Introduction of Hybrid Additive Manufacturing for Producing Multi-Material Artificial Organs for Education and In Vitro Testing

: The evolution of 3D printing has ushered in accessibility and cost-effectiveness, spanning various industries including biomedical engineering, education, and microfluidics. In biomedical engineering, it encompasses bioprinting tissues, producing prosthetics, porous metal orthopedic implants, and facilitating educational models. Hybrid Additive Manufacturing approaches and, more specifically, the integration of Fused Deposition Modeling (FDM) with bio-inkjet printing offers the advantages of improved accuracy, structural support, and controlled geometry, yet challenges persist in cell survival, interaction, and nutrient delivery within printed structures. The goal of this study was to develop and present a low-cost way to produce physical phantoms of human organs that could be used for research and training, bridging the gap between the use of highly detailed computational phantoms and real-life clinical applications. To this purpose, this study utilized anonymized clinical Computed Tomography (CT) data to create a liver physical model using the Creality Ender-3 printer. Polylactic Acid (PLA), Polyvinyl Alcohol (PVA), and light-bodied silicone (Polysiloxane) materials were employed for printing the liver including its veins and arteries. In brief, PLA was used to create a mold of a liver to be filled with biocompatible light-bodied silicone. Molds of the veins and arteries were printed using PVA and then inserted in the liver model to create empty channel. In addition, the PVA was then washed out by the final product using warm water. Despite minor imperfections due to the printer’s limitations, the final product imitates the computational model accurately enough. Precision adjustments in the design phase compensated for this variation. The proposed novel low-cost 3D printing methodology successfully produced an anatomically accurate liver physical model, presenting promising applications in medical education, research, and surgical planning. Notably, its implications extend to medical training, personalized medicine, and organ transplantation. The technology’s potential includes injection training for medical professionals, personalized anthropomorphic phantoms for radiation therapy, and the future prospect of creating functional living organs for organ transplantation, albeit requiring significant interdisciplinary collaboration and financial investment. This technique, while showcasing immense potential in biomedical applications, requires further advancements and interdisciplinary cooperation for its optimal utilization in revolutionizing medical science and benefiting patient healthcare.


Introduction
Additive manufacturing, with its wide-ranging applications across biomedical engineering, electronics, automotive sectors, education, and microfluidics, has evolved significantly.Initially characterized by high entry costs, recent advancements in printing Designs 2024, 8, 51 2 of 11 processes and materials have rendered 3D printing more accessible and cost-effective [1].The applications of 3D printing in biomedical engineering span from bioprinting tissues imitating natural structures to manufacturing affordable and custom-fit prosthetics [2].Additionally, the technology facilitates the creation of porous metal orthopedic implants, promoting better integration with natural bones and aiding pharmaceutical testing [3,4].In educational settings, 3D printing bridges the gap between theoretical concepts and physical manifestations, providing students with hands-on experiences for rapid prototyping.This technology enhances STEM education (Science, Technology, Engineering, and Mathematics) by enabling students to create models, scientific equipment, and replicas of historical artifacts [5].
Recent advances in 3D printing necessitate the development of biomaterials compatible with printing processes.These materials, especially for biomedical applications, require biocompatibility and structural integrity.They aim to create scaffolds for tissue engineering, drug screening, and regenerative medicine [6].Notable materials include natural polymers like chitosan, sodium alginate, and synthetic polymers [7,8].
Various hybrid 3D printing methods have emerged for biological materials: Challenges persist in enhancing cell survival rates, ensuring proper cell interactions, and facilitating nutrient delivery within printed structures.Researchers explore temperature-controlled bio-inkjet printing, precise cell placement, and microfluidic channels for improved viability and functionality [37][38][39][40].
There is a lot of discussion regarding the implementation of physical phantoms to study cell survival in organs and tissues after an irradiation procedure [41,42].Nevertheless, the cost for such applications needs a significant budget to be included in every laboratory, as well as a multidisciplinary approach combining many different types of expertise.Additionally, there is a need for physical phantoms for extensive practice with surgical approaches to optimize procedures, limiting the use of real tissues.Likewise, a recent surgical review highlighted the importance of understanding tissue mechanics [43].More specifically, recent studies have comprehensively explored the use of 3D printing for phantom fabrication, including discussions on material selection [44][45][46].A key finding is the limited availability of materials that can accurately mimic the full spectrum of tissue properties [44].However, the growing accessibility, affordability, and versatility of additive manufacturing techniques point towards its increased adoption in the medical field [46].With the ever-expanding array of tissue-mimicking materials (TMMs) presented in research and the ongoing surge in investigations, a pressing need exists to focus on the properties of these phantom materials across various imaging and therapeutic applications.
While digital or "computational" anthropomorphic phantoms offer a cost-effective alternative [47], physical phantoms remain the preferred choice for institution-specific acquisition and procedural protocols, which can vary depending on equipment manufacturers, models, and intended uses.The material selection, or the specific TMMs used within these physical phantoms, is crucial for their successful application [48].
This study aims to present a proof-of-concept for a low-cost implementation on combining the FDM technique with inkjet to produce soft-tissue structures, such as a small version of the human liver, using real clinical data (i.e., a CT dataset), following suggestions by the literature [43][44][45][46].Three different materials were utilized to fit the purpose of each structure, namely Polylactic Acid (PLA), Polyvinyl Alcohol (PVA), and light bodied, biocompatible silicone (Polysiloxane), which could incorporate living cells in a future extended implementation.
The following section (Section 2) includes a description of the materials and methods implemented, providing information on the materials and the techniques implemented to develop the whole procedure.Section 3 follows, providing the final printed products of this study and analysis of results.Section 4 provides a comprehensive analytical discussion of the results of this study and possible implementations, and Section 5 draws the final conclusions to summarize the whole study.

