Volume 11 - 2023 | https://doi.org/10.3389/fbioe.2023.1108396 The field of 3D bioengineering proposes to effectively contribute to the manufacture of artificial multicellular organ/tissues and the understanding of complex cellular mechanisms 3D cell cultures comprise a promising bioengineering possibility for the alternative treatment of organ function loss potentially improving patient life expectancies could benefit from treatment until organ transplantation or even undergo organ function restoration 3D bioprinters can produce tissues such as trachea cartilage or artificial skin Most low-cost 3D bioprinters are built from fused deposition modeling 3D printer frames modified for the deposition of biologically compatible material ranging between $13.000,00 and $300.000,00 the cost of consumables should also be considered as they can range from $3,85 and $100.000,00 per gram our report describes the first prototype of a significantly low-cost 3D bioprinter built from recycled scrap metal and off-the-shelf electronics We demonstrate the functionalized process and methodology proof of concept and aim to test it in different biological tissue scaffolds in the future using affordable materials and open-source methodologies thus democratizing the state of the art of this technology it stands to reason that low- and middle-income countries may be active participants in this revolution biology research equipment and medical devices continue to present high price tags and are hard to obtain due to issues related to the geographical distribution of suppliers we have developed an open source microextrusion bioprinter for tissue engineering using discarded computer materials Bioprinters built with low-cost parameters Technologies applied to 3D bioprinting: general pros and cons Components used to build the prototype described here Comparative list of commercially available bioprinters per mechanism and customized prototypes prices TABLE 5. Prices of materials to build the 3D bioprintera Access to these types of printers is not easy everywhere as their cost is still high for certain regions/institutions and logistics problems (importing costs make the technology difficult to reach outside the global north and cost-effective bioprinter should be amenable for construction with locally available materials and technologies The development of a bioprinter employing microextrusion-based bioprint is a necessity and also an opportunity to democratize the teaching of basic technologies To develop the printer, a Cartesian movement system (three orthogonal axes) was employed. Sourced material was obtained from scrap metal (Figure 1A). Based on available online repositories (DIY Micro Dispensing and Bio Printing, 2022) several screws and small metal “L” angle brackets were used to create a 3D positioning subsystem mechanical part Representative image of the recycled scrap material used to build the scrap metal-based low-cost bioprinter (B) Injection subsystem: Representative images of the equipment used for the injection subsystems within the extrusion system After assembly of the motorized mechanical structure (Figure 2), mechanical axes setup was performed (Figure 3), also including the printing table (Inset Figure 4), the optical limit switches (Figure 3), electronic boards (Arduino and Shield), power supply, wiring and limit switches were installed (Figure 4) Mechanical blocks to construct the three orthogonal axes (A) X-axis driver set (B) Y-axis driver set (C) Z-axis driver set Φ: Worm Thread; *: linear guides; Δ: stepper motor; Π: optical assembly support Mechanical setup of the axes in the 3D movement block (A) Steel cover plates from CD-ROM boxes used to prepare the main 3D printer structure A window was cut in the vertical plate to allow for the Y-axis movement (B) Setup of the X-axis block in the upper parts of the vertical plate An acrylic plate rectangle was glued onto the optical block support of the X-axis to attach the Z block (C) Setup of the Z-axis block with the rectangular acrylic plate glued onto the optical block support of the Z-axis with screws used to attach the needle support Limit switch installation with locations indicated in red squares Insert: Mechanical assembly of the printing table The representative image of all the components of this bioprinter is depicted in Figure 5 as a schematic design In order to begin building the bioprinter injection subsystem an insulin syringe needle was cut to 3 mm from the end of the barrel using a micro grinder diamond-cutting disc while the 1.2 mm diameter hypodermic needle was cut at a 30 mm distance from the barrel The plastic hose of the urethral tube was cut to a length of about 32 cm The end of the protective cap was coaxially drilled using a 2 mm diameter drill one end of the plastic hose was inserted into the 1.2 mm diameter needle until the barrel and tied using a few turns of steel wire tight enough so that the hose would not slip on the needle the end of the cannon and part of the tube up to the end of the needle were covered with a thick layer of epoxy glue with the hose inserted through the hole until covering the