A Comprehensive 3D-Molded Bone Flap Protocol for Patient-Specic Cranioplasty

We present a detailed step-by-step approach for the low-cost production and surgical implantation of cranial prostheses, aimed at restoring aesthetics, cerebral protection, and facilitating neurological rehabilitation. This protocol uses combined scan computed tomography (CT) cross-sectional images, in DICOM format, along with a 3D printing (additive manufacturing) setup. The in-house developed software InVesalius® is an open-source tool for medical imaging manipulation. The protocol describes image acquisition (CT scanning) procedures, and image post-processing procedures such as image segmentation, surface/volume rendering, mesh generation of a 3D digital model of the cranial defect and the desired prostheses, and their preparation for use in 3D printers. Furthermore, the protocol describes a detailed powder bed fusion additive manufacturing process, known as Selective Laser Sintering (SLS), using Polyamide (PA12) as feedstock to produce a 3-piece customized printed set per patient. Each set consists of a “cranial defect printout” and a “testing prosthesis” to assemble parts for precision testing, and a cranial “prostheses mold” in 2 parts to allow for the intraoperative modeling of the �nal implant cast using the medical grade Poly(methyl methacrylate) (PMMA) in a time span of a few min. The entire 3D processing time, including modelling, design, production, post-processing and quali�cation, takes approximately 42 h. Modeling the PMMA �ap with a critical thickness of 4 mm by means of Finite Element Method (FEM) assures mechanical and impact properties to be slightly weaker than the bone tissue around it, a safety design to prevent fracturing the skull after a possible subsequent episode of head injury. On a parallel track, the Protocol seeks to provide guidance in the context of equipment, manufacturing cost and troubleshooting. Customized 3D PMMA prostheses offers a reduced operating time, good biocompatibility, and great functional and aesthetic outcomes. Additionally, it offers greater than 15-fold cost advantage over the usage of other materials, including metallic parts produced by additive manufacturing.


PREAMBLE
The current protocol is a guidance tool, in the public domain, designed to assist practitioners in reconstructing large (> 50 cm 2 ) or complex skull defects in adult patients.Our aim, guided by standards of excellence, ethics and equity, remains to guide healthcare personnel in neurosurgical practice in limited-resource communities, reaching the unreachable, in low-and middle-income countries.
"It may be part of human nature to err, but it is also part of human nature to create solutions, nd better alternatives and meet the challenges ahead" (Kohn LT, Corrigan J and Donaldson MS) CRANIOPLASTY: BASIC CONSIDERATIONS Neurological disorders constitute a large and increasing share of the global burden of diseases studies by the WHO, with much of this burden originating in low-and middle-income countries (LMICs) (Feigin et al., 2019).Along with the categorization of neurological diseases, which account for as much of 25% of global death and disability, massive stroke, traumatic brain injury (TBI), brain tumors, congenital deformities and gunshot wounds frequently require large decompressive craniectomy (DC) to reduce intracranial pressure and cerebral edema.For patients who survive, subsequent cranioplasty (i.e., cranial repair) aims to provide cerebral protection, improve blood perfusion and cerebrospinal uid (CBF) dynamic restoring function and esthetics (see reviews by Hutchinson et al., 2019;, laccarino et al., 2020, 2021).However, varying shapes, sizes and complexities of defects observed in these patients present reconstructive challenges dispensing a customized solution to each individual case (Gil et al., 2019).As a result, cranial prostheses production is often pricy, becoming out of reach to patient populations that do not have the resources to defray the costs and that are not funded by public health systems in resourcede cient countries.Therefore, despite advancements in bioengineering and medicine, patients in resource-de cient countries continue to face wait times of years -or stand in line over their lifetime -to repair their skull to relieve psychological drawbacks and to increase both mental and social performances.
For centuries, several materials have been tested to repair cranial defects, ranging from coconut shells, allogenic and xenogenic bone grafts, metals to biosynthetic materials, such as ceramics and resins (Alkhaibary A et al., 2020;Aydin et al., 2001;Khader and Towler, 2016).The characteristics of an ideal prosthesis are resistance to mechanical deformation and to infection, inability to cause in ammatory responses, chemical inertness, non-carcinogenicity, nonconductive to heat/cold, capacity to be sterilized and to be molded into the desired shape, lightweight and low cost.Although there is no 'silver bullet', lowcost intraoperatively produced Poly(methyl methacrylate) (PMMA) bone cement, approved by regulatory agencies worldwide, has been increasingly used in cranial repair with good biocompatibility, and great cosmetic and functional results, presenting performance similar To other available materials and little related implant complications when done with proper techniques (Giese et al, 2019;Gil et al., 2019;Khader and Towler, 2016;Las et al., 2021).Because PMMA has strength comparable to the bone, our safety design in the current protocol assures mechanical and impact properties to be a little weaker than the bone tissue around it to prevent fracturing the skull after a subsequent episode of TBI (see Procedures).Thus, customized 3D PMMA cranial prostheses offers a greater cost-effective strategy for both patients and society, reducing operative time and presenting a maximum gain in overall well-being per unit of expenditure.
Here, we provide step-by-step instructions to help carry out TC imaging, computational work ow, operational additive manufacturing-and intraoperatively-procedures to perform cranioplasty using a PMMA customized implant.A list of reagents, equipment and troubleshooting is provided to ensure that prostheses manufacturing is replicated in a strongly-consistent manner on multiple machines and/or by groups with limited-resources.Reagents see 'Reagents and Equipment' (Table 6 -Suppl.Files) Equipment REAGENTS AND EQUIPMENT Economic implications of 3D printing.3D printing is heralded as one of the technologies that will enable smart, cost-effective, sustainable manufacturing.The possibility to establish a nearly wasteless 3D printing process is within reach.However, creating a successful Additive Manufacturing Unit that will not fail, while keeping the process cost-effective is challenging if you do not have the proper structure.Too often, engineers must scrap multiple parts before they nd the optimal build orientation and support strategy.For beginners, the initial investment required for producing a product using 3D manufacturing processes may exceed that of traditional methods.But, again, the investment on 3D printing is booming because this technology has the potential to resolve the "scale-scope" dilemma on the cost side and because there are no penalties associated with a higher degree of product variety.Below (see Table 6 -Suppl.Files), we provide a list of basic equipment and reagents required for 3D printing of cranial prostheses.Table 6 illustrates a quotation inquiry of 3D Systems for a basic infrastructure composed of an additive manufacturing equipment, related peripherals and basic consumables.Substitute products with similar technical aspects, at a lower cost or value added bene ts, may be offered by other companies like EOS, Germany.There are also additional desktop machines with competitive prices, but their main constraint is the maximum build size to accommodate the 3-piece customized printed set per patient.

