/home/opencode/cpanel/stomaeduj_hacked/uploads/2018/04/Article_5_4_5.pdf MAXILLOFACIAL SURGERY ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Review Article Simon Cleemput1a * , David Las2b , Reinhilde Jacobs1c , Constantinus Politis1d 1 OMFS-IMPATH research group, Department of Imaging & Pathology, KU Leuven & Oral and Maxillofacial Surgery, Faculty of Medicine, University Hospitals Leuven, BE-3000 Leuven, Belgium 2 University Hospital Brussels, Free University Brussels, BE-1090 Brussels, Belgium a MD b MD, DDS c DDS, MSc, PhD, Professor D MD, DDS, MHA, MM, PhD, Professor ABSTRACT DOI: 10.25241/stomaeduj.2018.5(4).art.5 OPEN ACCESS This is an Open Background: A series of conditions can leave the human mandible with a bony defect Access article under the CC BY-NC that is still difficult to compensate for with current clinical techniques. 3D bioprinting 4.0 license. (computer-controlled, highly organized deposition of bio-materials and stem cells into a Peer-Reviewed Article 3D structure) is a new tissue engineering strategy showing potential to contribute to the Citation: Cleemput S, Las D, Jacobs R, treatment of these defects. Politis C. Advances in 3D bioprinting for bony defects of the mandible. Stoma Edu J. Objective: The aim of this review is to give clinicians an idea of how 3D bioprinting works, 2018;5(4):243-253 where this technology is currently at and how it is developing towards clinical application Academic Editor: Gabi Chaushu in the field of maxillo-facial surgery. DMD, MSc, Professor, Head, The Maurice and Data sources: Bone tissue engineering literature was searched for articles that describe Gabriela Goldschleger School of Dental Medicine, Tel Aviv-University, Tel Aviv, Israel the use of additive manufacturing (collective term for layer-wise stacking of materials, including 3D printing) with use of biomaterials and stem cells. Received: November 06, 2018 Revised: November 14, 2018 Study selection: 3D bioprinting reviews and research articles presenting bone tissue Accepted: November 27, 2018 Published: November 29, 2018 constructs were selected. Data Extraction: Information on 3D bioprinting background, design, applied techniques *Corresponding author: Simon Cleemput, MD and used biomaterials for bone tissue were bundled. Research projects aiming at creating OMFS-IMPATH research group, Department viable bone constructs were selected. of Imaging & Pathology, Faculty of Medicine, University Hospitals Leuven, BE-3000 Leuven, Data Synthesis: This review presents a comprehensive summary of 3D bioprinting Belgium basics and shows how this technique is evolving towards bone tissue constructs with the Tel: 0032485310696, Fax: 003216332410, e- mail: simon.cleemput@student.kuleuven.be potential of clinical application in the management of bony mandibular defects. Keywords: tissue engineering, 3D printing, bioprinting, biomaterials, bone, mandible. Copyright: © 2018 the Editorial Council for the Stomatology Edu Journal. 1. Introduction Most of the cutting-edge research that applies this Previous methods of dealing with substantial bony tissue engineering strategy currently operates in the in defects in the maxillo-facial area (most often arising vitro-domain, with just a few experiments transferring from trauma, osteonecrosis, tumour removal or towards animal studies. Recent years however have congenital disorders) have sought to replace the brought incredible expansion of the spectrum of missing tissue with either artificial materials or with applicable materials and techniques. Researchers are tissue from elsewhere in the body. To this idea, metals creating extensive designs and customized processes and polymers have been used, as well as autogenic to unite the properties of all of these techniques parts of the fibula, radius bone, iliac bone and scapula. into constructs with a viability that was previously Though these modalities have proven themselves unknown outside of the body. worthy of performing over not treating the defect Following is an overview of the general workflow, the at all, they both present some major downsides and printing techniques, materials, properties and designs limitations. Artificial materials remain foreign objects of 3D bioprinting and some illustrative examples of to the body, lack regenerative capacities and suffer what is currently state of the art concerning bone from wear whilst autogenic transplants are never fully tissue constructs. compatible with the defect, require difficult moulding and provide donor site morbidity. Recent advancements in the fields of stem cell 2. Background research, biomaterial development and computerized Paraphrasing the introduction, “3D bioprinting” 3D printing have given birth to a new tissue is to simply put “3D printing with biocompatible engineering strategy named 3D bio-printing, aiming materials and with living cells”. It is thus a variant