needle The plunger of the insulin syringe was removed the rubber separated from the rod and the rubber end of the plunger pierced with the 2 mm drill bit so opposite end of the plastic hose is inserted into the hole from the side of the rod fitting The end of the hose lines was joined with the rubber end of the plunger (A) Frontal bioprint view indicating all prototype parts following a (B) schematic design indicating details at the bottom describing the infusion pump The plunger rubber was inserted into the syringe to about 1 cm below the handle flange The space to the flange was filled with epoxy glue (∼0.2 mL) and small balls of soft paper (∼3–4 mm in diameter) were inserted between the plastic hose and the syringe wall filling the space around the hose to act as a plunger for the glue The assembly was pushed until the rubber plunger touched the bottom of the syringe to minimize the volume between the hose opening and the syringe needle The overpressure sensor is a microswitch connected to the injector mechanism the increased pressure inside the syringe exerts an axial force in the mechanism and produces a small displacement of the driver screw against a spring enough to press the microswitch and abort the printing avoiding damages in the injection system due to excessive forces with the respective pin out identification (id with the function name of the pins in the same respective order of the corresponding pins in the Arduino (C) Stepping motor Allegro A4988driver module with the corresponding functional pin identifications (D) Transmissive photomicrosensor OMRON EE-SX1235A-P2 used as the limit switch where the block in the upper right part of the picture is the phototransistor and the block in the lower right is the LED (E) Wiring layout drawing of the limit switch connections (F) Typical photosensor connection as the limit switch and Z-axes and for the Injector (named “A axis” in the CNC board labels) (G) Circuit schematic of the overpressure sensor and Emergency Stop Switch the series of commands that will be interpreted by repetierHost to move the printer sequentially following the model initially created and extruding bioink The Y and X-axis parameterized with a one-step 10 µm resolution with a maximum non-printing move speed of 80 usual printing speed range of 2–10 and 36 mm axis range the Z-axis has a step resolution of 10 μm 40 mm/s of maximum non-printing move speed a usual speed rage of 2–10 and 20 mm axis range and subjected to at least 30 min of UV light decontamination prior to use Peracetic acid solution is used to decontaminate the pathway in which the bioink is strained for bioprinting The probe is soaked in a 0.2% peracetic acid aqueous solution for 10 min prior to use The probe is then washed three times with sterile deionized water a 5 mm × 5 mm x 2.5 mm (L × W × H) cuboid block The first model was designed in Wings3D and the others were drawn in Tinkercad exported to STL and imported into repetierHost where they were scaled and sliced into G-CODE using the Slic3r module In order to verify the accuracy of our prototype we printed a construct with pluoronic-F-127 (30%w/v) (#SLBG6026V United States) without cells and with the addition of an orange food coloring to improve visualization A 6% sodium alginate solution (4% w/v) (W201502-1 KG United States) was prepared in 100 mL of 0.9% w/v saline solution (Sorimax Stirring was maintained throughout the preparation The temperature was maintained at around 60°C to completely homogenize the alginate A 3-fold higher calcium chloride (CaCl2) (C50080-500G United States) concentration was prepared to maintain the final solution concentration A total of 0.03 g of CaCl2 were mixed with a 0.9% w/v saline solution Solutions I and ii were mixed at a 3:1 ratio resulting in a 4% sodium alginate solution containing 30 mM CaCl2 The solutions were then autoclaved and put to use 1 mL of the bioink was drawn with a 3 mL sterile syringe (990174 BD PlastipakTM) and placed in the bioprinter to begin the printing process 500 µL of a 5 mM CaCl2 aqueous solution was dropped on top of the construct for 3 min and the block was cultured at 37°C under a 5% CO2 atmosphere in an oven United States) (4% w/v) was prepared in 100 mL of a 0.9% w/v saline solution Stirring was continued throughout the preparation The temperature was maintained at around 60°C for complete alginate homogenization A total of 3 g or 4 g of gelatin (G8150-100G United States) were mixed with the solution mentioned above to prepare a 4% sodium alginate solution containing 3% or 4% gelatin solution we added cells to the printed construct assess biocompatibility Around 107 cell/mL HEPG2 lineage was bioprinted on a 35 mm × 10 mm Petri dish 500 µL of a 5 mM CaCl2 aqueous solution was dropped on top of the construct for 5 min and the block was cultured at 37°C under a 5% CO2 atmosphere in an oven It is important to note that all solutions can be stored for 1 month after preparation at 4°C Bioink temperatures of both the alginate and calcium chloride and alginate and gelatin solutions must be used at around 37°C as cells are maintained at that temperature The bioinks