** Table 6 **
As with any new piece of equipment, it can often be di cult to determine how quickly it will pay itself off.In this case, the answer is simple: it depends on what branch of healthcare you are in, ranging from a few to a large number of patients.3D printers may cost up to $1M.However, there are plenty of e cient printers in the mid-range ($100,000 -$250,000), which suits well mid-size medical groups with prostheses manufacturing needs.To work out your ROI ('return on investment'), take the current market prostheses costs, the potential number of patients that could bene t from this solution, and subtract the cost to print the corresponding parts to determine the savings.Take the price of the printer, divide by the cost of savings to determine the number of printed parts needed to pay off a speci c printer.Below is a guide to determined required equipment and reagents cost (Table 6 -Suppl Files).

Procedure
Our protocol provides clear, step-by-step instructions to help healthcare professionals carry out routine cranioplasty procedures, ranging from CT imaging acquisition, PMMA prostheses manufacturing to intraoperative ap implantation (Fig. 1).Given that a manufacturing facility is capable of printing a signi cant number of customized cranial prostheses without retooling, and that each printing can be performed without additional cost, establishment of a single central Manufacturing Additive Unit in lowincome economies could provide effective assistance (cost-effectiveness analysis) in a timely fashion to an increasing number of patients in need of cranioplasty procedures.purpose and photographs were obtained prior to the project's initiation.

• CT scanners
Multidetector computed tomography (MDCT) and volume computed tomography (VCT) have made it simple to obtain a volume data set that can be reconstructed as well as viewed with multiplanar reformation tools (MPR).Studies of the cranial and facial region, although viewed in both soft tissue and bone windows, render its use primarily for osseous and dental structures of the cranio-maxillofacial complex.
It is crucial for image quality to position the patient in the center of the scan eld.Patients lying in supine position, head rst into the gantry, should be assessed at rest, maintaining teeth occlusion, and must not swallow nor chew during the imaging acquisition process.The table height should be centered such that the external auditory meatus (EAM) is at the center of the gantry so that the desired cranial defect area lies within the boundaries of a selected eld of view (FOV).This is achieved by moving the patient's head in the FOV or moving the FOV over the patient's region of interest (ROI).Use the lowest head rest available to get the patient's head back su ciently so that the occlusal plane is vertical (see Table 1 -Suppl.Files).
It is easier to acquire high quality images for a particular ROI using a larger FOV.To reduce or avoid ocular lens exposure, the scan angle should be parallel to a line created by the supraorbital ridge and the inner table of the posterior margin of the foramen magnum -perform one or both of these maneuvers whenever possible.Finally, a helpful way to determine the orientation of a CT scan image is to use radiopaque markers.Remember: gantry tilt is available for sequence scanning, not for spiral scanning and that gantry tilt is not available for dual source scanners.i) DICOM-based CDs and DVDs -burning CDs or DVDs for each patient comes with its burden of costs.The cost of delivering the CD must also be taken into account.Also, CDs and DVDs can be easily lost, misplaced or get damaged.CD-RW (Compact Disc-ReWritable) often get scratches that ruin the data and are not recommended to be used.

ii)
Cloud-based DICOM solutions, such as the GoogleDrive (Google®), OneDrive (Microsoft ®) and AppleCloud (Apple ®).Most providers have free versions of their services.Enable users to access DICOM images without installing special software on their devices.Cloud-based DICOM viewers are usually 'zero footprint'.This means that the device somebody else use to view the images will not be affected by the viewer.This offers several advantages, among them: i) almost any device can work with cloud-based viewers; and, that, ii) most standalone DICOM viewers are compatible with only one particular type of operating system (OS), either Windows or Mac OS.Zero footprint viewers, however, work through the internet browser (such as Chrome, Firefox, or Safari) and, therefore, do not require a speci c kind of OS.Summary of advantages: greater accessibility, lower costs, security and safety.

iii)
Sending DICOM les via File Transfer Protocol (FTP) using WeTransfer.comservices-send up to 2 GBytes in one go for free.Send up to 20 GBytes in one go and allow for storage of 1 TByte (terabyte) for about U$ 120/year (WeTransfer Pro) -equivalent to four Windows or Mac laptops with 256 GBytes of storage.There are many others FTP services freely available.

iv)
The DICOM les can be also stored either in pendrives or in external hard disk drives, but the services above mentioned allow for simple and fast transferring of image les from a Hospital to an Additive Manufacturing Unit, which will demand a faster internet connection depending on the frequency and amount of data transferred.

Phase #2: 3D manufacturing
The DICOM le is received at the Additive Manufacturing Unit.At this point, the image data of the CT scanned area is a series of 2D images that need to be segmented, in order to separate the bone from other tissues, and converted into 3D mesh, to generate volumetric reconstruction (Fig. 2) ** Figure 2 ** 3D modelling and printing hardware Image processing is classi ed as a high performance computing task.This means that computers used for such applications must meet high requirements in terms of CPU, RAM, and graphical speci cations in order to achieve optimal performance.Su cient power supply and cooling must also be ensured for the server or workstation.We strongly recommend that the chosen hardware meets minimum requirement (Table 3 -Suppl.Files).