of to artificially recreate human tissue by cultivating stem 3D printing, a manufacturing technique which has cells applied onto (or even printed directly into) a 3D been around since 1984, when Charles Hull invented printed scaffold. an ‘‘Apparatus for making three-dimensional objects Stomatology Edu Journal 243 ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Review Article Figure 1. One of the earliest CT-based, plaster-milled study-models of a Figure 2. An STL formatted model of a modern surgical wafer, printed human skull (1987). Reprinted with permission by Dr. T. Lambrecht.) by stereolithography[1]. Reprinted with permission by Elsevier. Figure 3 Figure 4 Figure 5 Figure 3, 4, 5. The hydroxy-apatite-coated, laser-melted, titanium full mandible, produced by Xilloc® and implanted by Dr Poukens in 2012. Reprinted with permission by Xilloc®. by stereolithography’’. Steady development and then sterilized and applied during surgery. improvement have made 3D printing applicable And (often in collaboration with specialized 3D to several production processes in modern life and printing design and production firms), patients already useful in the maxillofacial clinic. For example, are even treated with 3D printed, patient-specific 3D printed study models (Fig. 1) have been around implants, such as custom titanium meshes to cover since 1987, proving themselves useful in evaluating up craniectomy sites and as of 2012 a fully functional, pathology or planning of surgery. Several maxillofacial titanium mandible (Fig. 3), successfully implanted in departments already use 3D printing to produce an 83-year-old suffering from severe osteomyelitis. acrylic wafers [1] or cutting guides (Fig. 2), which are 244 Stoma Edu J. 2018;5(4):243-253 http://www.stomaeduj.com ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Figure 6. Overview of the parameters affecting accuracy of medical AM constructs [5]. Overview of the process of creating personalized medical constructs by “additive manufacturing” (= collective term for production techniques based Review Article on layer-wise stacking of materials, including 3D printing methods) with parameters that can influence the accuracy of the constructs [5]. This re- search group found the differences between the imaged reality and 3D printed construct to range between 0.04 mm and 0.62 mm, when applying the most commonly used segmentation technique of “global thresholding”. Reprinted with permission by Elsevier. medical images (JPEG, TIFF, BMP, but mostly raw 3. From medical image to 3d (bio) printing design DICOM data) through 3D converting software to create It is remarkable how early on medicine jumped on a 3D surface model [2]. This digital process performing the wagon of 3D printing, with maxillofacial surgery this transition is named “segmentation” and the most apparently at its forefront. In part this reflects the great popular type “global thresholding”. The model thus demand of maxillofacial surgery for highly customized obtained can be a helpful tool by itself in evaluating constructs. On the other hand, the application of 3D existing pathology or planning surgery and can at this printing in medicine has benefited greatly from the stage also easily be digitally adjusted, in which case it readily available high quality medical imaging, which becomes a 3D CAD model. can serve as a reference for the 3D printing construct A second process named “slicing” converts the (at least for the gross contouring) when processed by obtained 3D surface model (or adjusted CAD model) appropriate computer programs. into structural guidance for the 3D printer. So Converting medical images into a structural reference called “slicer-software” will firstly reconstruct the for the 3D printer usually begins with feeding the unstructured surface of the 3D surface model into a Stomatology Edu Journal 245 ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Review Article Figure 7. Schematic drawing representing the major bioprinting techniques [12]. (A) Inkjet bioprinting (B) Laser-assisted bioprinting (C) Extrusion bioprinting (D) Stereolithography. Reprinted with permission by Wiley. standard tessellation language (.stl formatted) model sound waves (acoustic) or static electricity [7-11].The with a surface consisting of triangles (a process called first two of these methods are the most widely applied “triangulation”, most often performed by applying and even though heat generation would seem a risk the technique of “marching cubes”) [3]. Secondly the for cell viability, more difficulty is experienced with the software will help design an appropriate structural piezoelectric mechanism. framework to support the geometrically-approached surface and fill up the volume underneath. 