were placed in a water bath to complete melting we wait for temperature decreases at 37°C Cells at the concentration of interest were washed and placed in a polypropylene tube A total of 1 mL of the bioink was added slowly to avoid bubbles and then placed in a 1 mL syringe they were maintained at 4 °C for 1 h to improve polymerization A total of 500 µL of calcium chloride were added to the top of the blocks for 3 or 5 min The calcium chloride solution was removed with the aid of a pipette and 3 mL of high glucose DMEM (Sigma United States) medium supplemented with 10% fetal bovine serum (FBS - GIBCO The culture was performed as described previously Nebulization device (red square) coupled to the bioprinter equipment to solve submerged calcium chloride printing issues The materials used to carry out the first calibration injection tests were sodium alginate at 100 mg/mL and calcium chloride at 5 mM It is important to note that calcium chloride can improve alginate gelification while being biocompatible In order to better visualize the alginate print in these first tests The first verified parameter comprised alginate gelling efficiency. Dropping alginate into the calcium chloride solution directly from the extrusion needle produced stable and mechanically resistant-gelled alginate capsules approximately 2 mm in diameter (Figure 8) Alginate gelification in calcium chloride: (A) dripping and (B) appearance of the gelled capsules The first tests indicate some issues, as follows (Figure 9) - Problems with Petri dish alginate adherence with the alginate remaining attached to the injection needle; - Low precision of the table adjustment to needle movement coordinates Representative images of the problems caused by alginate gelification insufficiency: (A) Excess extruded alginate (B) Lack of extrudated alginate due to needle stickiness Several materials were tested to solve glass adherence issues, such as tracing paper, polypropylene, and acrylic (Figure 10) The best adherence material was glass with smaller contact angles The test was then carried out immersed in a calcium chloride solution with the needle tip moving to check for adhesion or trailing Adhesion tests employing different material surfaces Representative images of different materials used to verify alginate adhesion to surfaces from extrusion bioink the calcium mist minimized the adherence caused by the immersion printing although the needle still suffered from lumps formed by alginate gelation when extruded Gelled alginate lumps were observed at the end of the injection needle at the lowest calcium chloride ratio they spread the deposited alginate and ink the printing in addition to obstructing nozzle of injection and The lumps increased as the alginate was attracted by capillary action to the needle was coated with a protective hydrophobic membrane (e.g. assigned by vaseline also caused calcium chloride repulsion This can be minimized by adjusting the position of the chloride injection closer to the end of the alginate injection needle tests with a 3:1 alginate to calcium chloride ratio were performed and an insufficient amount of calcium chloride was observed the chosen model by the Wings3D builder for the tests presented in this report was a standardized 5 × 5 × 2.5 mm block 1:3 and 1:1 alginate to calcium chloride ratios were tested but excess calcium chloride was noted through higher alginate polymerization and the presence of two bioink phases the first test proved to be the best ratio Construct bioprinted with a mix of biopolymers at different concentrations Bioink comprising 4% alginate and 3% gelatin (A,B) 4% gelatin and post-printing crosslinking with 5 M calcium chloride (C,D) Scale bar: 1 cm (B,D); 2 cm (A,C) Accuracy test for resolution of the constructs printed by the bioprinter employing the following: Shape printed with Pluoronic-F127 15% w/v without cells and employing food coloring (A) Heart-shaped print dimensions 3 mm × 6 mm × 8 mm (B) 3D-shaped print dimension 3 mm × 13 mm × 6.95 mm (C) Star-shaped print dimensions 2 mm × 6.18 mm × 7 mm Right: rendering of models imported into RepetierHost and Left: Printed shape from our prototype Analysis of extrusion influence on bioprinted construct contains cells Bioprinted constructs employing alginate 4% and gelatin 3% on day 0 (A,B) and in culture after 7 days (C,D) after the bioprinting process Green: Calcein AM; Red: PI (Propidium iodide) View of Z stacks—AxioObserver Zeiss microscope Bioprinted construct viability and maintenance: 4% alginate and 3% gelatin with 107 cells/mL of the HepG2 bioprinted construct on day 0 (A–C) and in culture after 7 days (D–F) Construct size: 1.42 mm × 1.42 mm—(A–C) Z stack: 129 slices (640 μm) and (D–E) Z stack: 124 slices (356.21 1 μm) it is difficult to visualize the entire construct fluorescence which is why the PI fluorescence is almost non-existent Serial cuts of these constructs could be performed to build a broader image The confocal images indicate that the PI fluorescence is stronger on day 7 Further analyses are required to confirm that cultured cell viability decreases through time 3D bioprinters