** Table 3 **
The image segmentation process can be performed manually, automatically or using both algorithms and lters (Abdullah et al. 2019).There are several segmentation methods and algorithms for medical images, among them the threshold base, where a threshold value is chosen using the radiodensity of different tissues to select a range of pixels, which highlights and separates speci c structures in the image, making for easier identi cation of bones and other tissues (Fig. 2A-D).
The 3D model used in the computational simulation consists of the PMMA prosthesis and half of a skull.The cranial geometry was obtained from DICOM images of a cranium of a patient with bone defects.The 3D geometry of the cranium exhibiting defects was obtained by rendering images via InVesalius ® .
InVesalius® is an in-house free open-source 3D medical imaging reconstruction software that generates a 3D image from a sequence of 2D DICOM images (CT or MRI) (Fig. 2A-D; Fig. 3A,B).It has been developed by the Renato Archer Information Technology Center (CTI-RA) under the leadership of Dr Jorge Silva, Campinas/São Paulo, Brazil, supporting 24 languages (Fig. 3B,D).The software is compatible with Windows, Linux and macOS.The application of this widely used and reproduceable software has been bene ting research institutions and public hospitals in LMICs to produce accurate STL skull models for teaching, medical training or clinical purposes over the last eleven years (Fig. 3A,B).The 3D models produced by InVesalius® Using Magics®, the prosthetic geometry was generated in correspondence to the cranial defect.The .stl les of the skull and the prosthesis were processed through Geomagic Design X ® , in which the component mesh was optimized (Fig. 2E,F).Of note, the three-dimensional nite element method (3D-FEM) is limited to at one-time use, to resolve the general geometry stress analysis of the skull and to de ne the optimized thickness of the cranial prostheses, and the concept applied to all 3D modeling.Afterwards, the skull and prostheses images were exported to CAD Rhinoceros ® where the region of contact between components was adjusted, as well as where the solid model of the combined components was generated.Fig. 4 shows the prosthesis geometry (A), the skull geometry (B) and the combined assembly (C).As seen in Fig. 4A (left side) the prosthesis possesses variable thickness.The value in the region of least thickness is approximately 2.75 mm, the central region contains a value of approximately 3.95 mm and the thickest region is approximately 5 mm thick.The average thickness for the model is around 4 mm.Fig. 4A (right side) shows a circular region previously de ned by geometry for applying force in the computational model to be analyzed using the nite element method.** Figure 4 ** Afterwards, each component was saved through the extension.step,and the model was sent to the commercial software for nite elements, Hypermesh® for mesh optimization.Each component was built with a 2D mesh using triangular elements.Subsequently, a 3D mesh was generated from solids using rst order tetrahedral elements (CTETRA).Table 4 (Suppl.Files) presents the quantity of nodes and elements, 'backbones' of nite element analysis (FEA) [1], from the skull and prostheses.Fig. 5 presents the nite element mesh of the model components.[1] In FEA, the model is divided into small pieces, called Finite Elements (FE).Those elements connect all characteristic points (called Nodes), that lie on their circumference.This 'connection' is a set of equations termed shaped functions.Adjacent Elements share common Nodes (the ones on the shared edge).Thus, the shape functions of all the Elements in the model are tied by common Nodes.The properties of the skull and prosthetic material were assumed to be linearly elastic, homogenous and isotropic for analysis of resistance.The mechanical material properties of the PMMA and cortical bone are listed in

Mechanical contact characteristics
In mechanical systems, the contact between bodies is a non-linear problem that presents certain solution di culties.The formulation of a mathematical model that appropriately expresses the stress distribution and the displacement eld is one of the main di culties of mechanical contact between solid bodies.
In a nite element analysis, contact conditions are special classes of discontinuous constraints establishing that loadings are transmitted from one part of the model to another.In this Protocol, we used Hypermesh® software to establish a relationship between the 'glued' contact surfaces of the skull and the prosthesis, with the skull being the "master" and the prosthesis being the "slave".Therefore, it is expected that the customized prostheses and the bone are in perfect contact.