4.2. Extrusion-based After this, the software will effectively slice the obtained Similar to inkjet bioprinting, extrusion- based 3D model horizontally into layers and it will write bioprinting uses pressure to force the bioink out of “G-code” which will serve as computer commandos the nozzle of the printer but does this by applying for the 3D printer on the course it has to run the nozzle direct mechanical force or air pressure onto a plunger through while printing the digital model, layer on top in a syringe-type of depositor [7-10]. With this robust of layer in a CNC-like way [4]. depositing system, extrusion-based printers can handle more viscous types of bioink with higher cell densities (groups of cells, organoids), resulting in a 4. The different types of 3D bioprinters continuous cylindrical stream rather than droplets. The previous paragraph described a design process that is very similar for 3D printers as it is for 3D 4.3. Laser-assisted bioprinters. The outside look of a ‘regular’ 3D printer A third type of 3D bioprinting is based on the and a 3D bioprinter can also be very similar, as mechanism of laser-induced forward transfer of companies such as ‘Cellink’ are now selling desktop energy (LIFT). It uses the energy of a pulsed laser beam, models starting at approx. 5000 USD. However, most of focused and directed onto a specially designed 2-layer the cutting-edge 3D bioprinting research preformed plate (called “a ribbon”), consisting of an absorbing nowadays makes use of very specific biomaterials layer generating local heath and ultimately small and even more specific strategies of processing these high-pressure bubbles which force droplets of bioink materials. As a result, a lot of researchers modify to form from an underlaying plate of biomaterial existing 3D printers to these specific requirements, [9,11]. Since there is no (mechanical stress-inducing) creating dozens of unique 3D bioprinters. nozzle for the cells to pass through, cell viability is The exact method of delivering materials into a 3D relatively high and precise focussing of the laser beam structure however can always be narrowed down to can provide good resolution of the printed construct. one of 4 mechanisms, described below and illustrated The process of printing however is rather slow and the in Fig.7 [6]. 2-layered “ribbon” is an expensive component. 4.1. Inkjet bioprinting 4.4. Stereolithography This type of bioprinting relies on the formation of small This last category of 3D bioprinting also applies focused air bubbles to push droplets of bio-ink (the chosen UV light, but uses this to cure or selectively solidify a mixture of biomaterials and cells) out of the nozzle photosensitive biomaterial [9,13]. It is the oldest type of the printer. The bubbles can be generated by local of bioprinting and has yielded good resolutions with heat (thermal), current over a crystal (piezoelectric), polymers with high molecular weights. The direct 246 Stoma Edu J. 2018;5(4): 243-253 http://www.stomaeduj.com ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Review Article polymers natural - collagen - gelatine Present in humans - fibrin - hyaluronic acid - heparine - alginate Found in - chitosan bacteria, - (carboxy-methyl) cellulose fungi,… - pectine - silk fibre - chondrotine sulfaat Biomaterials used in 3D bioprinting - carrageenans - xanthan - dextran synthetic - PGA (poly-glycolic acid) - PLA (poly-lactic acid) - PCL (poly ε- caprolactone) - PPF (poly propylene fumarate) - PEG (poly ethylene glycol) - PU (polyether urethane) - PEEK (polyether ether ketone) ceramics - Hydroxyapatite - B- tricalcium phosphate - coralline - Bioglass - calcium-silicate composites Hybrid hydrogels + ceramics, + synthetic polymer fibres + peptides +… Table 1. Overview of popular biomaterials used in bioinks. UV lighting of the biomaterial however is known to the natural cell environment and thus providing induce stress, lowering cell viability. good bio-compatibility, osteo-conductivity and low immunogenicity [8]. These hydrogels can be printed at relatively low temperatures, which also favours 5. Combining biomaterials and cells into cell survival. The lack of intrinsic strength however the bioink almost always demands crosslinking of the polymer; Up to this point we have described the part of 3D for example, with Ca2+ or Mg2+ in the case of alginate bioprinting that consists of computer programming and NaOH in the case of chitosan. Crosslinking can be and printing apparatuses. The part that is “bio”, done by heat, chemicals or UV light, most often right consists of the cells that will be printed and the bio- after printing, but all of these are known to induce compatible materials which will accommodate stress on the printed cells. When implanted in the these cells. Together they form the bio-ink. To give body, the polymer construct would be degraded by an understanding of the spectrum of biomaterials, enzymes such as collagenase and the degradation table 1 provides a summarization categorizing them