Materials and Methods
Combining FDM with bio-inkjet printing involves creating a supportive FDM scaffold for bio-inkjet-deposited living cells.The utilization of Ultra-Violet (UV) light for curing forms the living tissue, allowing greater control over geometry and architecture.Its advantages include improved accuracy and structural support, enabling the creation of complex structures.Challenges of this approach encompass enhancing cell survival, promoting proper cell interactions, and ensuring efficient nutrient delivery within the printed structures [49][50][51][52].
To start with the formation of the physical phantom, clinical data are needed.For this study, anonymized (for ethical purpose) clinical Computed Tomography (CT) data, featuring a segmented liver including veins and arteries, was utilized.To make the physical phantom more accessible for the laboratory's equipment, as well as to accelerate the additive manufacturing procedure, the liver model was scaled down to 50% of its original size, as this is only a feasibility study.The 3D printing process was conducted using the Creality Ender-3 printer, as this is a widely used, low-cost, and accessible piece of equipment.
Three types of material were employed to assure accessibility, convenience, and biocompatibility, as presented in Table 1.More specifically, they are

•
PLA: Used for printing the liver mold to ensure mechanical integrity.Additionally, this material is flexible in the way that it is printed, ensuring that no special conditions are needed; • PVA: Employed as the mold for veins and arteries, allowing easy removal by washing with hot water.This was crucial as the delicate veins' geometry could not be extracted from a PLA mold without damage; • Polysiloxane: Medical-grade silicone used to fill the PLA molds.This silicone is harmless to living organisms, and its density is close to the density of organ tissue (~1.1 g/cm 3 ).For this reason, this material could also be a base in future applications to accommodate living cell populations.Figure 1 depicts a 3D model of the liver used, showcasing the veins in blue and arteries in red.The liver and its intricate network of veins and arteries were derived from anonymized CT data and were previously segmented.The TotalSegmentator tool [53] was considered for the segmentation procedure.The TotalSegmentator is able to segment a big portion of the human body, delineating 117 distinct organs and regions.TotalSegmentator is an AI model, developed and trained by the department of Research and Analysis at University Hospital Basel, on the Zenodo database [54], to be able to recognize human organs in any provided CT data.However, precise clinical implementation may necessitate evaluation and refinement by experienced clinicians.
(~1.1 g/cm 3 ).For this reason, this material could also be a base in future applications to accommodate living cell populations.1 depicts a 3D model of the liver used, showcasing the veins in blue and arteries in red.The liver and its intricate network of veins and arteries were derived from anonymized CT data and were previously segmented.The TotalSegmentator tool [53] was considered for the segmentation procedure.The TotalSegmentator is able to segment a big portion of the human body, delineating 117 distinct organs and regions.
TotalSegmentator is an AI model, developed and trained by the department of Research and Analysis at University Hospital Basel, on the Zenodo database [54], to be able to recognize human organs in any provided CT data.However, precise clinical implementation may necessitate evaluation and refinement by experienced clinicians.Utilizing 3D-Slicer v.5.6.2 software, the segmented data from DICOM files was exported into STL (Stereo-Lithography) file format, which is a commonly used file format in any CAD software.3D-Slicer is a free, open-source software for the visualization, processing, segmentation, registration, and analysis of medical, biomedical, and other 3D images and meshes; and planning and navigating image-guided procedures.Based on a large supporting community of users and developers, 3D-Slicer can solve advanced image computing challenges with a focus on clinical and biomedical applications.It allows one to build and deploy custom solutions for research and commercial products.