Contour and loading conditions
The prostheses resistance was evaluated using three different values of static loading: 50 N, 600 N and 1200 N. The 50 N load was based on the work performed by Ridwan-Pramana and collaborators (2016) and is an approximation of the reaction force induced in the skull (≈ 5 kg) when resting on a at surface without any other force acting on it.A 1200 N force was chosen to simulate an extreme impact condition in the prosthesis region.The 600 N load was chosen as an intermediate value between 50 and 1200 N.
Each load was applied perpendicularly to a circular region de ned in the center of the PMMA prostheses.The contour of half of the skullcap was xed in order to prevent translation and rotation on an xyzcoordinate axis.The objective of the simulation was to verify whether the thickness of the PMMA cranial prostheses would be su cient to support loads imposed on it.Fig. 7 shows the results obtained for the Von Mises equivalent stress when the applied force in the central region of the PMMA prostheses was 50 N (A), 600 N (B), and 1200 N (C).
As seen in Fig. 7, the stress distribution in the three models demonstrated the same behavior.In addition to the central region where the force was applied, it is evident that there was a tendency for stress to concentrate on the inferior edge that corresponds to the location of least prosthetic thickness.The values maximum stress were 4,25 MPa, 22,38 MPa and 44,83 MPa for loads of 50 N, 600 N and 1200 N, respectively.According to these values, the prostheses would not fail due to ow stress, since the PMMA ow stress is 72 MPa (Ridwan-Pramana et al., 2016, 2017).** Figure 7 ** Displacement analysis is important to verify the stability of the prostheses.Therefore, the total displacement observed in all of the conditions analyzed, remained acceptable and below 1 mm.The highest displacement was 0,83 mm and was observed in the 1200 N load simulation.The distribution of displacement levels in each model can be seen in Fig. 8.The nite element analysis showed that the thickness of the prostheses was su cient to support loads imposed on the PMMA structure.The resistance was also ensured by the safety factor (SF) attributed to the design of the thickness of the prostheses.To properly design a structure, it is necessary to restrict the stress imposed on a material to a level that is safe and stable.Thus, it is essential that the stress generated in the analyzed structure, labeled as the allowable stress (σadm), is less than the rupture stress of the material (σrup).Among the reasons for this relationship is the fact that the load for which the structure was designed for may be different from the actual loads applied to it.Another fact is that the projected measurements of the structure may not be exact, due to errors in manufacturing (Shigley et al., 2004).** Figure 8 ** A Safety Factor (SF) for the cranial prostheses was calculated considering the maximum loading applied to the model, which was 1200 N.For this, the maximum stress obtained was 44 MPa (σadm), considering a uniform prostheses thickness of 4 mm.Since the PMMA rupture stress is 72 MPa (σrup) (Table 5), the SF calculated according to the formula below it is 1.6, consistent with the literature (Shigley et al., 2004).SF for the prostheses: SF = σrup∕ σadm = 1.60 Therefore, considering the above-mentioned conditions for SF simulation, the cranial prostheses thickness of 4 mm was adopted in all prostheses produced by our group.It is worth mentioning, again, that this procedure of simulation is limited to a one-time event.
The molding of a personalized prostheses, in general, is done manually during surgical procedures (Maricevich et al. 2018(Maricevich et al. , 2019)).This can lead to variations in the thickness of the prostheses in addition to speci c defects, such as bubbles, which can form during the polymer curing process.The person responsible for the production of the prostheses should also ensure the homogeneity of the mixture (liquid and solid components).Such factors could in uence the nal resistance of the cranial prostheses.
According to Saura (2014), one of the requirements for materials for craniofacial bone reconstruction is that these materials shall have considerable order of magnitude in terms of mechanical resistance and deformation as the original bone.Thus, one of the reasons for choosing PMMA lies in the fact that the resistance prior to rupture is approximately 72 MPa (Table 5 -Suppl.Files) while that of the cortical bone of the skull is in the range of 65 MPa to 130 MPa (Boruah et al., 2020; Ridwan-Pramana et al., 2016).On the other hand, the use of metal as titanium alloy prostheses with resistance of 900 MPa (Shigley et al., 2004) can result in force transmission between implant and skull, at increased risk of fracture of bone tissue around the prostheses.

Thickness of the cranial prostheses -'bedside' (multiple episodes of TBI)
When evaluating the need to perform a large cranial repairment procedure (> 50 cm 2 ), the surgeon needs to be aware of the choice of the ideal material for reconstruction, adopting a safety design to prevent fracturing the skull again after multiple rather than a single episode of head trauma -as 'a lightning may de nitely strike the same place twice' (Fig. 9).** Figure 9 ** From bench to bedside: proof-of-concept Lack of complications following prosthetic replacement in a patient with subsequent episodes of TBI, under real life conditions, supported our computational strategy, using the nite element method, simulating the stress of static device placed onto the skull.From the obtained results, we con rmed that the 4 mm thickness PMMA prostheses is effective for cranioplasty.Therefore, the 4 mm-thickness is adequate to guarantee the mechanical protection of the skullcap considering the conditions imposed on the product -and also biomechanical properties similar to the bone tissue (see Fig. 12).Thus, in contrast to a range of materials available, comminuted fracture of the PMMA prostheses, associated with eventual subsequent episodes of head injury, spares the bone framework as predicted, e ciently preventing enlargement of the primary bone defect.
Autogenous bone grafts remain the gold standard for cranial reconstruction.While several problems remain that limit the wide utilization of such option, including regulatory requirements, high costs, comorbidity as well as method-speci c limitations, customized intraoperative PMMA implants manufactured over the rapid prototyping molds proved to be effective and feasible.Thus, although advances in tissue engineering and biomaterials technology are expected alternatives on the long-run, the current cranioplasty Protocol represents a realistic approach that support a safe and cost-effective delivery of care.

Troubleshooting
The Protocol presented here focuses on the 3D printing technique known as selective laser sintering (SLS), which affords high-quality results at relatively low costs.The current Protocol does not report on other types of printing (Negi et al., 2020), which, on the basis of years of experience are considered less satisfactory.
Since the actual printing process is directly in uenced by how the model is sliced, oriented, and lled, optimizing the results, systematic reviews highlight general problems and provide guidance for an effective problem-solving process (Martinez-Marquez et al., 2018, 2019, 2020; Szczykutowicz, 2020; Van der Molen et al. 2007).Looking at 'troubleshooting' with a different perspective, we focused on solving technical problems encountered in our operations, from the OR to intraoperative modelling of PMMA prostheses (Table 7 -Suppl.Files).

