- Email: [email protected]
3D printed scaffolds with random microarchitecture for bone tissue engineering applications: Manufacturing and characterization
Journal Pre-proof 3d printed scaffolds with random microarchitecture for bone tissue engineering applications: Manufacturing and mechanical characterization Raffaella Pecci, Silvia Baiguera, Pietro Ioppolo, Rossella Bedini, Costantino Del Gaudio PII:
S1751-6161(19)31144-0
DOI:
https://doi.org/10.1016/j.jmbbm.2019.103583
Reference:
JMBBM 103583
To appear in:
Journal of the Mechanical Behavior of Biomedical Materials
Received Date: 16 August 2019 Revised Date:
29 November 2019
Accepted Date: 4 December 2019
Please cite this article as: Pecci, R., Baiguera, S., Ioppolo, P., Bedini, R., Del Gaudio, C., 3d printed scaffolds with random microarchitecture for bone tissue engineering applications: Manufacturing and mechanical characterization, Journal of the Mechanical Behavior of Biomedical Materials (2020), doi: https://doi.org/10.1016/j.jmbbm.2019.103583. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
3D PRINTED SCAFFOLDS WITH RANDOM MICROARCHITECTURE FOR BONE TISSUE ENGINEERING APPLICATIONS: MANUFACTURING AND MECHANICAL CHARACTERIZATION
Raffaella Pecci a, Silvia Baiguera b, Pietro Ioppolo a, Rossella Bedini a, Costantino Del Gaudio c,d
a
National Centre of Innovative Technologies in Public Health, Istituto Superiore di Sanità, Rome,
Italy b
Center for Regenerative Medicine, University of Rome Tor Vergata; 00133 Rome, Italy
c
E. Amaldi Foundation, Via del Politecnico snc, 00133 Rome, Italy
d
Department of Mechanical and Aerospace Engineering, “Sapienza” University of Rome, Via
Eudossiana 18, 00184, Rome, Italy
Corresponding author: Costantino Del Gaudio, PhD E. Amaldi Foundation Via del Politecnico snc, 00133 Rome, Italy Tel. +39-6-8567645 Email: [email protected]
1
Abstract
Additive manufacturing for tissue engineering applications offers the possibility to design scaffolds characterized by a fine and detailed microarchitecture. Several fabrication technologies are currently available which allow to prepare tailored structures with a large selection of materials for restoring and healing tissues. However, 3D printed scaffolds are generally collected by assembling repetitive geometrical units or reproducing specific patterns in the layering direction, leading to a highly ordered architecture that does not mimic the morphology of the natural extracellular matrix (ECM), one of the main goals to be reached for an effective therapeutic approach. It is usually stated in the tissue engineering field that a scaffold has to be considered a temporary ECM, resembling all the peculiar clues as close as possible and, in this regard, an ordered microstructure cannot be usually observed within biological tissues and organs. With the aim to overcame this limitation and offer a potential approach for bone tissue applications, the present study proposes a design methodology to fabricate 3D printed scaffolds characterized by a random microarchitecture which can be repeatedly reproduced thanks to the intrinsic controllable process of additive manufacturing. In this framework, four different models in polylactic acid were fabricated by means of fused deposition modelling, including a three-dimensional random distribution of spherical pores of 400, 500, and 600 µm for the first three cases, and a randomly varied distribution in the range 400-600 µm for the fourth case. A detailed assessment by means of microcomputed tomography and mechanical evaluation was then carried out in order to fully analyse the resulting scaffolds, providing both morphological and quantitative data.
Keywords: 3D printing, bone scaffolds, random microarchitecture, mechanical testing, micro-CT analysis
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1. Introduction
Additive manufacturing (AM) is a broad collection of different 3D printing technologies, constantly gaining interest for several industrial and research applications. The main characteristic of this fabrication approach is related to the precise control in each stage of the manufacturing process, starting from the design to the production of the final component. In this regard, the proper selection of the material(s), the inner architecture and the resulting mechanical properties, just to mention the most evident features, allow to deal with a tailored structure for the desired application. In the field of tissue engineering and regenerative medicine, AM is regarded as an alternative and potential manufacturing technique for innovative scaffolds and fused deposition modelling (FDM) is one of the most popular process for this aim. Nowadays, there are many commercial FDM printers, comprised in a wide technological range, that can suitably replicate anatomical districts or collect specific substrates. The fine control of the microstructure to be sliced is certainly one of the main advantages, and this usually leads to reproduce regular patterns with the possibility to modify the orientation of two consecutive layers or the distance between two adjacent strands on a single layer. These occurrences usefully contribute to define the expected response of the scaffold either from a bioengineering point of view (e.g., morphology, stiffness) and a biological one (e.g., cell viability, proliferation or differentiation when dealing with stem cells). Moreover, it is often reported that a scaffold with a regular pattern can be a valuable solution as it allows to control the expected functionality and cell distribution (Wüst et al., 2011). Xia et al. (2018) observed that 3D printed hydrogels for cartilage regeneration with a 50% infill density showed higher chondrocyte seeding efficiency and more homogeneous distribution compared with 30% and 70% groups. 3D printed scaffolds made of polylactic acid and treated with methacrylamide-modified gelatin were assessed as hybrid substrates for mouse calvaria-derived preosteoblast cells, finding cell uniform distribution and high seeding efficiency (Markovic et al., 2015). Kumar et al. (2016) reported that interconnected pore network, predefined pore size and architecture support vascularization in 3
porous scaffolds and ensure long term implants success, conversely from contorted path that may limit the growth of blood capillaries and the supply of oxygen and nutrients during tissue remodelling. The spatial control provided by the inner morphology could be of interest, but it should be critically revised as well, since a scaffold for tissue engineering applications has to be considered as a temporary substitute of the natural extracellular matrix (ECM) of the tissue to be regenerated, closely mimicking its microarchitecture (Dutta and Dutta, 2009; Kim et al., 2016; Gilpin and Yang, 2017; Jun et al., 2018; Bittner et al., 2018). Generally, ECM is a complex structure not characterized by a regular and repetitive assembling of the key-elements, such as collagen or elastin, and this pattern should be therefore resembled to favour a proper integration between the scaffold and the surrounding biological district to be healed. In order to be compliant with this statement, a random microarchitecture could be a suitable alternative. Several techniques can be considered for this aim, e.g. solvent casting/salt leaching, freeze drying, but most of them are not fully controllable thus leading to a result that is not strictly repeatable in terms of scaffold production. This might be an apparent contradiction, but cell response is dictated by scaffold morphology and a fine tuning of this feature should be exerted in order to limit the number of possible technical variables affecting the outcome. Therefore, with the aim to perform a biological assay just based on the cell-scaffold interaction with a specific and defined random substrate architecture, a tailored experimental approach should be designed. In this regard, FDM can be evaluated as a potential means as allows to design and realize scaffolds characterized by a random microstructure, which is also repeatable and reproducible for each fabrication session being the output of a fixed generated G-code script. The present study reports an alternative approach to design 3D printed scaffolds characterized by random microarchitecture, mimicking natural ECM. FDM was selected as fabrication technique to process a random distribution of virtual porogens, as representative and constitutive elements of the resulting scaffolds. With the aim to infer on the role of the fabrication parameters to resemble the 4
microstructure of a human iliac crest bone sample, a detailed experimental analysis was carried out by means of 3D microcomputed tomography (3D micro-CT) evaluating all the relevant features to fully characterize the resulting architecture. In addition, a mechanical characterization was also performed in order to assess the structural properties for potential applications in the bone tissue engineering field.