The produced STL files of the model were then transformed into G-code files using slicers.G-code is a programming language for CNC (Computer Numerical Control) machines.G-code stands for "Geometric Code" and is used to tell a machine what to do or how to do something, translating the path that needs to be followed.UltiMaker Cura v5.6.0 was employed to generate the initial G-code, which was later converted into a non-planar version for this study.For the non-planar implementation, the CurviSlicer (March 2020 build) was employed.The CurviSlicer is a recently developed tool that is able to receive STL files and generate the G-code, including non-planar movement.Nevertheless, the code was not able to run directly with the printer.The next step was to obtain this G-code file and compare it to another file that can be used by the printer, to manually apply modi-Designs 2024, 8, 51 5 of 11 fications in the initial and final part of the G-code, making it recognizable by the printer.In this implementation, a simple G-code file was generated using the Cura software and then used as reference for manual corrections on the non-planar G-code file generated by CurviSlicer.The header part of the file needed to be copied by the Cura G-code file, to make the G-code compatible with the 3D printer.Additionally, the end part of the G-code file produced through CurviSlicer needed to be copied by the Cura G-code file, to help the printer conclude the procedure without problems.
The 3D printing process utilized in this study involved the use of a specialized material known as condensation Polysiloxane, specifically Protolast PF [55].This material is characterized by its light-based consistency and finds extensive application as a precision impression material in the field of dentistry.It must be stated that this type of silicone could be integrated with living cells, if such an implementation is a target.In our case, as this study is only proof-of-concept, no such step was followed.
We now delve into the methodology in greater detail.The procedure commenced with the precise printing of veins and arteries using PVA.Subsequently, these printed vascular structures were incorporated into the corresponding positions in the liver mold to create areas that would stop the diffusion of the light-bodied silicone.These areas could act as channels that could be filled with liquids to simulate blood circulation.The liver mold was printed in a second step using PLA to ensure the integrity of the mold, as it was completely empty on the inside.The walls of this piece were 0.8 mm, as this thickness was enough for both its integrity and easy removal after being filled with silicone.
It needs to be stated that the injection of the silicone was performed manually by the researchers, during the 3D printing process, using corresponding syringes, that are used in dentistry for this type of silicone, which is both a flexible and durable material.At this stage, the objective was to demonstrate that the integration of these different operations is feasible, and a valid part could be achieved, thus proving the success of the proposed method.To further extend the hybrid procedure, the syringes could be merged with the printer using an extra motor, as well as advanced hardware and software.Nevertheless, additive manufacturing is about prototyping and as such different methods can be applied, meaning automated and semi-automated.Regardless, in many industrial or lab applications it is common ground to perform combinatory processing and manufacturing by implementing robotic arms, thus precisely simulating operations performed by trained personnel who can operate simultaneously or serially with other processes, thus performing Hybrid Manufacturing on the produced part, as it is carried out in the same build space and with the purpose of creating a single part.
After filling the liver mold with the PVA arteries and veins, as well as with the Polysiloxane, an intricate washing process was undertaken to remove the PVA material entirely, leaving behind the final silicone liver including the channels.This process resulted in the creation of a quite detailed and lifelike model: a replica of a liver characterized not only by its external appearance but also by the presence of accurately reproduced arteries and veins.The intricacies of this hybrid additive manufacturing technique are visually demonstrated in Figure 2a-c, showcasing the molds that were created through this innovative process, using both the PLA and the PVA materials.
It is worth noting that the printing of the liver involved the utilization of a non-planar edited G-code.Despite the complexity introduced by this non-planar approach, the final product did not exhibit noticeable differences compared to the model printed with the standard technique.This is due to the complexity and the different levels of the surface of liver geometry.This means that it is not a simple aero-foil, but a more complex shape.For such a case, it was more convenient to use the standard technique for the mold and implement a non-planar approach for the infill of the model, which is the procedure that was finally selected in this study.