product would not be toxic. Variation in locale enzyme as polymers, ceramics and composite materials concentration however would make the degradation [6,8,14,15]. rate hard to control. The group of natural polymers consists of organic Using polymers that are instead synthetic linear polysaccharides that are spontaneously formed by aliphatic polyesters, would eliminate some of the organisms in nature. Some of these appear in humans, problems of unpredictable characteristics associated such as collagen and gelatine. Others are of fungal or with naturally occurring saccharides. Their molecular bacterial origin, such as chitosan [16] or alginate [17]. weight and size distribution are known and can stably Since they attract lots of water, these polymers can be controlled and reproduced [14]. Their intrinsic be easily made into hydrogels, closely resembling strength is much higher than that of the natural Stomatology Edu Journal 247 ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Review Article Figure 8. 3D printed gelatine mandibular condyle mold [24]. Figure 9. 3D-printed, porous PCL scaffolds of a mandible at 40% infill Reprinted with permission by Elsevier. density[4]. Reprinted with permission by Wiley. polymers; thus they allow constructs with a more made suitable for 3D bioprinting when mixed into complex architecture. Unfortunately, liquefying these composite materials, such as: synthetic polymers for printing requires temperatures • Polycaprolactone crosslinked to alginate, resulting between 60° and 200°C [18]. Degradation of these in improved strength[22]. constructs, though spontaneous and predictable • Polycaprolactone, added as a microfiber to hybrid by simple hydrolysis, results in local accumulation of hydrogels of alginate and gelatine, or to collagen, acids. Both of these characteristics negatively impact resulting in accelerated new bone formation, even cell survival. in in vivo experiments [23]. Bio-ceramics are also a category of biomaterials used in These are just a few examples of biomaterial- 3D bioprinting and they can rely on a longer tradition configurations that show potential for 3D bioprinting, of clinical application, as some of them already exist as they are able to unite viable conditions for living as injectables and are approved by the FDA. They cells with processability and favourable characteristics surpass synthetic polymers in compressive strength, towards scaffold design. Strictly, 3D bioprinting can be and the often-high calcium content and porous done by printing these combinations of biomaterials microstructure provide good osteo-conductivity. (without any cells) and allowing the printed construct However, as they are processed as a sludge, directly to be colonised by cells, either in vitro or upon printing them together with cells is difficult and has implantation. Some examples of this “indirect” 3D not yet yielded high cell survival. bioprinting are listed below. It is almost impossible to present an exhaustive list, In 2013 Lee et al. bioprinted a mandibular condyle out as the category of composite materials is by far the of gelatine (Fig. 8), with an outer surface and anatomical largest as it is made up of combinations of materials. shape based on patient specific imaging and an inner Not only combinations of components from the first structure of a regular (cuboid) lattice with tubes of two categories, but also combinations of biomaterials 1.3 mm diameter and pores of 1.7 mm. By infiltrating and bioactive compounds are currently in use. As the structure thus obtained with a PCL or chitosan mentioned above, most 3D bioprinting research solution and washing away the gelatine afterwards, is not performed by standard printers, but by the same construct was obtained in PCL and chitosan. custom-tailored variants. This is because researchers These constructs were also successfully seeded with constantly try to unite biomaterial properties, printing mouse bone marrow stromal cells (mBMSC’s), which techniques and tissue engineering designs into showed good spread and proliferation, especially projects with viability. Some examples are described when coating the construct with hydroxyapatite [24]. below. We have mentioned the good cell viability of In 2014, Temple et al. managed to bioprint a complete natural polymer-based hydrogels such as alginate human mandible (Fig. 9) and maxilla directly out of and gelatine, and their lack of intrinsic strength. PCL, using a self-designed 3D bio-printer (a converted Researchers went by this and have made mixtures of: CNC, able to melt and extrude PCL through a nozzle • Gelatine and acryl, resulting in methacrylated of 470 µm at a speed of 2.7 mm/s). They based the gelatin hydrogels [14]. design on patient specific imaging and used a slicer • Alginate, gelatine and calcium phosphate, showing programme which filled up the inner structure with improved