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2. Materials and Methods
2.1. Design procedure for 3D printed scaffolds A random three-dimensional distribution of spheres, i.e. virtual porogens, was created by means of custom-made script within a defined volume of fixed shape that resembles the maximum size of the scaffold to be fabricated (Matlab, MathWorks, Natick, USA). As a case study, sphere was selected as a suitable model to mimic a pore, the final scaffold porosity being related to the number of spheres generated and their size. In this framework, four different conditions were evaluated, consisting of 3000 spheres differing for the size, i.e., three occurrences characterized by fixed diameters of 400, 500 and 600 µm, and one by a random diameter distribution in the range 400-600 µm, the pore sizes matching those previously considered for bone tissue engineering (Saska et al. 2018; Mota et al., 2017; Heo et al. 2019). The lower dimensional limit was selected being the diameter of the nozzle of the 3D printer subsequently used to fabricate the scaffolds. The resulting matrix with the location of the centers of the spheres and their diameters was exported for each case and processed in a CAD environment in order to realize the final model by subtracting the cloud of spheres from the representative volume of the scaffold (i.e., 10 x 10 x 3 mm). The resulting model is then exported as a STL file to be processed by a software for the 3D printer.
2.2. Fabrication of 3D printed scaffolds Scaffolds were fabricated in polylactic acid (PLA filament; Formfutura BV, The Netherlands) by means of the N2 3D printer (Raise 3D Inc., Irvine, CA, USA). Each model file was imported in ideaMaker (Raise 3D Inc., Irvine, CA, USA) and then sliced at 0.1 mm, 0.25 mm, and 0.4 mm in the Z direction. The temperature of the nozzle (0.4 mm diameter) was set at 205 °C while that of the build platform at 40 °C.
2.3. Scaffold Characterization 6
2.3.1. Thermal analysis Differential scanning calorimetry (DSC) was performed to assess the thermal properties of 3D printed PLA scaffolds. Specimens were located in aluminium pans and heated at 10 °C/min, in nitrogen atmosphere, in the temperature range 20–200 °C (Nestch DSC 200 PC). An empty aluminium pan was used as reference. The crystallinity degree χ was calculated as the ratio between the sample enthalpy and the melting enthalpy of 100% crystalline PLA, which was assumed to be 93 J/g (Battegazzore et al., 2011).
2.3.2. Micro-CT analysis The quantitative and qualitative analysis of the 3D printed PLA scaffolds was performed by means of a micro-CT system. The analysis of each 3D printed PLA scaffold consisted of approximately 2 h scanning and 2 h reconstruction procedures. A desktop micro-CT scanner (SkyScan 1072; Bruker microCT, Kontich, Belgium) was used to scan all the specimens. The acquisition stage was carried out setting a pixel size of 14.65 x 14.65 µm, which corresponds to a 20x magnification with 1.7 s exposure time and 0.45° rotation step (for a total rotation of 180°) applying a voltage of 40 kV with a current of 248 µA. The obtained acquisition data were subsequently elaborated and reconstructed (NRecon V1.7.0; Bruker microCT, Kontich, Belgium), providing axial cross sections with a pixel size of 14.65 x 14.65 µm for the total height of 3D printed PLA scaffolds. The distance between each cross section was 14 µm. All the files of each specimen were resliced stepwise setting a unitary slice spacing factor in vertical cross section, using a computer software analysis system (micro-CT-Analyser V1.16; Bruker microCT, Kontich, Belgium). The Percent scaffold volume (Percent bone volume), the Structure thickness (Trabecular thickness), the Structure linear density (Trabecular number), the Scaffold surface/volume ratio (Surface/volume ratio), and the Total porosity (%) were calculated. For the evaluation of these features, the grey level images were binarized implementing a global threshold method which is based on only one 7
parameter (i.e., the grey level value) to be set by the user (micro-CT-Analyser V1.16; Bruker microCT, Kontich, Belgium; Cengiz et al., 2018).
2.3.3. Mechanical analysis Compression tests were carried out on selected specimens, those moistly resembling the microstructure of the reference bone sample, as resulted from the µCT investigation. For this aim, box-shaped samples (5 x 5x 10 mm3) were deposited with a layer height of 0.25 and 0.4 mm, preserving the same porogen to volume ratio of the scaffolds previously considered. Mechanical properties were evaluated by applying a deformation value of 25% of the initial length at 1 mm/min by means of a universal testing machine equipped with a 5 kN load cell (M30K, Lloyd Instruments Ltd., UK). Ten specimens were tested for both manufacturing conditions.
2.4. Statistical analysis Data are expressed as mean ± standard deviation. Statistical analysis was performed by means of ANOVA test (Matlab, MathWorks, Natick, USA). Significant level was set at p < 0.05.
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3. Results and Discussion
Additive manufacturing can provide a number of different options to prepare tailored scaffolds for tissue engineering. Referring to bone applications, several studies investigated the collected output in terms of, e.g., structural resolution, materials and cell response (Pereira et al., 2018; Zhang et al., 2019), but dealing with an effective biomimetic scaffold is a crucial issue that still needs to be addressed. This topic was critically revised also considering the role of hydroxyapatite, a wellknown filler especially targeted for bone tissue engineering (Milazzo et al., 2019). However, most of the already proposed technical solutions are characterized by a regular microstructure which unlikely match that of bone architecture. In order to develop specific fabrication approaches to be as compliant as possible to the tissue engineering paradigm, a potential solution was presented by Gòmez et al. (2016), implementing a Voronoi tessellation to design porous scaffolds to be 3D printed after numerical characterization and optimization. Based on the same design approach, Ti6Al4V scaffolds were fabricated by means of selective laser melting and mechanically tested to correlate the resulting behaviour to the morphological features (Wang et al., 2018). In addition, several thick scaffolds for bone tissue engineering have been realized so far by means of a number of experimental methods, such as foaming or replication of natural structures (Meskinfam et al., 2018; Clarke et al., 2016), that do not allow, however, to exert the characteristic fine control of 3D printers in the manufacturing stage. Referring to the possible materials selection, PLA is one of the most common polymers often considered for bone tissue applications. A number of suitable characteristics, e.g. biocompatibility, biodegradability, mechanical properties, and its versatility to be processed as a neat material or a composite scaffold, allows to consider PLA a valuable option for scaffolding (Diomede et al., 2018; Chou et al., 2017; Narayanan et al., 2016; Rakovskyet al., 2014; D’Angelo et al., 2012; Bianco et al., 2011). It should be underlined that different fabrication techniques, e.g. stereolithography, could provide a better resolution than FDM to reproduce the fine structure of the bone ECM, but a number of 9
drawbacks can limit the expected outcome. Possible issues can be related to the need to deal with photocrosslinkable polymers, photoinitiators can be toxic and unreacted monomers can be undesired by-products, resulting scaffolds can be affected by low mechanical strength and material gradients are difficult to produce with good fidelity (Egan, 2019; Moreno Madrid et al., 2019). Selective laser sintering can also provide a good accuracy, but the process is carried out at high temperatures and laser intensity can induce polymer degradation, as observed by Gayer et al. (2019) processing polylactide/calcium carbonate composites. It is also possible to consider 3D printing by means of binder materials to process powder layers which however requires post-processing procedures in order to deal with the final scaffold that, for instance, restricts the possibility to incorporate biomolecules (Moreno Madrid et al., 2019). In this regard, a fabrication approach based on material extrusion, such as FDM, has been developed to collect bone ECM-like scaffolds made of PLA to assess the potential of the here presented proposal.