comprise cutting edge technology and will certainly become a tool for the improvement of several clinical procedures as therapeutic approaches. However, they are still expensive, keeping them from being broadly adopted and accessed by labs and hospitals worldwide. It is important to note that many proposed low-cost models are high maintenance (Tong et al., 2021) We proposed the construction of a really low cost it is important to analyze the functioning and mechanisms behind the assembly and execution of this task comparing them to the most employed bioprinter models which strongly affects the cost of bioprinters stable 3D printing while also maintaining high cell viability for this technology to be applied in transplants or even in toxicological analyses The prototype reported herein paves the way for the assembly of low-cost equipment and wide access to laboratories with little funding subsidies were accumulated for the detailing and improvement of the 3D bioprinter prototype project the software and equipment designs are both freely available under open source licenses democratizing access to these technologies Our proof of concept indicates cell viability at day 0 and day 7 More studies are required to verify how long the constructs remain viable in culture as a low-cost bioprinter option and an important tool for in vitro bioprinted 3D tissue applied for future transplantations or drug toxicity tests presenting itself as an alternative to the use of animals in research All data are available in our github repository (https://github.com/Laboratory-of-Cellular-Communication/E-Waste-3D-bioprinter) as well as in this supplementary material of this manuscript LA and JC originally conceived the present article RA and JC provided bioprinter methodological building LA and AC provided critical reviews to the manuscript All authors contributed to the article and approved the submitted version “Redes de Pesquisa em Saúde no Estado do Rio de Janeiro”—Grant number: E-26/010.002422/2019 CNPq and to FAPERJ (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Rio de Janeiro) for their support of this study 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 All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1108396/full#supplementary-material Alginate-based hydrogels as drug delivery vehicles in cancer treatment and their applications in wound dressing and 3D bioprinting PubMed Abstract | CrossRef Full Text | Google Scholar PubMed Abstract | CrossRef Full Text | Google Scholar 3-D bioprinting technologies in tissue engineering and regenerative medicine: Current and future trends CrossRef Full Text | Google Scholar Principles of open source bioinstrumentation applied to the poseidon syringe pump system PubMed Abstract | CrossRef Full Text | Google Scholar Influence of calcium ions on cell survival and proliferation in the context of an alginate hydrogel CrossRef Full Text | Google Scholar DIY Micro Dispensing and Bio Printing (2022). 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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 distribution or reproduction is permitted which does not comply with these terms *Correspondence: Luiz Anastacio Alves, YWx2ZXNsYWE0MEBnbWFpbC5jb20= †These authors have contributed equally to this work Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Thanks for visiting The use of software that blocks ads hinders our ability to serve you the content you came here to enjoy We ask that you consider turning off your ad blocker so we can deliver you the best experience possible while you are here This website is using a security service to protect itself from online attacks The action you just performed triggered the security solution There are several actions that could trigger this block including submitting a certain word or phrase You can email the site owner to let them know you were blocked Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page Brazilians cite corruption and lousy government services as key reasons that more than 1 million citizens are protesting Millions of voices cry out in unison on the streets of Brazil For about two weeks these people have been protesting against the prices of public transportation the conditions of public healthcare and education There are too many issues being discussed at once there is no obvious focus or specific demand Only days ago our president allowed government officials to use public money to travel and use hotels to comfortably watch the soccer games of the Confederations Cup she promises to solve the healthcare problem by importing doctors This is as helpful as hiring cooks to work in an empty building without a sink people need to know why they are on the streets and what exactly needs to change first because only specific issues can be solved one at a time Vaccine hesitancy is a phenomenon that can interfere with the expansion of vaccination coverage and is positioned as one of the top 10 global health threats. Previous studies have explored factors that affect vaccine hesitancy, how it behaves in different locations, and the profile of individuals in which it is most present. However, few