Results
The FDM-printed mold, crafted using the 3D printing technique, closely resembles the original design depicted in the STL file.However, owing to the limitations of the 3D printer's resolution and precision, some minor roughness and imperfections are discernible along the edges of the printed mold.These slight discrepancies, attributed to the inherent constraints of a low-quality 3D printer, marginally affect the fidelity of the final output when compared to its digital design.Updating the hardware and software of our system could greatly improve the quality of the product.
Subsequently, the final printed product, following the injection of the gel polymer into the FDM-printed mold and subsequent extraction, exhibits a considerable reduction in size compared to the initial FDM mold.This dimensional variation is an anticipated outcome owing to the presence of the polymer shell, which contributes to a fractional decrease in the overall size of the produced item.However, this disparity in dimensions can be effectively compensated for, by factoring in the thickness of the shell during the design phase.By incorporating adjustments to accommodate the shell's effect on the final product's size, such as modifying the initial STL file dimensions, precise calibration can be achieved, minimizing discrepancies between the digital design and the physical outcome.It needs to be mentioned that the size difference between the 3D printed product and the STL file was less than 10% in all dimensions (i.e., x, y, and z axes, or non-orthogonal axes).The calculated volume also differed by less than 8% when measured in the STL file (~156 mL) and as water volume (~145 mL) inside the PLA liver mold.
Moreover, to further enhance the surfacing and extraction of the final product from the mold, the utilization of a thin layer of Vaseline could augment the surface quality of the final product, which is shown in Figure 3.This additional step aids in optimizing the release of the printed product, ensuring a smoother surface finish, and facilitating a more efficient extraction process.Incorporating such refinements into the production process significantly contributes to achieving a final output that aligns closely with the intricacies of the initial digital design, bridging the gap between the STL file's precision and the physical manifestation of the printed product.

Discussion
In summary, this study showcases a novel low-cost additive manufacturing methodology, combining advanced materials and intricate techniques to produce anatomically accurate models.Through the meticulous integration of silicone veins and arteries into a