3.1. Scaffold characterization The fabricated PLA scaffolds are shown in Fig.1, the influence of the four investigated process parameters is clearly visible. The porosity of the respective CAD models was about 27% for a porogen size of 400 µm, 46% for a porogen size of 500 µm, 65% for a porogen size of 600 µm, and 35% for a porogen size distribution of 400 - 600 µm. An example of the CAD model and the related sliced version of the PLA scaffold to be printed is reported in the Supplementary Information. The thermal characterization was carried out by means of DSC analysis. Significant differences of PLA before and after scaffolds fabrication were not assessed (Fig. 2), collected results are resumed in Table I. The processing conditions did not induce any modification in the material which is not always verified, being for instance dependent on the selected polymer (Del Gaudio, 2019).
3.2. Tomographic investigation
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The micro-CT analysis was carried out to finely investigate the microarchitecture of all the fabricated scaffolds. Fig. 3 shows a representative slice for each scaffold to visualize the internal structure, while Table II summarizes the main values of specific morphometric features, generally computed to quantify bone characteristics. The effect of virtual porogen size can be clearly observed on the computed values for scaffold volume, surface to volume ratio, and porosity. The latter parameter was in good agreement with that evaluated from the CAD models, allowing to estimate, or to design, this relevant characteristic for an effective scaffold for tissue engineering applications. Clearly, porosity is just a partial information since pore size is directly implied to support cell migration and mass transport within the three-dimensional environment of the fabricated substrate. This critical issue was here addressed, considering several conditions in order to optimize the scaffold architecture before to move towards biological assays. The micro-CT assessment of a reference bone sample, i.e. human iliac crest bone, was therefore performed as a complementary analysis to verify the proposed approach. The acquired result is shown in Fig. 4, which also summarizes the analogous quantitative values considered for the 3D printed PLA scaffolds. A direct comparison of the data led to select two cases that mostly match the bone microstructure, being the scaffolds printed at 0.25 and 0.4 mm height layer considering the virtual porogen size of 600 µm. This output was also supported by literature. For instance, Liu et al. (2010) investigating the human distal tibia bone collected an average value for the trabecular thickness of 0.15 mm, and a trabecular number of 1.18 mm-1. The µCT analysis of the human mandibular condylar bone, as reported by Renders et al. (2007), showed that the trabecular thickness was in the range 0.11-0.22 mm, the trabecular number within 1.16-1.67 mm-1, and the surface to volume ratio within 12.05-22.46 mm-1. Nakashima et al. (2019) investigated human cadaveric vertebrae to be related to bone strength parameters, finding that the average values for the trabecular thickness and the trabecular number were 0.328 mm and 0.876 mm-1, respectively.
3.3. Mechanical analysis 11
The 3D printed PLA scaffolds that more closely resembled the bone microstructure were mechanically assessed by means of compression tests. As shown in Fig. 5, representative stressstrain curves were characterized by two linear regions which can be ascribed to the compaction of the porous internal structure, before the toe region, and to the compression of the resulting configuration, as the steepest increase of the stress-strain curve clearly highlighted. The compression moduli in the first region were 27.8 ± 3.5 MPa and 25.3 ± 1.5 MPa for a scaffold layer height of 0.25 mm and 0.4 mm, respectively (significant differences were not computed). Past the toe region, the compression moduli were 1.3 ± 0.2 GPa and 1.6 ± 0.2 GPa for a layer height of 0.25 mm and 0.4 mm, respectively (p < 0.05). PLA scaffolds, and composites, fabricated by stacking layers in regular patterns considering the FDM technique were previously evaluated, showing a wide range for the computed compression modulus which is obviously dependent on the morphological strands arrangement and testing conditions. A similar compression response was observed for 3D printed scaffolds made of polylactic acid/poly-caprolactone/hydroxyapatite which were characterized by a compression modulus in the range 0.5 -1.5 MPa, depending on the polymer ratio and hydroxyapatite content (Hassanajili et al., 2019). Differently, 3D printed PLA scaffolds including nano- and microhydroxyapatite were characterized by a Young’s modulus from 1.6 GPa to 2.6 GPa, before and after cultural medium incubation (Niaza et al., 2017). Comparable values were also measured for 3D printed PLA scaffolds including 15% hydroxyapatite, being in the range 1.3 – 2.0 GPa with respect to 1.2 – 1.4 GPa for the neat case (Senatov et al., 2016). Martin et al. (2019) measured mechanical values comprised in the range 100 – 200 MPa, reporting a substantial match with the variability of the Young modulus of the trabecular bone. The influence of the FDM fabrication conditions were also investigated by Naghieh et al. (2016), showing that it was possible to modulate the elastic modulus from about 210 to 1000 MPa. However, a large variability can be also found when testing human bones. Trabecular bones from different anatomical locations were characterized by an average compression modulus of 597.9 ± 401.6 MPa (Rincón-Kohli and Zysset, 2009), while the 12
modulus of human cortical bone evaluated with different techniques and, again, from different locations spanned from 15.7 ± 3.5 GPa to 25.8 ± 0.7 GPa, as reviewed by Libonati and Vergani (2016). The same mechanical parameter was measured by Singh et al. (2019), resulting to be 3.73 ± 1.27 GPa for the humerus, 3.05 ± 1.03 for the ulna, and 2.75 ± 0.71 for the radius. As already stated, the dependence of the mechanical properties with the testing conditions contributes to the wide range of the collected published data and allows to observe that the herein presented results are clearly comprised within these findings. This statement obviously implies that biological assays are needed to fully verify the aim of this proposal, but the mechanical assessment allowed to support that the developed scaffolds can be potentially considered for structural and bearing purposes.
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5. Conclusions
With the aim to design and fabricate scaffolds for tissue engineering applications, close resembling the natural ECM (e.g., the bone microarchitecture in this study), a specific experimental procedure was developed. The proposed methodology is not related to the materials and to the 3D printing technique to be selected. The approach can be generally applied when a random microstructure needs to be replicated within a tissue engineered scaffold. In this respect, pore size and overall dimensions of the structure depend on the resolution of the manufacturing technique and the final design can be therefore tailored based on the 3D printer technical characteristics. The here presented investigation is the first step towards the definition of a different procedure to design and fabricate ECM-like 3D printed scaffolds. Several experimental conditions were compared to a real human bone sample in order to select those mostly matching the related morphometric parameters. However, it should be also underlined that this comparison was carried out considering only one biological reference case and this could be reasonably a limitation, even if a good agreement was observed, further supported by literature data. Clearly, this is just the starting point of a systematic approach that already provided interesting findings that allow to consider the proposed scaffolds for potential tissue engineering applications, being planned as a future development.
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References
Battegazzore D, Bocchini S, Frache A. Crystallization kinetics of poly(lactic acid)-talc composites. eXPRESS Polym Lett. 2011; 5:849–58. Bianco A, Bozzo BM, Del Gaudio C, Cacciotti I, Armentano I, Dottori M, D’Angelo F, Martino S, Orlacchio A, Kenny JM. Poly (L-lactic acid)/calcium-deficient nanohydroxyapatite electrospun mats for bone marrow stem cell cultures. J Bioact Compat Polym 2011;26(3):225-41. Bittner SM, Guo JL, Melchiorri A, Mikos AG. Three-dimensional printing of multilayered tissue engineering scaffolds. Mat Today. 2018;21:861-74. Bruker microCT. CT-analyser - The user’s guide. Kontich (Belgium): Skyscan Ed.; 2013. Cengiz IF, Oliveira JM, Reis RL. Micro-CT - a digital 3D microstructural voyage into scaffolds: a systematic review of the reported methods and results. Biomater Res. 2018;22:26. Chou YC, Lee D, Chang TM, Hsu YH, Yu YH, Chan EC, Liu SJ. Combination of a biodegradable three-dimensional (3D) - printed cage for mechanical support and nanofibrous membranes for sustainable release of antimicrobial agents for treating the femoral metaphyseal comminuted fracture. J Mech Behav Biomed Mater. 2017;72:209-218. Clarke SA, Choi SY, McKechnie M, Burke G, Dunne N, Walker G, Cunningham E, Buchanan F. Osteogenic cell response to 3-D hydroxyapatite scaffolds developed via replication of natural marine sponges. J Mater Sci Mater Med. 2016;27(2):22. D'Angelo F, Armentano I, Cacciotti I, Tiribuzi R, Quattrocelli M, Del Gaudio C, Fortunati E, Saino E, Caraffa A, Cerulli GG, Visai L, Kenny JM, Sampaolesi M, Bianco A, Martino S, Orlacchio A. Tuning multi/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats. Biomacromolecules. 2012;13(5):1350-60. Del Gaudio C. A feasibility study for a straightforward decoration of a 3D printing filament with graphene oxide. Fuller Nanotub Car N. 2019;27:607-12.