adhesion and cell proliferation when a cuboid lattice and automatically created extra printed with bone-related Saos-2 cells [19]. supporting structures, which were trimmed away • Alginate crosslinked with gelatine, combined with after printing. The 3D bio-printed mandible however Bioglass [20] or Hydroxyapathite [21], showing was not seeded with stem cells [4]. These examples improved mechanical strength, as well as improved demonstrate that reconstruction of a human mandible proliferation and mineralisation when printed with (or at least fragments) with an accurate shape and with Saos-2 cells. some sort of viability has been lying within the interest Synthetic polymers are generally stronger than natural field of 3D bioprinting research for some time already. polymers, but less bio-compatible. They too can be Implanting these structures however would seem 248 Stoma Edu J. 2018;5(4): 243-253 http://www.stomaeduj.com ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Review Article Figure 10. The ITOP system [36]. (a) The ITOP system consists of three major units: (i) 3-axis stage/controller, (ii) dispensing module including multi-cartridge and pneumatic pressure controller and (iii) a closed acrylic chamber with temperature controller and humidifier. (b) Illustration of basic patterning of 3D architecture including multiple cell-laden hydrogels and supporting PCL polymer. Reprinted with permission by Nature America. insensible, as we would all sense they would have to functional grading and with known characteristics, mimic the human mandible much closer if they should more closely mimicking the complexity of tissue [25]. replace it, survive in its environment, be subject to the Supposedly it is due to the small scale of the research forces it withstands. and focus on cell-viability that these more complex algorithms have not yet been applied to in vitro 3D bio-printing studies, because most likely they would 6. 3D bioprinting design not cause executional problems. It is clear that more elaborate designs for 3D bioprinting are still to precede possible attempts at implantation. 6.2. Choice of cells Executing these proceedings, many researchers Many researchers favour the direct printing of cell- are now focussing on creating smaller patches of laden bioinks, as it allows for niche formation and tissue, with more realistic viability. Incredible ingenuity a level of interaction with the scaffold that cannot has already led to several successful constructs be matched when colonizing the scaffold from the and in doing so, several 3D bioprinting parameters outside. The cells that would then be embedded in have been studied. The obtained insights are also the bioink can either be functional primary cells with broadening the view on how tissue engineering a supporting cells (osteoblasts, osteoclasts and perhaps load-bearing structure, such as required for bone osteocytes) for bone tissue, or stem cells (adipose tissue should be approached. A few key features derived or bone marrow derived mesenchymal). Stem contributing to a successful 3D bioprinting design for cells require stimulation towards differentiation but bone tissue are described below. contain much more regenerative capacities and are clearly the preference in 3D bioprinting. Survival of the 6.1. Configuration of the construct (design of the construct when implanted however, would unlikely scaffold) succeed if there were no additional colonisation of Current approaches towards load-bearing bone cells from the outside of the construct. range from loose configurations of hydrogels already containing stem cells, to rigid, volumetric and 6.3. Pore size morphologically adequate printed scaffolds, later To allow such cells to enter and continuously recolonize to be seeded with stem cells. Generally, constructs the 3D bioprinted construct and to allow them to are more bio-compatible and supporting of self- proliferate and function, the construct needs to offer organising capacities of the cells as they are more appropriate passages-spaces. This is also necessary to natural polymer based, and more structured and allow nutrients to reach the cells within the construct mechanically strong as they contain more synthetic by diffusion. Keeping in mind the µm size of pre- polymers or bio-ceramics. It must be noted that the osteoblasts, it was established that the appropriate majority of all 3D bioprinting research currently makes pore size for a vascularized bone matrix would be use of the more organised approach. 