** Table 7 ** INTRAOPERATIVE TIPS
Intraoperative modeling of the PMMA ap requires a modest but well thought out pre-planned room setup.The prosthetist needs a small, but dedicated area (table/bench) in a work space free of cords, suction tubing and other OR equipment, and must be able to move easily throughout the operating room in order to avoid contamination while preparing the PMMA prostheses, adjust the PMMA ap to facilitate prosthetic tting, drill holes on the fabricated ap, side to scrub (sterile surgical scrub brush) the ap, wash the prostheses with saline, dry it and pass suture threads through the drill holes in order to anchor the dura and to x the scalp to the ap to help prevent seromas (Maricevich et al., 2018) Bone cement preparation: self-curing (polymerizing) PMMA cements are available in medical applications in the form of a two-component system (PMMA formulation pack), consisting of a powder (PMMA) and a liquid (MMA) that are mixed (Fig. 11A,B).Mixing of the components is followed by polymerization of the liquid monomer becoming a solid mass ('dough-like) after 3-5 min: the lower the heat of polymerization, the longer the setting time for the cement to harden; the greater the heat of polymerization, the shorter the setting time."Cement' is formed by way of polymerization of the MMA initiated by free radicals produced by the reaction of the BPO, present in the powder, with DMPT, present in the liquid.Notably, PMMA is currently available with different viscosities, different peak reaction temperature and different hardening temperature.Therefore, mixing formulations from different brands or mixing PMMA subtypes from the same brand is not recommended.** Figure 11 ** The reaction starts with the addition of the liquid to the powder, and ends when the dough appears homogeneous (time to place it into the molding) (Fig. 11A,B).The cement 'dough-like' consistency and time point parameters depend on the mixing speed and time, ranging from 3-5 min to reach the setting point.Mix thoroughly, but for better consistency, the prosthetist should maintain routine pace, from start to nish, to obtain similar end results.At the end of the 'mixing phase', the mixture should be a homogeneous and sticky mass, displaying a consistency similar to 'cookie-dough'.The mixing phase is then followed by swelling of the beads, leading to an increase in viscosity.Polymerization proceeds and the cement mixture turns into a sticky dough.The beginning of the 'modelling phase' occurs when the cement mixture is no longer sticky.At this stage, apply Dersani ® Oil, an essential thermal stable fatty acid, to the inner faces of the molding (both upper and lower moldings) -this simple procedure will facilitate detachment of the cranial prostheses from the molding after the heat curing of the PMMA.That is the key time to start spreading the cement mixture homogeneously along the concave surface of the molding (Fig. 11C) -spreads 'like butter on bread'.Remember: the ideal thickness of the prostheses is 4 mm; thus, do not overload the molding shell with PMMA-dough.Firmly hold the molding shells containing the PMMA-dough with both hands, engaging the two complementary halves of the mold (Fig. 11E).
Polymerization continues and the viscosity increases inside the molding as exothermic reaction associated with polymerization leads to generation of heat (up to 90°C).Keep rmly holding the molding shells containing the PMMA-dough.An estimate of the ideal curing time of the compressed PMMA inside the moldings can be obtained, in parallel, by feeling the increase in hardness of small leftover 'dough balls' (≈1 cm 3 ) with gloved ngers every 30 sec until it reaches the desired consistency.Cranial prostheses were xed with 4 titanium bridge miniplates, each held down with 2 titanium screws.
Post-cranioplasty procedures: A procedure-control CT scan was performed within the rst 12 hr after surgery (Figs.11G, 12D).The drain was withdrawn with a ow rate <50 ml in the last 12 hr and hospital discharge usually occurred within 48 hr.** Figure 12 ** Advice -feedback from staff from HR (Dr.Maricevich) and from staff and residents from HMMC (Drs Monteiro, Schiavini, Pilon, Bertani and Moreno) lead to an improved response to cranial repair delivery (Fig. 11F,H): * Monitor if healthcare workers are keeping the settings (i.e., temperature and time of procedure) established for sterilization of the 3 piece PA set.* Volumetric reconstruction of the cranial ap requires about 2 commercial packs of PMMA (each pack contains liquid in an ampoule and powder in a bottle) -do not mix different brands of bone cement or different viscosities formulations from the same brand in order to produce a single ap.* Expose the border of the bone defect widely in order to facilitate the insertion, proper tting and xation of the PMMA prostheses.
•* Make ≥ 6 drill holes oriented perpendicular to the cranial prostheses.* To minimize risks of epidural hematoma formation (optional) (Pascual JM and Prieto R , 2012; Salcman et al., 2004;Winston, 1999): low-tension, dural tact-sutures can be applied throughout the extension of the craniotomy, mainly on its edges, adopting a distance of 1.5 to 2.0cm between ori ces.These ori ces can be drilled with 1mm drills.plane (i.e., frontal, temporal and parietal-occipital) to run dural tent and subcutaneous suturing.The threads are kept isolated from one another (Fig. 14A-D The average time to produce the proposed 3-piece customized set, consisting of a PA "cranial defect printout", a "testing prosthesis" and a cranial "prostheses mold", is about 42 hr, of which the majority of the time is automated/unattended printing (71.5%) and with an average 'hands-on' labor time of about 12 hrs (Table 8 -Suppl.Files).The sequence of steps in the whole 3D approach is illustrated in Fig. 10.

** Table 8 **
The cost for general consumables is relatively modest, except for SLS-DuraForm® PA, which has an average cost of about U$ 130.00 per kg.Assuming that an average 3.3 kg PA is laid down on the print bed/set, and that only about 1/3 (≈ 1 kg) of the mass is sintered by the SLS per cycle to produce a 3piece customized set, 69.7% of the powder load will not be used during a typical cycle.Thus, recycling of PA represents one of the most dynamics areas in 3D manufacturing of cranial prostheses.Recycling PA is both economically and environmentally bene cial, not only providing opportunities to reduce costs, but also to reduce quantities of waste.In our Protocol, 'leftover' PA was puri ed from the degraded components and was reintroduced into the 3D process 6-7 additional times by combing it with virgin PA powder.Including other consumables and personnel salaries, the 3-piece customized set costs ≈U$ 1,800 to manufacture (without scale manufacturing).Although the initial setup costs for 3D printing is relatively high compared to hand-crafted prosthetic modelling, this is a one-time xed cost.Additionally, additive manufacturing mold-based prostheses have the advantage over intraoperative molding of reduced surgical time, better t geometry, less distortion, better mechanical behavior, lower blood loss and infection rate, and, nally and equally important, great aesthetic results.Also, polymerization of PMMA bone cement undergoes an exothermic reaction, during which the cement hardens and the temperature increases.In order to prevent tissue necrosis, the ISO 58833 ( 2002