15
Diomede F, Gugliandolo A, Cardelli P, Merciaro I, Ettorre V, Traini T, Bedini R, Scionti D, Bramanti A, Nanci A, Caputi S, Fontana A, Mazzon E, Trubiani O. Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair. Stem Cell Res Ther. 2018;9(1):104 Dutta RC, Dutta AK. Cell-interactive 3D-scaffold; advances and applications. Biotechnol Adv. 2009;27(4):334-9. Egan PF. Integrated design approaches for 3D printed tissue scaffolds: review and outlook. Materials (Basel). 2019;12(15). pii: E2355 Gayer C, Ritter J, Bullemer M, Grom S, Jauer L, Meiners W, Pfister A, Reinauer F, Vučak M, Wissenbach K, Fischer H, Poprawe R, Schleifenbaum JH. Development of a solvent-free polylactide/calcium carbonate composite for selective laser sintering of bone tissue engineering scaffolds. 2019;101:660-673. Gilpin A, Yang Y. Decellularization strategies for regenerative medicine: from processing techniques to applications. Biomed Res Int. 2017;2017:9831534. Gómez S, Vlad MD, López J, Fernández E. Design and properties of 3D scaffolds for bone tissue engineering. Acta Biomater. 2016;42:341-350. Hassanajili S, Karami-Pour A, Oryan A, Talaei-Khozani T. Preparation and characterization of PLA/PCL/HA composite scaffolds using indirect 3D printing for bone tissue engineering. Mater Sci Eng C. 2019;104:109960 Heo SY, Ko SC, Oh GW, Kim N, Choi IW, Park WS, Jung WK. Fabrication and characterization of the 3D-printed polycaprolactone/fish bone extract scaffolds for bone tissue regeneration. J Biomed Mater Res B Appl Biomater. 2019;107(6):1937-1944. Jun I, Han HS, Edwards JR, Jeon H. Electrospun fibrous scaffolds for tissue engineering: viewpoints on architecture and fabrication. Int J Mol Sci. 2018;19(3). pii: E745. Kim Y, Ko H, Kwon IK, Shin K. Extracellular matrix revisited: roles in tissue engineering. Int Neurourol J. 2016;20(Suppl 1):S23-29. 16
Kumar A, Nune KC, Misra RD. Biological functionality of extracellular matrix-ornamented threedimensional printed hydroxyapatite scaffolds. J Biomed Mater Res A. 2016;104(6):1343-51. Libonati F, Vergani L. Understanding the structure–property relationship in corticalbone to design a biomimetic composite. Compos Struct. 2016;139:188–198. Liu XS, Zhang XH, Rajapakse CS, Wald MJ, Magland J, Sekhon KK, Adam MF, Sajda P, Wehrli FW, Guo XE. Accuracy of high-resolution in vivo micro magnetic resonance imaging for measurements of microstructural and mechanical properties of human distal tibial bone. J Bone Miner Res. 2010;25(9):2039-50. Markovic M, Van Hoorick J, Hölzl K, Tromayer M, Gruber P, Nürnberger S, Dubruel P, Van Vlierberghe S, Liska R, Ovsianikov A. Hybrid tissue engineering scaffolds by combination of three-dimensional
printing
and
cell
photoencapsulation.
J
Nanotechnol
Eng
Med.
2015;6(2):0210011-210017 Martin V, Ribeiro IA, Alves MM, Gonçalves L, Claudio RA, Grenho L, Fernandes MH, Gomes P, Santos CF, Bettencourt AF. Engineering a multifunctional 3D-printed PLA-collagenminocycline-nanoHydroxyapatite scaffold with combined antimicrobial and osteogenic effects for bone regeneration. Mater Sci Eng C Mater Biol Appl. 2019;101:15-26. Meskinfam M, Bertoldi S, Albanese N, Cerri A, Tanzi MC, Imani R, Baheiraei N, Farokhi M, Farè S. Polyurethane foam/nano hydroxyapatite composite as a suitable scaffold for bone tissue regeneration. Mater Sci Eng C Mater Biol Appl. 2018;82:130-140. Milazzo M, Contessi Negrini N, Scialla S, Marelli B, Farè S, Danti S, Buehler MJ. Additive manufacturing approaches for hydroxyapatite-reinforced composites. Adv Funct Mater. 2019, 1903055. Moreno Madrid AP, Vrech SM, Sanchez MA, Rodriguez AP. Advances in additive manufacturing for bone tissue engineering scaffolds. Mater Sci Eng C Mater Biol Appl. 2019;100:631-644.
17
Mota C, Wang SY, Puppi D, Gazzarri M, Migone C, Chiellini F, Chen GQ, Chiellini E. Additive manufacturing
of
poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate]
scaffolds
for
engineered bone development. J Tissue Eng Regen Med. 2017;11(1):175-186. Naghieh S, KaramoozRavari MR, Badrossamay M, Foroozmehr E, Kadkhodaei M.Numerical investigation of the mechanical properties of the additive manufactured bone scaffolds fabricated by FDM: The effect of layer penetration and post-heating. J Mech Behav Biomed Mater. 2016;59:241-250. Nakashima D, Ishii K, Nishiwaki Y, Kawana H, Jinzaki M, Matsumoto M, Nakamura M, Nagura T. Quantitative CT-based bone strength parameters for the prediction of novel spinal implant stability using resonance frequency analysis: a cadaveric study involving experimental micro-CT and clinical multislice CT. Eur Radiol Exp. 2019;3(1):1. Narayanan G, Vernekar VN, Kuyinu EL, Laurencin CT.Poly (lactic acid)-based biomaterials for orthopaedic regenerative engineering. Adv Drug Deliv Rev. 2016;107:247-276. Niaza K,Senatov F, Anisimova N, Kiselevskiy M, Kaloshkin S. Effect of co-incubation with mesenchymal stromal cells in cultural medium on structure and mechanical properties of polylactide-based scaffolds. BioNanoSci. 2017;7:712. Pereira T, Prendergast ME, Solorzano R. State of the art biofabrication technologies and materials for bone tissue engineering. J 3D Print Med.2018;2:103-13. Rakovsky A, Gotman I, Rabkin E, Gutmanas EY.β-TCP-polylactide composite scaffolds with high strength and enhanced permeability prepared by a modified salt leaching method. J Mech Behav Biomed Mater. 2014;32:89-98. Renders GA, Mulder L, van Ruijven LJ, van Eijden TM. Porosity of human mandibular condylar bone. J Anat. 2007;210(3):239-48. Rincón-Kohli L, Zysset PK. Multi-axial mechanical properties of human trabecular bone. Biomech Model Mechanobiol. 2009;8(3):195-208.