200-300 µm in diameter [26]. A larger pore size seems The scaffolds created are most often regular shaped, to be met with more vascular differentiation of stem supported by space-filling lattices (regular cubes or cells whilst a smaller one seems to favour osteogenic honeycomb pattern (e.g. Fig 8). This is despite the fact differentiation [27]. The established porosity percentage that algorithms and even libraries and tools have been of 90% for bone tissue is left redundant as many developed to create more complex scaffolds with researchers have achieved favourable results with Stomatology Edu Journal 249 ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Review Article Figure 11. Mandible bone reconstruction [36]. (a) 3D CAD model recognized a mandible bony defect from human CT image data. (b) Visualized motion program was generated to construct a 3D architecture of the mandible bone defect using CAM software developed by our laboratory. Lines of green, blue and red colors indicate the dispensing paths of PCL, Pluronic F-127 and cell-laden hydrogel, respectively. (c) 3D printing process using the integrated organ printing system. The image shows patterning of a layer of the construct. (d) Photograph of the 3D printed mandible bone defect construct, which was cultured in osteogenic medium for 28 d. (e) Osteogenic differentiation of hAFSCs in the printed construct was confirmed by Alizarin Red S staining, indicat- ing calcium deposition. Reprinted with permission by Nature America. smaller percentages [4,26]. Also, there is an increased Examples include slowing down degradation of bio- interest in creating a pore size gradient throughout ceramics (like Wollastonite; CaMgSiO3) to provide a constructs, as this could allow to control growth and steady release of ions, which serves as a bio-cue for mineralisation rates and would fit in the strategy of bone forming cells [31], adding protein residues such making scaffold give time-dependant instructions as a cyclic arg-gly-asp chain to bio-gels to stimulate to the stem cells (Time, often referred to as the osteogenic differentiation [32], as mentioned above, “4th dimension in 3D printing”). or even adding plasmid DNA complexes to PLLA/ collagen scaffolds to stimulate BMP-2 expression [33]. 6.4. Cell adhesion Porosity of the construct also contributes to cell 6.6. Ability to develop vasculature adhesion, albeit more on a micro-level (pores in the As mentioned above, tissue-engineered bone tissue scaffold surface = micro-roughness of the scaffold). could not succeed in viability without proper supply To this idea, scaffolds have been conjugated with of nutrients and oxygen throughout the construct, porogens like F-127 [28], NanoHA and NH4HCO3 + Mg hence the need for pores. When dealing with a larger [29], often slightly compromising the strength, but bone construct, it would be hard to imagine adequate improving cell adhesion. Polarity and surface tension supply without development of vasculature in the of the construct also play a pivotal role in cell adhesion construct. as they determine the hydrophility of the construct, Actually, this need for vasculature within the which accounts for cell adhesion throughout protein engineered bone tissue is presumed indispensable binding to the scaffold. Strategies of reducing the and this is reflected by the considerable amount of often very negative surface tension (expressed as researchers simultaneously evaluating bone- and “surface zeta” value; ex. PLA = -40 mV) include coating vasculature-formation in their 3D printed scaffolds [4]. the surface with dopamine [28], PEG or Bioglass [30], Most research truly focussing on (micro)vessel effectively reducing contact-angles of PLA from 131.2° development within 3D bio-printed bone have to 51.9°, making cell-adhesion much easier. either (architecturally) created spaces for endothelial cells to arrange into tubes or have aimed to attract 6.5. Ability of the scaffold to send biological cues/ (micro)vessel-infiltration from the supporting outside interact with stem cells environment. Various researchers have experimented with methods Examples include silicate bio-ceramics printed into of mimicking the interaction between cells and their hollow tubes releasing angiogenesis-inducing ions micro-environment. like Mg, Ca, and Si [34], porous CaP scaffolds releasing 250 Stoma Edu J. 2018;5(4): 243-253 http://www.stomaeduj.com ADVANCES IN 3D BIOPRINTING FOR BONY DEFECTS OF THE MANDIBLE Review Article Figure 12. Fabrication of bone mimetic 3D architecture containing osteogenic and vasculogenic niches [37]. (a) Schematic illustration of complex bone tissue structure. (b) Illustration of the bioprinting strategy for fabricating complex bone tissue architecture. A perfusable vascular lumen lined with HUVECs can be fabricated within a pyramidal bioprinted construct by arranging individual rods of VEGF-functionalized GelMA bioinks with different mechanical strengths. The hMSCs-laden three outer layers of cylinders were loaded with silicate nanoparticles to induce osteogenic differentiation of hMSCs into bone tissue. The VEGF was covalently conjugated into the three outer layers of the cylindrical hydrogels. The concentrations of conjugated VEGF were determined with ELISA as 