, the literature is scant on rabbit models for RFIs, and the influence of such infections on fracture healing and local tissue environments remains under-explored. Establishing a stable rabbit model is thus pivotal for examining the role of infections in rib fracture prognosis and will pave the way for future preventative research in this field.

Of course, there are limitations in this study. An animal model always remains imperfect, and additional translational research is necessary before using Ti-MoS₂-Ag₃PO₄ implants in humans. The sample size was small. In addition, the actual healing of the rib fractures was not evaluated in the present study. Only one concentration of MoS₂-Ag₃PO₄ was used, and the impact of different concentrations remains unknown. Although S. aureus is the most common culprit for infections after fracture and instrumentation (46), other bacteria can also be responsible. The effect of MoS₂-Ag₃PO₄ should be tested on other strains as well. Finally, no molecular experiments were performed to determine the mechanisms of MoS₂-Ag₃PO₄ against S. aureus.

Conclusions

A standard rabbit RFI model was successfully established, and the S. aureus concentration of 1×10⁶ CFU/mL was a suitable bacterial concentration. The Ti-MoS₂-Ag₃PO₄ implants showed good biocompatibility and no major organ toxicity. The antibacterial effectiveness of the MoS₂-Ag₃PO₄ coating in vivo was confirmed in vivo.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2024-2214/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2024-2214/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2024-2214/prf

Funding: This study was supported by grants from the Tianjin Youth Medical Talents Fund (grant No. TJSQNYXXR-D2-142 to H.X.) and the Tianjin Hospital Science and Technology Fund of Tianjin (grant No. TJYYQ2403 to H.G.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2024-2214/coif). H.G. reports that this study was supported by grant from the Tianjin Hospital Science and Technology Fund of Tianjin (grant No. TJYYQ2403). H.X. reports that this study was supported by grant from the Tianjin Youth Medical Talents Fund (grant No. TJSQNYXXR-D2-142). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The experiments were approved by the Animal Use Ethics Committee of Tianjin University Tianjin Hospital (approval #2020-064), in compliance with Tianjin University Tianjin Hospital guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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