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Saska S, Pires LC, Cominotte MA, Mendes LS, de Oliveira MF, Maia IA, da Silva JVL, Ribeiro SJL, Cirelli JA. Three-dimensional printing and in vitro evaluation of poly(3-hydroxybutyrate) scaffolds functionalized with osteogenic growth peptide for tissue engineering. Mater Sci Eng C Mater Biol Appl. 2018;89:265-273. Senatov FS, Niaza KV, Zadorozhnyy MY, Maksimkin AV, Kaloshkin SD, Estrin YZ. Mechanical properties and shape memory effect of 3D-printed PLA-based porous scaffolds. J Mech Behav Biomed Mater. 2016;57:139-48. Singh D, Rana A, Jhajhria SK, Garg B, Pandey PM, Kalyanasundaram D. Experimental assessment of biomechanical properties in human male elbow bone subjected to bending and compression loads. J Appl Biomater Funct Mater. 2019;17(2):2280800018793816. Wang G, Shen L, Zhao J, Liang H, Xie D, Tian Z, Wang C. Design and compressive behavior of controllable irregular porous scaffolds: based on Voronoi-tessellation and for additive manufacturing. ACS Biomater Sci Eng. 2018;4:719−27. Wüst S, Müller R, Hofmann S. Controlled positioning of cells in biomaterials-approaches towards 3d 3D tissue printing. J Funct Biomater. 2011;2(3):119-54. Xia H, Zhao D, Zhu H, Hua Y, Xiao K, Xu Y, Liu Y, Chen W, Liu Y, Zhang W, Liu W, Tang S, Cao Y, Wang X, Chen HH, Zhou G. Lyophilized scaffolds fabricated from 3d3D-printed photocurable natural hydrogel for cartilage regeneration. ACS Appl Mater Interfaces. 2018;10(37):31704-31715. Zhang L, Yang G, Johnson BN, Jia X. Three-dimensional (3D) printed scaffold and material selection for bone repair. Acta Biomater. 2019;84:16-33.
19
FIGURES
Fig. 1. 3D printed PLA scaffolds. Virtual porogen size: 500 µm (A), 400 µm (B), 600 µm (C), 400 – 600 µm (D)
20
Fig. 2. Calorimetric curves of PLA before and after 3D printing
21
Fig. 3. Slices of the 3D printed PLA scaffolds collected from the micro-CT analysis
22
Fig. 4. Slice of a specimen from human iliac crest bone and the related morphometric parameters as computed from the micro-CT analysis
Fig. 5. Compression curves for the scaffolds fabricated setting a height layer of 0.25 and 0.4 mm
23
Supplementary Information
An example of the STL file representative of a random pore distribution of 500 µm diameter is shown in Fig. S1, while a picture of the sliced version to be 3D printed is reported in Fig. S2. A random PLA scaffold was obtained, even if a close match with the model cannot be observed by visual inspection.
Fig. S1 – The CAD model of the scaffold characterized by a random pore distribution of 500 µm diameter
Fig. S2 – The sliced PLA scaffold starting from the model shown in Fig. S1
24
Table I - Thermal properties of PLA before and after 3D printing. Tg: glass transition temperature, Tm: melting temperature, ∆H: meting enthalpy; χ: crystallinity degree
Tg [°C]
Tm [°C]
∆H [J/g]
χ [%]
PLA Filament
63.68
151.54
19.43
21.0
PLA Scaffold
65.38
156.36
17.82
19.2
Table II – micro-CT morphological evaluation of 3D printed scaffolds
400 µm
500 µm
600 µm
400 - 600 µm
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
0.1 mm
0.25 mm
0.4 mm
0.1 mm
0.25 mm
0.4 mm
0.1 mm
0.25 mm
0.4 mm
0.1 mm
0.25 mm
0.4 mm
76.75
75.86
71.83
55.83
61.73
58.35
40.02
39.65
39.77
70.76
67.00
67.66
0.43
0.46
0.51
0.28
0.41
0.44
0.27
0.35
0.41
0.34
0.36
0.40
1.77
1.64
1.40
1.97
1.50
1.33
1.46
1.12
0.98
2.06
1.87
1.67
6.15
5.77
5.24
12.54
8.25
7.81
14.41
10.77
10.77
8.37
10.22
8.45
23.25
24.14
28.17
44.16
38.26
41.65
60.00
60.34
60.23
29.23
33.00
32.34
Percent scaffold volume (%) Structure thickness (mm) Structure linear density (1/mm) Scaffold surface/volume ratio (1/mm) Total porosity (%)
Conflict of interest
The authors have no conflict of interest
Author statement
No competing financial interests
S1751-6161(19)31144-0
DOI:
https://doi.org/10.1016/j.jmbbm.2019.103583
Reference:
JMBBM 103583
To appear in:
Journal of the Mechanical Behavior of Biomedical Materials
Received Date: 16 August 2019 Revised Date:
29 November 2019
Accepted Date: 4 December 2019
Please cite this article as: Pecci, R., Baiguera, S., Ioppolo, P., Bedini, R., Del Gaudio, C., 3d printed scaffolds with random microarchitecture for bone tissue engineering applications: Manufacturing and mechanical characterization, Journal of the Mechanical Behavior of Biomedical Materials (2020), doi: https://doi.org/10.1016/j.jmbbm.2019.103583. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
3D PRINTED SCAFFOLDS WITH RANDOM MICROARCHITECTURE FOR BONE TISSUE ENGINEERING APPLICATIONS: MANUFACTURING AND MECHANICAL CHARACTERIZATION
Raffaella Pecci a, Silvia Baiguera b, Pietro Ioppolo a, Rossella Bedini a, Costantino Del Gaudio c,d
a
National Centre of Innovative Technologies in Public Health, Istituto Superiore di Sanità, Rome,
Italy b
Center for Regenerative Medicine, University of Rome Tor Vergata; 00133 Rome, Italy
c
E. Amaldi Foundation, Via del Politecnico snc, 00133 Rome, Italy
d
Department of Mechanical and Aerospace Engineering, “Sapienza” University of Rome, Via
Eudossiana 18, 00184, Rome, Italy
Corresponding author: Costantino Del Gaudio, PhD E. Amaldi Foundation Via del Politecnico snc, 00133 Rome, Italy Tel. +39-6-8567645 Email: [email protected]
1
Abstract
Additive manufacturing for tissue engineering applications offers the possibility to design scaffolds characterized by a fine and detailed microarchitecture. Several fabrication technologies are currently available which allow to prepare tailored structures with a large selection of materials for restoring and healing tissues. However, 3D printed scaffolds are generally collected by assembling repetitive geometrical units or reproducing specific patterns in the layering direction, leading to a highly ordered architecture that does not mimic the morphology of the natural extracellular matrix (ECM), one of the main goals to be reached for an effective therapeutic approach. It is usually stated in the tissue engineering field that a scaffold has to be considered a temporary ECM, resembling all the peculiar clues as close as possible and, in this regard, an ordered microstructure cannot be usually observed within biological tissues and organs. With the aim to overcame this limitation and offer a potential approach for bone tissue applications, the present study proposes a design methodology to fabricate 3D printed scaffolds characterized by a random microarchitecture which can be repeatedly reproduced thanks to the intrinsic controllable process of additive manufacturing. In this framework, four different models in polylactic acid were fabricated by means of fused deposition modelling, including a three-dimensional random distribution of spherical pores of 400, 500, and 600 µm for the first three cases, and a randomly varied distribution in the range 400-600 µm for the fourth case. A detailed assessment by means of microcomputed tomography and mechanical evaluation was then carried out in order to fully analyse the resulting scaffolds, providing both morphological and quantitative data.