studies have analyzed the volatility of vaccine hesitancy. Identify the volatility of vaccine hesitancy manifested in social media. Twitter’s academic application programming interface was used to retrieve all tweets in Brazilian Portuguese mentioning the COVID-19 vaccine in 3 months (October 2020, June 2021, and October 2021), retrieving 1,048,576 tweets. A sentiment analysis was performed using the Orange software with the lexicon Multilingual sentiment in Portuguese. The feelings associated with vaccine hesitancy were volatile within 1 month, as well as throughout the vaccination process, being positioned as a resilient phenomenon. The themes that nurture vaccine hesitancy change dynamically and swiftly and are often associated with other topics that are also affecting society. People that manifest the vaccine hesitancy present arguments that vary in a short period of time, what demand that government strategies to mitigate vaccine hesitancy effects be agile and counteract the expressed fear, by presenting scientific arguments. Infectious Diseases: Epidemiology and Prevention Volume 11 - 2023 | https://doi.org/10.3389/fpubh.2023.1192155 Background: Vaccine hesitancy is a phenomenon that can interfere with the expansion of vaccination coverage and is positioned as one of the top 10 global health threats Previous studies have explored factors that affect vaccine hesitancy and the profile of individuals in which it is most present few studies have analyzed the volatility of vaccine hesitancy Objective: Identify the volatility of vaccine hesitancy manifested in social media Methods: Twitter’s academic application programming interface was used to retrieve all tweets in Brazilian Portuguese mentioning the COVID-19 vaccine in 3 months (October 2020 A sentiment analysis was performed using the Orange software with the lexicon Multilingual sentiment in Portuguese Results: The feelings associated with vaccine hesitancy were volatile within 1 month as well as throughout the vaccination process being positioned as a resilient phenomenon The themes that nurture vaccine hesitancy change dynamically and swiftly and are often associated with other topics that are also affecting society Conclusion: People that manifest the vaccine hesitancy present arguments that vary in a short period of time what demand that government strategies to mitigate vaccine hesitancy effects be agile and counteract the expressed fear Vaccine hesitancy is a global phenomenon. Still, public acceptance of the vaccine varies depending on the region (10). Countries in Asia have higher rates of vaccine acceptance (Malaysia with an acceptance of 94.3%, Indonesia of 93.3%, and China of 91.3%) compared to Europe (France with an acceptance of 58.9%, Poland of 56.3%, and Italy of 53.7%) and the United States (acceptance of 56.9%), which have lower levels of vaccine acceptance (11) the findings of this research make up a contribution to the mosaic of international studies that address VH caveats must be taken when generalizing their findings because they are characteristic of the Brazilian population The exported data had issues with character encoding (accents and other special formats) and provided a date formatting presented as year hour (including minutes and seconds) that is different from the Brazilian standards it was necessary to use the R software to help edit the data since the database had over 1 million observations were removed all duplicate tweets and the same tweet from the same user the data were exported from R as a.csv file there are no independent or dependent variables since the paper focused on text sentiment analysis The.csv data was imported into the Orange Data Mining software to conduct the sentiment analysis an unsupervised text mining technique (without an initial reference/classification as the initial input into the software) that aims to extract the emotion present in a message the text corpus was pre-processed to remove stop words (prepositions Only whole words without numbers or other characters (Regexp) are maintained Specific features of the Twitter language (for example the use of hashtag and at signs) are identified and URLs removed from the text The sentiment analysis of the VH was performed in three different periods of the vaccination process which are presented in charts divided into 1-week periods This distribution was planned to observe the sentiment of VH in short periods (week) within the same month and in longer intervals (over 4 months) The analysis relied on the selection of 1.047.018 tweets adhering to the topic of vaccine hesitancy the overall sentiment of the analyzed tweets for October 2020 was −0.0438 The overall sentiment was therefore slightly negative in all periods It is noteworthy that the sentiment analysis is focused on VH and not on other aspects pointed out in the tweets The sentences that exemplify the sentiment identified and presented in the first columns of the tables were translated from Portuguese to English to present the research by the authors Sentiment analysis of the COVID-19 vaccine in October 2020 The data show the onset of vaccine