Discussion
In summary, this study showcases a novel low-cost additive manufacturing methodology, combining advanced materials and intricate techniques to produce anatomically accurate models.Through the meticulous integration of silicone veins and arteries into a PLA mold, the research team successfully created a liver model that not only captures the external features of the organ but also replicates its internal vascular network with precision.This innovative approach holds immense promise for various applications in the fields of medical education, research, and surgical planning.
One of the most significant implications lies in the field of medical education and training.Clinical scientists could utilize this advanced technique for injection training, allowing medical professionals to optimize their skills with highly detailed and realistic in vitro simulations.This also minimizes the use of animals as well as the need for human tissue, offering a more ethical and advanced platform for medical training while preparing future healthcare professionals for complex clinical scenarios.Moreover, innovation should not stop here; this technology could be expanded upon to explore more sophisticated robotic methods for intricate injections, opening doors to previously unattainable medical procedures.
Furthermore, this technique holds immense promise in the realm of personalized medicine.By refining the process and investing in higher-grade materials, it could contribute to the creation of complete, personalized anthropomorphic phantoms.These phantoms, akin to the RTsafe devices [56], could revolutionize radiation therapy quality assurance, ensuring that treatments are precisely calibrated and tailored to individual patients.The potential benefits for patients' well-being and treatment outcomes are immeasurable.
However, the true marvel of this technology lies in its potential for organ transplantation.With further advancements and investment in cutting-edge materials, this technique could be integrated with living cells, pushing the boundaries of medical science.Functional living organs could be artificially created by the exploitation of such a low-cost technique, offering hope to countless patients on transplant waiting lists.Such an endeavor, though, demands an interdisciplinary collaboration of epic proportions and a substantial financial commitment.The fusion of expertise from various fields, including biology, materials science, and engineering, is essential to navigate the complexities of producing living organs in a controlled environment.
It is crucial to acknowledge the challenges and avenues for future research in this groundbreaking study.Rigorous testing-including pressure assessments, radiation scanning, and dose measurements-is imperative to ensure the safety and efficacy of the developed product.While the initial indications from the existing literature are promising, comprehensive experimentation and validation are necessary steps in this pioneering journey.As the research community delves deeper into this innovative technique, the potential to transform the landscape of medical education, personalized medicine, and organ transplantation becomes increasingly tangible.The ongoing dedication to exploration and refinement will undoubtedly pave the way for a future where the boundaries of medical science are pushed further than ever before, bringing hope and healing to millions around the globe.
Moreover, while this study successfully demonstrates the feasibility of hybrid 3D printing using biocompatible materials, it is imperative to note that the assessment of cell cultures and the viability of cells throughout the complete printing procedure remains an area requiring further investigation.The interaction between printed structures and living cells is a crucial aspect in biomedical applications, necessitating in-depth analyses to ensure optimal cell survival, proliferation, and functionality post-printing.
Future research endeavors should focus on comprehensive studies involving cell cultures to evaluate the biocompatibility and cytocompatibility of the printed structures.Assessing cell behavior, including adhesion, proliferation, and differentiation, throughout the printing process and post-printing stages is crucial for the development of functional tissue constructs or biomedical devices.Furthermore, exploring innovative bioinks and refining printing parameters to enhance cell viability within the printed constructs is fundamental.
Assessing such approaches will enable the scientific community to study the survivability of cells and tissues, even when such population of cells is irradiated.This way, radiation therapy procedures could be highly improved.Addressing these aspects will not only advance our understanding of the interaction between printed materials and cells but also pave the way for the utilization of hybrid additive manufacturing techniques in various biomedical applications, ranging from tissue engineering to drug delivery systems.Drug delivery studies on such phantoms will improve the understanding of Physiologically Based Pharmacokinetic Modeling (PBPK) that will allow for the optimization of therapeutic procedures.

Conclusions
In conclusion, this study presents a promising proof-of-concept for the hybrid 3D printing of biocompatible materials, highlighting the successful integration of two distinct steps: FDM printing utilizing PLA and PVA, and inkjet printing employing gel polymers.While the implemented procedure was not automated, the groundwork laid in this research provides a foundational framework for potential automation in the future [57].Achieving full automation would necessitate a multidimensional approach encompassing advancements in hardware, software evolution, and cross-disciplinary collaborations across fields such as physics, mechanics, biology, and software development.The envisioned automated process holds immense potential, offering efficiency and scalability in the fabrication of biocompatible structures.Overall, this study underscores the feasibility and potential avenues for the development of an automated hybrid 3D printing procedure, marking a significant stride towards innovative applications in biocompatible material fabrication.This study showcases a groundbreaking methodology in 3D printing, successfully integrating advanced materials and intricate techniques to produce highly detailed and anatomically accurate organ models.The potential applications include medical education, research, and surgical planning, emphasizing the promise and utility of this innovative approach.

Figure 1 .Figure 1 .
Figure 1.3D representation of the liver model.Colors differentiate arteries (red) from veins (blue) and the liver (yellow).Letters indicate the anatomical orientation of the organ in the human body: S is for Superior, I for Inferior, L for Left, R for Right, P for Posterior and A for Anterior.

Table 1 .
Printing and material parameters used during the hybrid additive manufacturing procedure.

Table 1 .
Printing and material parameters used during the hybrid additive manufacturing procedure.