Keywords: 3D printing, bone scaffolds, random microarchitecture, mechanical testing, micro-CT analysis
2
1. Introduction
Additive manufacturing (AM) is a broad collection of different 3D printing technologies, constantly gaining interest for several industrial and research applications. The main characteristic of this fabrication approach is related to the precise control in each stage of the manufacturing process, starting from the design to the production of the final component. In this regard, the proper selection of the material(s), the inner architecture and the resulting mechanical properties, just to mention the most evident features, allow to deal with a tailored structure for the desired application. In the field of tissue engineering and regenerative medicine, AM is regarded as an alternative and potential manufacturing technique for innovative scaffolds and fused deposition modelling (FDM) is one of the most popular process for this aim. Nowadays, there are many commercial FDM printers, comprised in a wide technological range, that can suitably replicate anatomical districts or collect specific substrates. The fine control of the microstructure to be sliced is certainly one of the main advantages, and this usually leads to reproduce regular patterns with the possibility to modify the orientation of two consecutive layers or the distance between two adjacent strands on a single layer. These occurrences usefully contribute to define the expected response of the scaffold either from a bioengineering point of view (e.g., morphology, stiffness) and a biological one (e.g., cell viability, proliferation or differentiation when dealing with stem cells). Moreover, it is often reported that a scaffold with a regular pattern can be a valuable solution as it allows to control the expected functionality and cell distribution (Wüst et al., 2011). Xia et al. (2018) observed that 3D printed hydrogels for cartilage regeneration with a 50% infill density showed higher chondrocyte seeding efficiency and more homogeneous distribution compared with 30% and 70% groups. 3D printed scaffolds made of polylactic acid and treated with methacrylamide-modified gelatin were assessed as hybrid substrates for mouse calvaria-derived preosteoblast cells, finding cell uniform distribution and high seeding efficiency (Markovic et al., 2015). Kumar et al. (2016) reported that interconnected pore network, predefined pore size and architecture support vascularization in 3
porous scaffolds and ensure long term implants success, conversely from contorted path that may limit the growth of blood capillaries and the supply of oxygen and nutrients during tissue remodelling. The spatial control provided by the inner morphology could be of interest, but it should be critically revised as well, since a scaffold for tissue engineering applications has to be considered as a temporary substitute of the natural extracellular matrix (ECM) of the tissue to be regenerated, closely mimicking its microarchitecture (Dutta and Dutta, 2009; Kim et al., 2016; Gilpin and Yang, 2017; Jun et al., 2018; Bittner et al., 2018). Generally, ECM is a complex structure not characterized by a regular and repetitive assembling of the key-elements, such as collagen or elastin, and this pattern should be therefore resembled to favour a proper integration between the scaffold and the surrounding biological district to be healed. In order to be compliant with this statement, a random microarchitecture could be a suitable alternative. Several techniques can be considered for this aim, e.g. solvent casting/salt leaching, freeze drying, but most of them are not fully controllable thus leading to a result that is not strictly repeatable in terms of scaffold production. This might be an apparent contradiction, but cell response is dictated by scaffold morphology and a fine tuning of this feature should be exerted in order to limit the number of possible technical variables affecting the outcome. Therefore, with the aim to perform a biological assay just based on the cell-scaffold interaction with a specific and defined random substrate architecture, a tailored experimental approach should be designed. In this regard, FDM can be evaluated as a potential means as allows to design and realize scaffolds characterized by a random microstructure, which is also repeatable and reproducible for each fabrication session being the output of a fixed generated G-code script. The present study reports an alternative approach to design 3D printed scaffolds characterized by random microarchitecture, mimicking natural ECM. FDM was selected as fabrication technique to process a random distribution of virtual porogens, as representative and constitutive elements of the resulting scaffolds. With the aim to infer on the role of the fabrication parameters to resemble the 4
microstructure of a human iliac crest bone sample, a detailed experimental analysis was carried out by means of 3D microcomputed tomography (3D micro-CT) evaluating all the relevant features to fully characterize the resulting architecture. In addition, a mechanical characterization was also performed in order to assess the structural properties for potential applications in the bone tissue engineering field.
5
2. Materials and Methods
2.1. Design procedure for 3D printed scaffolds A random three-dimensional distribution of spheres, i.e. virtual porogens, was created by means of custom-made script within a defined volume of fixed shape that resembles the maximum size of the scaffold to be fabricated (Matlab, MathWorks, Natick, USA). As a case study, sphere was selected as a suitable model to mimic a pore, the final scaffold porosity being related to the number of spheres generated and their size. In this framework, four different conditions were evaluated, consisting of 3000 spheres differing for the size, i.e., three occurrences characterized by fixed diameters of 400, 500 and 600 µm, and one by a random diameter distribution in the range 400-600 µm, the pore sizes matching those previously considered for bone tissue engineering (Saska et al. 2018; Mota et al., 2017; Heo et al. 2019). The lower dimensional limit was selected being the diameter of the nozzle of the 3D printer subsequently used to fabricate the scaffolds. The resulting matrix with the location of the centers of the spheres and their diameters was exported for each case and processed in a CAD environment in order to realize the final model by subtracting the cloud of spheres from the representative volume of the scaffold (i.e., 10 x 10 x 3 mm). The resulting model is then exported as a STL file to be processed by a software for the 3D printer.
2.2. Fabrication of 3D printed scaffolds Scaffolds were fabricated in polylactic acid (PLA filament; Formfutura BV, The Netherlands) by means of the N2 3D printer (Raise 3D Inc., Irvine, CA, USA). Each model file was imported in ideaMaker (Raise 3D Inc., Irvine, CA, USA) and then sliced at 0.1 mm, 0.25 mm, and 0.4 mm in the Z direction. The temperature of the nozzle (0.4 mm diameter) was set at 205 °C while that of the build platform at 40 °C.
2.3. Scaffold Characterization 6
2.3.1. Thermal analysis Differential scanning calorimetry (DSC) was performed to assess the thermal properties of 3D printed PLA scaffolds. Specimens were located in aluminium pans and heated at 10 °C/min, in nitrogen atmosphere, in the temperature range 20–200 °C (Nestch DSC 200 PC). An empty aluminium pan was used as reference. The crystallinity degree χ was calculated as the ratio between the sample enthalpy and the melting enthalpy of 100% crystalline PLA, which was assumed to be 93 J/g (Battegazzore et al., 2011).
2.3.2. Micro-CT analysis The quantitative and qualitative analysis of the 3D printed PLA scaffolds was performed by means of a micro-CT system. The analysis of each 3D printed PLA scaffold consisted of approximately 2 h scanning and 2 h reconstruction procedures. A desktop micro-CT scanner (SkyScan 1072; Bruker microCT, Kontich, Belgium) was used to scan all the specimens. The acquisition stage was carried out setting a pixel size of 14.65 x 14.65 µm, which corresponds to a 20x magnification with 1.7 s exposure time and 0.45° rotation step (for a total rotation of 180°) applying a voltage of 40 kV with a current of 248 µA. The obtained acquisition data were subsequently elaborated and reconstructed (NRecon V1.7.0; Bruker microCT, Kontich, Belgium), providing axial cross sections with a pixel size of 14.65 x 14.65 µm for the total height of 3D printed PLA scaffolds. The distance between each cross section was 14 µm. All the files of each specimen were resliced stepwise setting a unitary slice spacing factor in vertical cross section, using a computer software analysis system (micro-CT-Analyser V1.16; Bruker microCT, Kontich, Belgium). The Percent scaffold volume (Percent bone volume), the Structure thickness (Trabecular thickness), the Structure linear density (Trabecular number), the Scaffold surface/volume ratio (Surface/volume ratio), and the Total porosity (%) were calculated. For the evaluation of these features, the grey level images were binarized implementing a global threshold method which is based on only one 7
parameter (i.e., the grey level value) to be set by the user (micro-CT-Analyser V1.16; Bruker microCT, Kontich, Belgium; Cengiz et al., 2018).