CLINICAL DATA
The current open-source Protocol describes the step-by-step guidelines for conducting 3D manufacturing of cranial prostheses.It illustrates what should be made in 3D manufacturing by explaining each essential part of the cranioplasty procedure, from head CT imaging acquisition criteria to skull ap implantation, including optional suggestions and tips to prep for surgery.All cranioplasty procedures ended without any unexpected events.The patients were hospitalized for about 2 days and discharged after a single drain was removed to continue their care as outpatients.
Here, we describe the eligibility of the participants, the outcome of the surgeries and the related functional tests in accordance with humanitarian principles, so as to enable patients to lead a life of dignity and respect.
Sterilization and cleaning of the 3-piece PA customized 3D printed set Steam sterilization has limited industrial application but is frequently used in hospital facilities.A steam sterilizer (i.e.,'autoclave') uses saturated steam at 121°C.A typical standard for steam sterilization in our Protocol is achieved about 15-30 min under a pressure of 106 kPa (1 atm) once all parts have reached 121°C.Steam sterilization has many advantages.It is a simple, rapid, effective, safe, environment-friendly and low-cost sterilization method.
Damage on polymers can vary from a little oxidation to complete distortion and melting, depending on polymer composition and properties.It is important to mention here that some polymers, among them PA, can be safely sterilized by steam at 121°C up to 1 hr, or at 134°C for 5 min (Maricevich et al., 2019).Using the circumstances described we never observed distortion or melting of any of the 3-piece PA customized 3D printed set.
CUTOMIZED CRANIAL REPAIR AND PATIENT BASELINE CHARACTERISTICS: A total of fty-four (54) 3piece PA customized 3D printed sets were manufactured.The sample included randomly selected surgical patients aged ≥18 years old presenting a Glasgow Coma Scale (GCS) score of 15.Table 9 (Suppl.Files) and Fig. 12 show/illustrate baseline characteristics of the patients admitted to this study at HMMC, Rio de Janeiro, and at HR, Recife, Brazil.

** Table 9 **
The mean age of the participants at HMMC was 44 ± 12 years (ranging from 19 to 60 yr) as compared to that performed in patients at HR with a mean age of 33 yr, sex ratio (female:male) 1:2.8 (HMMC) and 1:6.8 (HR).Indications for the primary craniectomy at HMMC were tumor resection, infection and stroke in contrast to that of closed traumatic brain injury (TBI) in civilian settings at the HR.The cranial repair study at HMMC also included 3 military policemen (38 ± 4 years old) who underwent decompressive craniectomy (DC) following gunshot wounds to the head (links F,G).Interval between previous surgery and PMMA cranioplasty ranged between 6 months and 8 years.The operative time ranged from ranged from 90 to 180 min (mean 112 ± 40 min).The average cranial defect measured 137.98 ± 40.63 cm 2 (estimated using Materialise Magics® reconstruction) and the estimated prosthetic volume 52.03 ± 14.35 cm 3 (estimated modelling the PA ap using Materialise Magics®, version 22.01), with 4 mm thickness.
Patients were evaluated prior (T0), at 1 month (T1), 3 months (T3), 6 months (T6) and at 12 months (T12) after surgery, combining cognitive and neuropsychological testing, 'Trephine Syndrome' questionnaire, neuroimaging to assess functional anatomy (fMRI) (liquor ux), transcranial doppler assessment (cerebral perfusion in gunshot wound patients), EEG tests and qEEG assessments (pattern of slow waves).An evaluation of the signs and symptoms was performed through a 'Trephine Syndrome' questionnaire, for all patients, at T0 and T6.Common reported complaints in both cohorts (HMMC and HR) were local discomfort, headache, dizziness, tinnitus, insomnia, fatigue, irritability, depression, insecurity, paresis, dysphasia, dyspraxia, attention de cit, memory de cit, and worsening symptoms in orthostatic position.Seizures, associated to TBI, were frequently reported at the cohort conducted at HR (TBI patients), but rarely observed in the cohort developed at HMMC -and thus were considered a nonspeci c implant-related complication.At T6, patients were evaluated regarding aesthetic result of their cranial repair (excellent, very good, good, regular, and bad).Complications within the follow-up period were assessed as: grade #1: no invasive treatment required; grade #2: invasive treatment required, but not intensive care unit admission (ICU); grade #3: invasive treatment required and ICU admission; grade #4: death].All patients assisted at HR and HMMC reported enhanced self-esteem and built con dence after surgery.Participants also reported increase in cognitive ability, physical capacity and performance after reconstruction of their skull bone.The proposed PMMA cranial repair Protocol, in addition to reestablishing the cranial aesthetic contour, protected the underlying cerebrum by promptly restoring the brain vascular compliance (Fig. 13A-C).Improvement in cerebral hemodynamics was further con rmed by fMRI tests on patients up to 12 months after cranioplasty (Fig. 13D-F).The Chi-square test for independence was used to test relationships between categorical variables between the cohorts, considered signi cant for P <0.05.** Figure 13 ** Seromas may develop where any skin break occurs (De La Peña et al., 2018;Maricevich et al., 2018).The formation of seroma in cranial repair frequently raises questions about the need for implant fenestration, aspiration and/or drainage (Maricevich et al., 2018).Studies have suggested that seroma production in implant-based to reconstruct the cranium varies with different types of matrices (De La Peña et al., 2018) and biomaterials, namely metals ('titanium mesh erosion'), ceramics ('associated infections'), synthetic and natural polymers, as well as composite materials.However, lack of standard cranial-based guidance prostheses guidelines, including sterilization, intraoperative cleaning and manipulation of the ap and associated antibiotics regimen, does not explicitly preclude clinical success of any of the abovementioned biomaterials.
Previous studies demonstrated the effectiveness of the tacking sutures applied to cranial reconstructions with PMMA prostheses in reducing the incidence of seroma (Maricevich et al., 2018).Therefore, we adopted this intervention in the current protocol (Fig. 14A-D).The outlook for using 3D printing manufacturing PMMA cranial prostheses is bright, with rapidly progressing intraoperative adjustments.Continuing efforts to improve intraoperative performance will facilitate translation of the technology and design methods.Following are some organizing strategies that are being developed to manage to handle the PMMA dough.
Handling sticky PMMA dough: a simple guide to plastic molding You have carefully prepared and assembled the customized PPMA prostheses.After a tantalizing 5 min wait for the curing process of the PMMA-ap, you are now ready to take it off the molding set.However, as you go to slide the prostheses off, you notice that it is stuck to the mold.What do you do?You will know how stressful of an experience it can be, since you can't physically pry the prostheses off and leaving bits of it behind.
Any type of dough, including PMMA, tends to be fairly sticky.This is why we routinely greased the molds with inert oil (e.g., Dersani) to facilitate detachment of the PMMA cranial prostheses from the PA molding.
Alternatively, if circumstances permit, other methods, including making a heated plastic vacuum-mold making and silicone (Fig. 15, Fig. 16), may be used to form a 'coat barrier' between the PMMA prostheses and the PA mold.
At rst glance, this is an ideal solution, leaving less of a mess than oil to deal with during the intraoperative period.Some advocates argue silicone is somewhat of a hybrid between a synthetic rubber and a synthetic plastic polymer, which means it is still a plastic, no matter how it is spun.Regardless of your choice, the important step is to weigh the advantages/ disadvantages of each method to effectively prevent attachment of the PMMA prostheses to the PA mold, reducing the intraoperative time and without causing formation of reactive metabolites.