topic within the spectrum of solutions for the COVID-19 pandemic in Brazil In the first insertion the VH phenomenon is already observed focusing on the efficacy of the vaccines that are being proposed The data show an alternation between negative Sentiment analysis of the COVID-19 vaccine in June 2021 Sentiment analysis of the COVID-19 vaccine in October 2021 The data for October 2021 portray a period of a more advanced stage of the population vaccination process This period showed a greater VH manifestation probably due to the availability of the vaccine for everyone encouraging those who are more resistant to the vaccine to manifest with greater intensity The data show an alternation between negative and neutral sentiments with little occurrence of positive sentiments This indicates that almost a year after the beginning of the vaccination process VH still exists should be the object of planning and actions aimed at its mitigation The tweets also indicate the expectation for the end of the COVID-19 pandemic added to other factors such as the reduction of infection with the potential interaction among these factors have set an important milestone for people to interpret the period as the end of the pandemic that these characteristics observed in the tweets as well as the brands of vaccines available the period and priority of vaccination of the population are related to characteristics of the vaccination process in Brazil thus presenting intrinsic limitations of generalization of the findings of this research Previous studies have revealed that vaccine hesitancy (VH) is a dynamic phenomenon influenced by many variables that impact it in a positive or negative way (9, 11). The variables that influence VH operate differently in each region depending on characteristics of the society (10, 11). Residents of sub-Saharan Africa, for example, feared being victims of vaccine experimentation (43) The inappropriate manifestation of politicians regarding the vaccine strengthens VH impacting trust in the government and in the COVID-19 vaccine This suggests that politicians should base their communications on evidence provided by the experts and not on their own feelings about vaccination The second block of data analyzed concerns the month of June 2021 which is marked by a significant portion of the Brazilian population having already received at least the first dose of the vaccine VH was expressed as negative sentiments toward the vaccine three phenomena associated with the VH in relation to the COVID-19 vaccine stood out: pregnant women the Copa América being held in Brazil (soccer championship between the national teams of South American countries) The suitability of the COVID-19 vaccine for pregnant women was a dominant theme throughout the period. VH was expressed in questioning the suitability of vaccines in general as well as the suitability of one of the vaccines available (50) it is possible to ponder that doubting the suitability of the vaccine for pregnant women may cause this negative sentiment toward the vaccine to overflow to the society This block of analysis (June 2021) shows the possibility of observing multiple social factors interfering with VH at short time intervals (1 month) This period is also a turning point as the pandemic is concerned namely the proximity of the end of the crisis the beginning of manifestations alluding to the approach of the end of the COVID-19 pandemic are identified and most of them relate this event to the COVID-19 vaccination process there is a positive feeling toward vaccination and its influence on the end of the crisis one can also observe the manifestation of regret for having taken the vaccine This characteristic establishes an important point of future investigation as it constitutes a new attitude of people that differs from hesitation before the fact – the “vaccine regret” (VR) The VR can affect the VH and drive people to avoid getting booster doses This perspective validates VH as a social process under the influence of multiple meanings and logics circumscribed to the individual and collectivity during the pandemic the population increased its confidence in health professionals and in their recommendations for the vaccination the unspoken vaccine hesitancy of HCW is identified as an element of attention of health systems as it can influence the reduction of confidence in the vaccine by the public Two relevant aspects of the participation of HCWs in the HV mitigation process are thus identified: the first is that they influence people’s decision to take the vaccine so this aspect should be used in favor of awareness campaigns The second aspect indicates that HCW may have unspoken vaccine hesitancy which requires health system managers to establish awareness programs that include autonomy and freedom to express their hesitations This study defines vaccine hesitancy as a complex and dynamic social phenomenon Among the considerations and findings of this research are the arguments presented by society for not getting vaccinated actions aimed at reducing vaccine hesitancy must be agile in capturing the concerns that permeate society and in presenting information that encourages people to take the vaccine vaccine hesitancy studies are still in the initial phase so this research presents itself as a strength This characteristic establishes limitations when applying the knowledge developed in this study in countries with cultures other than Brazilian The origin of the data did not allow raising variables common to other studies this study presents additional information on vaccine hesitancy the data from this study refer to the Brazilian population so caveats should be established in generalizing the findings of this research to other populations The datasets presented in this study can be found in online repositories. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Celso Machado Júnior, Y2Vsc28uanVuaW9yQG9ubGluZS51c2NzLmVkdS5icg== we saw that Eric was taken into the woods by the Curupiro and we didn’t know if he would survive his injuries or not so Ines used her powers and asked the water to heal him who told him that his journey was still not over and that his life had a greater purpose let’s find out what that higher purpose was that Eric was destined to serve in his life Castro belonged to one of the most influential families in town which was also infamously known for carrying out illegal mining operations in the Marangatu forests who was a member of the indigenous tribe and who could transform into a snake Honorato was apparently the key to a treasure that Castro and Debora had wanted to find and put up for auction Danilo got frustrated when even after repeatedly asking Honorato he was not ready to tell him the location of the treasure though they hadn’t been able to get even a single breakthrough even after wandering for two years Luna had got to know about a witch named Matinta Perre who was known to fulfill the wishes of the people and make them get what they truly desired Matinta told Luna that she could fulfill her desire to be with her father provided she was ready to give her what she wanted Luna accepted the offer because she desperately wanted to find her father Eric had woken up in the river in the Marangatu area where Castro and his people were trying to find the treasure Eric was present on the scene when Danilo killed Honorato An old man named Lazo was held captive at the illegal mining site as he had the power to locate the gold that otherwise wasn’t visible to the naked eye Lazo and others like him who possessed the powers considered it a sin rather than a blessing he realized that Eric had some extraordinary powers that could release him from his curse Danilo came chasing him and Eric set the entire place on fire and severely injured himself Eric opened his eyes and found himself in the hospital as he couldn’t decipher where he was or how long it had been since the incidents in the cedar forest where he had encountered the Curupira who was around the same age as his daughter and who claimed that he knew the whereabouts of Luna Eric went with him and finally found his daughter He was elated and relieved to find that she was safe and he hoped that they could go back to living their normal lives But fate had something else planned for them Danilo knew that he couldn’t tell his bosses that he had killed the only man who knew where the treasure of Marangatu was hidden so he put the blame on Eric and told Castro and Debora that the gringo had put the entire facility on fire and killed Honorato had figured out another way of getting to the treasure as she had met Metinta Perres and made a deal with her just like Luna did Eric was just getting a grasp of things after meeting Ines when Metinta arrived out of nowhere in their makeshift house and took Luna with her and Eric realized that due to some unknown reason he was able to take the powers of other entities Metinta told Ines in Episode 2 of “Invisible City” Season 2 that messing with the natural order of things had its consequences and she had signaled the possibility of Eric turning into something evil the powers of the latter had been transferred into the former Eric found that Debora and Castro were holding a private auction where the stakeholders would bid for the gold that they had assumed they were going to have in their possession in the future and she was forced to help the family take advantage of the loopholes in the legal system and make their illegal activities look like they were being performed within the ambit of the law as she didn’t want to be with Castro and wanted to start a new life with a priest named Venancio Clarice told Eric that they would help him rescue his daughter if he removed her powers from within her and gave her freedom from her curse they went to the auction disguised as stakeholders Debora had transformed into a snake and bitten Luna Ines told Eric that there was only one person who could save Luna’s life:  an old woman named Jaciara who was a member of the indigenous tribes and a powerful sorceress and also a soothsayer It was Jaciara who revealed to Ines that Eric had transformed into something evil and couldn’t be trusted She made Eric wait outside while she performed the ritual on Luna