2.3.3. Mechanical analysis Compression tests were carried out on selected specimens, those moistly resembling the microstructure of the reference bone sample, as resulted from the µCT investigation. For this aim, box-shaped samples (5 x 5x 10 mm3) were deposited with a layer height of 0.25 and 0.4 mm, preserving the same porogen to volume ratio of the scaffolds previously considered. Mechanical properties were evaluated by applying a deformation value of 25% of the initial length at 1 mm/min by means of a universal testing machine equipped with a 5 kN load cell (M30K, Lloyd Instruments Ltd., UK). Ten specimens were tested for both manufacturing conditions.
2.4. Statistical analysis Data are expressed as mean ± standard deviation. Statistical analysis was performed by means of ANOVA test (Matlab, MathWorks, Natick, USA). Significant level was set at p < 0.05.
8
3. Results and Discussion
Additive manufacturing can provide a number of different options to prepare tailored scaffolds for tissue engineering. Referring to bone applications, several studies investigated the collected output in terms of, e.g., structural resolution, materials and cell response (Pereira et al., 2018; Zhang et al., 2019), but dealing with an effective biomimetic scaffold is a crucial issue that still needs to be addressed. This topic was critically revised also considering the role of hydroxyapatite, a wellknown filler especially targeted for bone tissue engineering (Milazzo et al., 2019). However, most of the already proposed technical solutions are characterized by a regular microstructure which unlikely match that of bone architecture. In order to develop specific fabrication approaches to be as compliant as possible to the tissue engineering paradigm, a potential solution was presented by Gòmez et al. (2016), implementing a Voronoi tessellation to design porous scaffolds to be 3D printed after numerical characterization and optimization. Based on the same design approach, Ti6Al4V scaffolds were fabricated by means of selective laser melting and mechanically tested to correlate the resulting behaviour to the morphological features (Wang et al., 2018). In addition, several thick scaffolds for bone tissue engineering have been realized so far by means of a number of experimental methods, such as foaming or replication of natural structures (Meskinfam et al., 2018; Clarke et al., 2016), that do not allow, however, to exert the characteristic fine control of 3D printers in the manufacturing stage. Referring to the possible materials selection, PLA is one of the most common polymers often considered for bone tissue applications. A number of suitable characteristics, e.g. biocompatibility, biodegradability, mechanical properties, and its versatility to be processed as a neat material or a composite scaffold, allows to consider PLA a valuable option for scaffolding (Diomede et al., 2018; Chou et al., 2017; Narayanan et al., 2016; Rakovskyet al., 2014; D’Angelo et al., 2012; Bianco et al., 2011). It should be underlined that different fabrication techniques, e.g. stereolithography, could provide a better resolution than FDM to reproduce the fine structure of the bone ECM, but a number of 9
drawbacks can limit the expected outcome. Possible issues can be related to the need to deal with photocrosslinkable polymers, photoinitiators can be toxic and unreacted monomers can be undesired by-products, resulting scaffolds can be affected by low mechanical strength and material gradients are difficult to produce with good fidelity (Egan, 2019; Moreno Madrid et al., 2019). Selective laser sintering can also provide a good accuracy, but the process is carried out at high temperatures and laser intensity can induce polymer degradation, as observed by Gayer et al. (2019) processing polylactide/calcium carbonate composites. It is also possible to consider 3D printing by means of binder materials to process powder layers which however requires post-processing procedures in order to deal with the final scaffold that, for instance, restricts the possibility to incorporate biomolecules (Moreno Madrid et al., 2019). In this regard, a fabrication approach based on material extrusion, such as FDM, has been developed to collect bone ECM-like scaffolds made of PLA to assess the potential of the here presented proposal.
3.1. Scaffold characterization The fabricated PLA scaffolds are shown in Fig.1, the influence of the four investigated process parameters is clearly visible. The porosity of the respective CAD models was about 27% for a porogen size of 400 µm, 46% for a porogen size of 500 µm, 65% for a porogen size of 600 µm, and 35% for a porogen size distribution of 400 - 600 µm. An example of the CAD model and the related sliced version of the PLA scaffold to be printed is reported in the Supplementary Information. The thermal characterization was carried out by means of DSC analysis. Significant differences of PLA before and after scaffolds fabrication were not assessed (Fig. 2), collected results are resumed in Table I. The processing conditions did not induce any modification in the material which is not always verified, being for instance dependent on the selected polymer (Del Gaudio, 2019).
3.2. Tomographic investigation
10
The micro-CT analysis was carried out to finely investigate the microarchitecture of all the fabricated scaffolds. Fig. 3 shows a representative slice for each scaffold to visualize the internal structure, while Table II summarizes the main values of specific morphometric features, generally computed to quantify bone characteristics. The effect of virtual porogen size can be clearly observed on the computed values for scaffold volume, surface to volume ratio, and porosity. The latter parameter was in good agreement with that evaluated from the CAD models, allowing to estimate, or to design, this relevant characteristic for an effective scaffold for tissue engineering applications. Clearly, porosity is just a partial information since pore size is directly implied to support cell migration and mass transport within the three-dimensional environment of the fabricated substrate. This critical issue was here addressed, considering several conditions in order to optimize the scaffold architecture before to move towards biological assays. The micro-CT assessment of a reference bone sample, i.e. human iliac crest bone, was therefore performed as a complementary analysis to verify the proposed approach. The acquired result is shown in Fig. 4, which also summarizes the analogous quantitative values considered for the 3D printed PLA scaffolds. A direct comparison of the data led to select two cases that mostly match the bone microstructure, being the scaffolds printed at 0.25 and 0.4 mm height layer considering the virtual porogen size of 600 µm. This output was also supported by literature. For instance, Liu et al. (2010) investigating the human distal tibia bone collected an average value for the trabecular thickness of 0.15 mm, and a trabecular number of 1.18 mm-1. The µCT analysis of the human mandibular condylar bone, as reported by Renders et al. (2007), showed that the trabecular thickness was in the range 0.11-0.22 mm, the trabecular number within 1.16-1.67 mm-1, and the surface to volume ratio within 12.05-22.46 mm-1. Nakashima et al. (2019) investigated human cadaveric vertebrae to be related to bone strength parameters, finding that the average values for the trabecular thickness and the trabecular number were 0.328 mm and 0.876 mm-1, respectively.