CLINICAL DATA
The current open-source Protocol describes the step-by-step guidelines for conducting 3D manufacturing of cranial prostheses.It illustrates what should be made in 3D manufacturing by explaining each essential part of the cranioplasty procedure, from head CT imaging acquisition criteria to skull ap implantation, including optional suggestions and tips to prep for surgery.All cranioplasty procedures ended without any unexpected events.The patients were hospitalized for about 2 days and discharged after a single drain was removed to continue their care as outpatients.
Here, we describe the eligibility of the participants, the outcome of the surgeries and the related functional tests in accordance with humanitarian principles, so as to enable patients to lead a life of dignity and respect.
Sterilization and cleaning of the 3-piece PA customized 3D printed set Steam sterilization has limited industrial application but is frequently used in hospital facilities.A steam sterilizer (i.e.,'autoclave') uses saturated steam at 121°C.A typical standard for steam sterilization in our Protocol is achieved about 15-30 min under a pressure of 106 kPa (1 atm) once all parts have reached 121°C.Steam sterilization has many advantages.It is a simple, rapid, effective, safe, environment-friendly and low-cost sterilization method.
Damage on polymers can vary from a little oxidation to complete distortion and melting, depending on polymer composition and properties.It is important to mention here that some polymers, among them PA, can be safely sterilized by steam at 121°C up to 1 hr, or at 134°C for 5 min (Maricevich et al., 2019).Using the circumstances described we never observed distortion or melting of any of the 3-piece PA customized 3D printed set.
CUTOMIZED CRANIAL REPAIR AND PATIENT BASELINE CHARACTERISTICS: total of fty-four (54) 3piece PA customized 3D printed sets were manufactured.The sample included randomly selected surgical patients aged ≥18 years old presenting a Glasgow Coma Scale (GCS) score of 15.Table 9 (Suppl.Files) and Fig. 12 show/illustrate baseline characteristics of the patients admitted to this study at HMMC, Rio de Janeiro, and at HR, Recife, Brazil.Patients were evaluated prior (T0), at 1 month (T1), 3 months (T3), 6 months (T6) and at 12 months (T12) after surgery, combining cognitive and neuropsychological testing, 'Trephine Syndrome' questionnaire, neuroimaging to assess functional anatomy (fMRI) (liquor ux), transcranial doppler assessment (cerebral perfusion in gunshot wound patients), EEG tests and qEEG assessments (pattern of slow waves).An evaluation of the signs and symptoms was performed through a 'Trephine Syndrome' questionnaire, for all patients, at T0 and T6.Common reported complaints in both cohorts (HMMC and HR) were local discomfort, headache, dizziness, tinnitus, insomnia, fatigue, irritability, depression, insecurity, paresis, dysphasia, dyspraxia, attention de cit, memory de cit, and worsening symptoms in orthostatic position.Seizures, associated to TBI, were frequently reported at the cohort conducted at HR (TBI patients), but rarely observed in the cohort developed at HMMC -and thus were considered a nonspeci c implant-related complication.At T6, patients were evaluated regarding aesthetic result of their cranial repair (excellent, very good, good, regular, and bad).Complications within the follow-up period were assessed as: grade #1: no invasive treatment required; grade #2: invasive treatment required, but not intensive care unit admission (ICU); grade #3: invasive treatment required and ICU admission; grade #4: death].All patients assisted at HR and HMMC reported enhanced self-esteem and built con dence after surgery.Participants also reported increase in cognitive ability, physical capacity and performance after reconstruction of their skull bone.The proposed PMMA cranial repair Protocol, in addition to reestablishing the cranial aesthetic contour, protected the underlying cerebrum by promptly restoring the brain vascular compliance (Fig. 13A-C).Improvement in cerebral hemodynamics was further con rmed by fMRI tests on patients up to 12 months after cranioplasty (Fig. 13D-F).Previous studies demonstrated the effectiveness of the tacking sutures applied to cranial reconstructions with PMMA prostheses in reducing the incidence of seroma (Maricevich et al., 2018).Therefore, we adopted this intervention in the current protocol (Fig. 14A-D).The outlook for using 3D printing manufacturing PMMA cranial prostheses is bright, with rapidly progressing intraoperative adjustments.Continuing efforts to improve intraoperative performance will facilitate translation of the technology and design methods.Following are some organizing strategies that are being developed to manage to handle the PMMA dough.
Handling sticky PMMA dough: a simple guide to plastic molding You have carefully prepared and assembled the customized PPMA prostheses.After a tantalizing 5 min wait for the curing process of the PMMA-ap, you are now ready to take it off the molding set.However, as you go to slide the prostheses off, you notice that it is stuck to the mold.What do you do?You will know how stressful of an experience it can be, since you can't physically pry the prostheses off and leaving bits of it behind.
Any type of dough, including PMMA, tends to be fairly sticky.This is why we routinely greased the molds with inert oil (e.g., Dersani) to facilitate detachment of the PMMA cranial prostheses from the PA molding.
Alternatively, if circumstances permit, other methods, including making a heated plastic vacuum-mold making and silicone (Fig. 15, Fig. 16), may be used to form a 'coat barrier' between the PMMA prostheses and the PA mold.
At rst glance, this is an ideal solution, leaving less of a mess than oil to deal with during the intraoperative period.Some advocates argue silicone is somewhat of a hybrid between a synthetic rubber and a synthetic plastic polymer, which means it is still a plastic, no matter how it is spun.Regardless of your choice, the important step is to weigh the advantages/ disadvantages of each method to effectively prevent attachment of the PMMA prostheses to the PA mold, reducing the intraoperative time and without causing formation of reactive metabolites.