and tried to save her life and strains of evil had started becoming more and more evident in his behavior At the end of Episode 4 of “Invisible City,” Season 2 Ines realized that she needed to keep Luna away from her father as he was no longer in control of his powers and couldn’t be trusted to do the right thing at that moment In the fifth episode of “Invisible City,” Season 2 we got to know how humans had penetrated Marangatu and started doing what they were best at It was Castro’s father who had brought Lazo to Marangatu so that he could use his powers and let him know where the gold was hidden he brought his men to the place and started plundering the area Marangatu was a sacred place for the indigenous people they had been able to maintain the delicate balance and allow the ecosystem to thrive without any sort of disturbance We got to know that Debora was Honoratu’s sister and their mother had made them wear the “muiraquitas” i.e. so that her home could be protected from the invaders Debora was brought back to her village by Daniel and Telma in the hope that she would be able to remember her roots and her mission in life and she realized that her mother had been killed by Castro’s father before he had taken her with him Ines was taking Luna to Marangatu to fulfill her destiny and return the guardian entity that had been given to her by her mother but he didn’t realize that he had become a menace to everyone’s safety and had ceased to be the savior that he thought himself to be Eric encountered Bento and Lazo on his way and asked them the direction in which Ines and Luna had gone but he used his powers to make Bento tell him that they were headed toward Marangatu Lazo told him that Marangatu was a sacred place for them and Ines got to know through her psychic powers that Eric had become uncontrollable and that she needed to stop him and buy some time for Luna Ines told Luna that it was about time they parted ways as she would have to go back to stop Eric Ines told Luna that she had the utmost faith in her abilities and that she had been ready for the challenge for quite some time now and just like she did in “Invisible City” Season 1 she once again showed him what had actually happened in the past and how Luna and Debora’s fates were intertwined Eric saw in his hallucinatory visions that Jaciara had realized that there was nobody to protect their sacred land after the death of Honoratu she realized that she was wearing the guardian entity that Debora’s mother had once given her Eric saw in his vision that Gabriela had come to Marangatu sometime in the past and met Honoratu It was Gabriela who had found the guardian entity near the river and Honoratu told her that it belonged to his sister Jaciara had told Gabriela to keep the amulet and give it back to Debora when the time came Gabriela didn’t understand how she could return the amulet when she didn’t even know where Debora was or what she looked like and the amulet would find a way back to its real owner and that’s how the little one had become a pivotal part of this entire mystery Contrary to what Eric had believed throughout the entire “Invisible City ” season 2 and she had even taken a stand for him in front of Matinta Ines was ready to sacrifice her powers to bring Eric back on track even though Matinta had warned her about the dire implications that it could have Eric begged Jaciara to let him out of his own head he had done nothing but protect the environment Eric came back to his senses in the real world The 200-year-old Cacu’s physical body aged and transformed right in front of Eric’s eyes and she took her last breath with the hope that Eric would now save the forest and fight for the cause of the indigenous people Eric finally found Luna and told her that he was ready to do the right thing Eric sacrificed his life and returned the powers of each and every entity that he had forcefully taken earlier and he got to know that Debora and others had lost their powers He took Lazo and Bento to find Luna so that he could take all the gold for himself though he didn’t know how much they would be able to help his cause without their powers Lazo was trying to deceive them and lead them astray when all of a sudden he realized that his powers had come back to him due to the sacrifice that Eric had made Castro knew that he had to run for his life as Bento and others would have also gotten their powers back and would be coming to hunt them down Although Castro was able to save his life and escape from Marangatu Talem was waiting for him in the court of law back in the city The court found Castro junior guilty of contamination of the water supply Catro’s family had ruined her entire life Eric had traversed into the parallel realm and he had sacrificed his mortal life to save the forest and its inhabitants The possibility of Eric returning to the mortal realm couldn’t be completely denied as there are a lot of higher powers at play that are still unknown to us and maybe if the need arises in the future Please include what you were doing when this page came up and the Cloudflare Ray ID found at the bottom of this page.