17.1, 34.2, and 68.5 ng mL−1. (c) Scheme of the 3D printing procedure of independent cell-laden cylinders using an automatized and computer-controlled bioprinter. Reprinted with permission by Wiley. angiogenic growth factors, up to scaffolds with a tube 3D bio-printed bone construct was creatively met by alignment that creates interconnected channels with Batzaya et al. in their 2017 publication (Fig. 12) in which vascular-like flow patterns [35]. they presented their pyramidal construct of 28 bio- We would like to conclude this design-section with 2 printed tubes with varying compositions of stem cells, examples of the latest wave of 3D bioprinting research, gelatine-methacryloyl, VEGF and Si-nanoparticles. making use of various, combined techniques to unite A commercially available 3D bio-printer was used the characteristics described above and bundling it all to lay down a central tube of HUVEC- and hMSC into a direct 3D bioprinted result. The ITOP (integrated -laden gelatine with low methacryolyl substitution tissue-organ printer) by Kang et al. (Figs. 10 and 11), (gelMaLOW), which would later gradually degrade to made use of several different cartridges, simultaneously a central open channel, surrounded by 3 layers of 3D printing combinations of PCL, cell-laden hydrogels of bio-printed tubes of hMSC-laden gelatine with high various compositions and a pluronic F-127 component methacryolyl substitution (gelMaHIGH) and a gradient (to stabilize the printing process) into “vascularized of covalently bound VEGF and Si-nanoparticles. After cellular constructs of clinically relevant size, shape and 7 days in culture, the central tube had become a structural integrity”. Amongst their productions was a perfusable lumen with an inner surface of HUVEC’s and mandible tissue construct (Fig. 11) consisting of PCL/ an outer surface of supporting hMSC’s, differentiated TCP, F-127 and a human amnionic fluid derived stem into supporting smooth muscle cells. cell (hAFSC)-laden hydrogel consisting of 35 mg/mL The construct was then perfused with an osteogenic of gelatine (to allow liquification above 37°C), 20 mg/ medium for 5 days, which supported proliferation and mL fibrinogen (for stability, cell conductivity and cell osteogenic differentiation of hMSC’s in the outer tubes, proliferation) and 10% glycerol (to prevent nozzle which, 21 days after printing, showed formation of clogging). After printing, the fibrinogen was directly mature bone niches, supported by micro-vasculature crosslinked with thrombin for stabilization after which [37]. the rest of the hydrogel components (except for the stem cells of course) were washed away. The outline of the mandible construct was CT image- and CAD 7. Conclusion model-based and the inner architecture consisted of The sample of recent studies listed above gives us tubes of PCL/TCP (of 130 µm in diameter) and tubes some idea of what is currently being investigated of hAFSC-laden hydrogel, creating micro-channels of in 3D bioprinting research focussed on bone tissue 500 x 300 µm2. Cell viability throughout printing (1 day engineering. It seems clear that obtaining viable 3D after printing) was shown to be 91 ± 2%, proving this printed bone constructs will require a combination complex printing process did not adversely influence of techniques and bio-materials with different cell viability and after 28 days in culture. 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[Full text links] [PubMed] Google Scholar(19) Scopus(16) Simon CLEEMPUT MD OMFS IMPATH Research Group Department of Imaging & Pathology Faculty of Medicine KU Leuven, Leuven, Belgium CV Dr. Cleemput graduated Medicine in 2018 (MD). He is currently studying Dentistry and is enlisted in a Postgraduate Programme in Biomedical Engineering at KU Leuven, Belgium . Questions 1. “Inkjet bioprinting” is: qa. The first developed type of bioprinting; qb. The most precise type of bioprinting (highest resolution); qc. A bioprinting technique that generates droplets of bio-ink; qd. A jet-laser-based bioprinting technique. 2. Inducing bio-ceramics printed into hollow tubes releasing ions like Mg, Ca, and Si was found to: qa. Increase the strength of the porous CaP scaffolds; qb. Increasing the E-Modulus of porous CaP scaffolds; qc. Stimulate osteogenic differentiation of hMSC’s; qd. Create interconnected channels with vascular-like flow patterns in porous CaP scaffolds. 3. The first 3D printer was invented by: qa. Charles Hull; qb. Jules Pouckens; qc. Huan Wook Kang; qd. Thomas Lambrecht. 4. An appropriate pore size for vascularized bone matrix was established at: qa. 20-30 µm, which is within reach of modern 3D bioprinters; qb. 20-30 µm, which is NOT within reach of modern 3D bioprinters; qc. 200-300 µm, which is within reach of modern 3D bioprinters; qd. 200-300 µm, which is NOT within reach of modern 3D bioprinters. Stomatology Edu Journal 253