Compression Molding
A heated plastic is placed into a heated mold and then pressed into a speci c shape (Fig. 15A,D) (link H).The plastic comes either in sheets or in bulk.Once the plastic is compressed into the right shape, the heating process ensures that the plastic retains maximum strength.The nal steps in this process involve cooling, trimming, and then removing the plastic part from the mold.The automotive industry frequently uses the compression molding technology to manufacture resistant and durable nal products.
Cost-effectiveness: the cost of each individual part is low at high quantities.This methodology provides an alternative choice to the intraoperative use of oil in facilitating detachment of the PMMA cranial prostheses from the PA molding.to assemble parts for precision testing, and a cranial "prostheses mold" in 2 parts to allow for the intraoperative modeling of the nal implant cast using the medical grade Poly (methyl methacrylate) (PMMA) in a span time of a few min (H).Illustration of nite element mesh of the components in the model using Hypermesh.
Location of the application of forces to the prostheses and the constraint of movement in the skull contour.
Page 41/50 Compression molding: heated plastic vacuum-mold making.(A) Vacuum thermoforming machines allows fast heating of a plastic sheet, which is then dropped over a mold (B,C) whilst a vacuum is applied (C,D).The mold is then allowed to cool before it is ejected from the mold using a gentle reverse pressure.Model used in this assay: Vacuum forming compacta 41x41.
Fig. 6 shows the application of the force perpendicular to the prostheses and the location of the skull xation.** Figure 6 ** Thickness of the cranial prostheses -'bench' (modeling and simulation) ). * The prostheses are installed and xed with titanium mini bone plates (n=3 or 4) and screws (2 per plate) to the surrounding host bone tissue.* The next step is performed anchoring the dura (n≥3 suture anchors) to the cranial vault.A surgical silicone drain is placed subgalealy, then tunneled and delivered through a separate stab incision and attached to a closed drain system to gravity.* At last, suture anchors (n≥3) are performed to x the subcutaneous tissue to the prostheses, and surgical wound plane closure is executed.** Figure 13 ** ** Figure 14 ** Time Taken TIME TAKEN AND COST/UNIT ) places a limit of 90°C on the hardening of bone cements.** Figure 10 ** Anticipated Results follow-up for the detection of seroma should continue for at least three months.II) instances of seroma -the patient should be evaluated at least 2x/week until resolution of the seroma with emptying and compressive dressing.Seromas should be addressed only by the scar (less likely site of vascular injury) with aspirated and drained using a 40 × 12 needle and a 10 ml syringe (Maricevich et al., 2018).** Figure 14 ** HOW CAN WE IMPROVE GUIDELINES USE?
follow-up for the detection of seroma should continue for at least three months.II) instances of seroma -the patient should be evaluated at least 2x/week until resolution of the seroma with emptying and compressive dressing.Seromas should be addressed only by the scar (less likely site of vascular injury) with aspirated and drained using a 40 × 12 needle and a 10 ml syringe (Maricevich et al., 2018).** Figure 14 ** HOW CAN WE IMPROVE GUIDELINES USE?

Figure 2 Low
Figure 2

Figure 4 A
Figure 4

Figure 9 Effects
Figure 9

Figure 14 The
Figure 14
** Table 5 ** Indications for the primary craniectomy at HMMC were tumor resection, infection and stroke in contrast to that of closed traumatic brain injury (TBI) in civilian settings at the HR.The cranial repair study at HMMC also included 3 military policemen (38 ± 4 years old) who underwent decompressive craniectomy (DC) following gunshot wounds to the head (links F,G).Interval between previous surgery and PMMA cranioplasty ranged between 6 months and 8 years.The operative time ranged from ranged from 90 to 180 min (mean 112 ± 40 min).The average cranial defect measured 137.98 ± 40.63 cm 2 (estimated using Materialise Magics® reconstruction) and the estimated prosthetic volume 52.03 ± 14.35 cm 3 (estimated modelling the PA ap using Materialise Magics®, version 22.01), with 4 mm thickness.
(De La Peña et al., 2018)Maricevich et al., 2018)test relationships between categorical variables between the cohorts, considered signi cant for P <0.05.**Figure13**Seromas may develop where any skin break occurs(De La Peña et al., 2018;Maricevich et al., 2018).The formation of seroma in cranial repair frequently raises questions about the need for implant fenestration, aspiration and/or drainage(Maricevich et al., 2018).Studies have suggested that seroma production in implant-based to reconstruct the cranium varies with different types of matrices(De La Peña et al., 2018)and biomaterials, namely metals ('titanium mesh erosion'), ceramics ('associated infections'), synthetic and natural polymers, as well as composite materials.However, lack of standard cranial-based guidance prostheses guidelines, including sterilization, intraoperative cleaning and manipulation of the ap and associated antibiotics regimen, does not explicitly preclude clinical success of any of the abovementioned biomaterials.