3.3. Mechanical analysis 11
The 3D printed PLA scaffolds that more closely resembled the bone microstructure were mechanically assessed by means of compression tests. As shown in Fig. 5, representative stressstrain curves were characterized by two linear regions which can be ascribed to the compaction of the porous internal structure, before the toe region, and to the compression of the resulting configuration, as the steepest increase of the stress-strain curve clearly highlighted. The compression moduli in the first region were 27.8 ± 3.5 MPa and 25.3 ± 1.5 MPa for a scaffold layer height of 0.25 mm and 0.4 mm, respectively (significant differences were not computed). Past the toe region, the compression moduli were 1.3 ± 0.2 GPa and 1.6 ± 0.2 GPa for a layer height of 0.25 mm and 0.4 mm, respectively (p < 0.05). PLA scaffolds, and composites, fabricated by stacking layers in regular patterns considering the FDM technique were previously evaluated, showing a wide range for the computed compression modulus which is obviously dependent on the morphological strands arrangement and testing conditions. A similar compression response was observed for 3D printed scaffolds made of polylactic acid/poly-caprolactone/hydroxyapatite which were characterized by a compression modulus in the range 0.5 -1.5 MPa, depending on the polymer ratio and hydroxyapatite content (Hassanajili et al., 2019). Differently, 3D printed PLA scaffolds including nano- and microhydroxyapatite were characterized by a Young’s modulus from 1.6 GPa to 2.6 GPa, before and after cultural medium incubation (Niaza et al., 2017). Comparable values were also measured for 3D printed PLA scaffolds including 15% hydroxyapatite, being in the range 1.3 – 2.0 GPa with respect to 1.2 – 1.4 GPa for the neat case (Senatov et al., 2016). Martin et al. (2019) measured mechanical values comprised in the range 100 – 200 MPa, reporting a substantial match with the variability of the Young modulus of the trabecular bone. The influence of the FDM fabrication conditions were also investigated by Naghieh et al. (2016), showing that it was possible to modulate the elastic modulus from about 210 to 1000 MPa. However, a large variability can be also found when testing human bones. Trabecular bones from different anatomical locations were characterized by an average compression modulus of 597.9 ± 401.6 MPa (Rincón-Kohli and Zysset, 2009), while the 12
modulus of human cortical bone evaluated with different techniques and, again, from different locations spanned from 15.7 ± 3.5 GPa to 25.8 ± 0.7 GPa, as reviewed by Libonati and Vergani (2016). The same mechanical parameter was measured by Singh et al. (2019), resulting to be 3.73 ± 1.27 GPa for the humerus, 3.05 ± 1.03 for the ulna, and 2.75 ± 0.71 for the radius. As already stated, the dependence of the mechanical properties with the testing conditions contributes to the wide range of the collected published data and allows to observe that the herein presented results are clearly comprised within these findings. This statement obviously implies that biological assays are needed to fully verify the aim of this proposal, but the mechanical assessment allowed to support that the developed scaffolds can be potentially considered for structural and bearing purposes.
13
5. Conclusions
With the aim to design and fabricate scaffolds for tissue engineering applications, close resembling the natural ECM (e.g., the bone microarchitecture in this study), a specific experimental procedure was developed. The proposed methodology is not related to the materials and to the 3D printing technique to be selected. The approach can be generally applied when a random microstructure needs to be replicated within a tissue engineered scaffold. In this respect, pore size and overall dimensions of the structure depend on the resolution of the manufacturing technique and the final design can be therefore tailored based on the 3D printer technical characteristics. The here presented investigation is the first step towards the definition of a different procedure to design and fabricate ECM-like 3D printed scaffolds. Several experimental conditions were compared to a real human bone sample in order to select those mostly matching the related morphometric parameters. However, it should be also underlined that this comparison was carried out considering only one biological reference case and this could be reasonably a limitation, even if a good agreement was observed, further supported by literature data. Clearly, this is just the starting point of a systematic approach that already provided interesting findings that allow to consider the proposed scaffolds for potential tissue engineering applications, being planned as a future development.
14
References
Battegazzore D, Bocchini S, Frache A. Crystallization kinetics of poly(lactic acid)-talc composites. eXPRESS Polym Lett. 2011; 5:849–58. Bianco A, Bozzo BM, Del Gaudio C, Cacciotti I, Armentano I, Dottori M, D’Angelo F, Martino S, Orlacchio A, Kenny JM. Poly (L-lactic acid)/calcium-deficient nanohydroxyapatite electrospun mats for bone marrow stem cell cultures. J Bioact Compat Polym 2011;26(3):225-41. Bittner SM, Guo JL, Melchiorri A, Mikos AG. Three-dimensional printing of multilayered tissue engineering scaffolds. Mat Today. 2018;21:861-74. Bruker microCT. CT-analyser - The user’s guide. Kontich (Belgium): Skyscan Ed.; 2013. Cengiz IF, Oliveira JM, Reis RL. Micro-CT - a digital 3D microstructural voyage into scaffolds: a systematic review of the reported methods and results. Biomater Res. 2018;22:26. Chou YC, Lee D, Chang TM, Hsu YH, Yu YH, Chan EC, Liu SJ. Combination of a biodegradable three-dimensional (3D) - printed cage for mechanical support and nanofibrous membranes for sustainable release of antimicrobial agents for treating the femoral metaphyseal comminuted fracture. J Mech Behav Biomed Mater. 2017;72:209-218. Clarke SA, Choi SY, McKechnie M, Burke G, Dunne N, Walker G, Cunningham E, Buchanan F. Osteogenic cell response to 3-D hydroxyapatite scaffolds developed via replication of natural marine sponges. J Mater Sci Mater Med. 2016;27(2):22. D'Angelo F, Armentano I, Cacciotti I, Tiribuzi R, Quattrocelli M, Del Gaudio C, Fortunati E, Saino E, Caraffa A, Cerulli GG, Visai L, Kenny JM, Sampaolesi M, Bianco A, Martino S, Orlacchio A. Tuning multi/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats. Biomacromolecules. 2012;13(5):1350-60. Del Gaudio C. A feasibility study for a straightforward decoration of a 3D printing filament with graphene oxide. Fuller Nanotub Car N. 2019;27:607-12.
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Diomede F, Gugliandolo A, Cardelli P, Merciaro I, Ettorre V, Traini T, Bedini R, Scionti D, Bramanti A, Nanci A, Caputi S, Fontana A, Mazzon E, Trubiani O. Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair. Stem Cell Res Ther. 2018;9(1):104 Dutta RC, Dutta AK. Cell-interactive 3D-scaffold; advances and applications. Biotechnol Adv. 2009;27(4):334-9. Egan PF. Integrated design approaches for 3D printed tissue scaffolds: review and outlook. Materials (Basel). 2019;12(15). pii: E2355 Gayer C, Ritter J, Bullemer M, Grom S, Jauer L, Meiners W, Pfister A, Reinauer F, Vučak M, Wissenbach K, Fischer H, Poprawe R, Schleifenbaum JH. Development of a solvent-free polylactide/calcium carbonate composite for selective laser sintering of bone tissue engineering scaffolds. 2019;101:660-673. Gilpin A, Yang Y. Decellularization strategies for regenerative medicine: from processing techniques to applications. Biomed Res Int. 2017;2017:9831534. Gómez S, Vlad MD, López J, Fernández E. Design and properties of 3D scaffolds for bone tissue engineering. Acta Biomater. 2016;42:341-350. Hassanajili S, Karami-Pour A, Oryan A, Talaei-Khozani T. Preparation and characterization of PLA/PCL/HA composite scaffolds using indirect 3D printing for bone tissue engineering. Mater Sci Eng C. 2019;104:109960 Heo SY, Ko SC, Oh GW, Kim N, Choi IW, Park WS, Jung WK. Fabrication and characterization of the 3D-printed polycaprolactone/fish bone extract scaffolds for bone tissue regeneration. J Biomed Mater Res B Appl Biomater. 2019;107(6):1937-1944. Jun I, Han HS, Edwards JR, Jeon H. Electrospun fibrous scaffolds for tissue engineering: viewpoints on architecture and fabrication. Int J Mol Sci. 2018;19(3). pii: E745. Kim Y, Ko H, Kwon IK, Shin K. Extracellular matrix revisited: roles in tissue engineering. Int Neurourol J. 2016;20(Suppl 1):S23-29. 16
Kumar A, Nune KC, Misra RD. Biological functionality of extracellular matrix-ornamented threedimensional printed hydroxyapatite scaffolds. J Biomed Mater Res A. 2016;104(6):1343-51. Libonati F, Vergani L. Understanding the structure–property relationship in corticalbone to design a biomimetic composite. Compos Struct. 2016;139:188–198. Liu XS, Zhang XH, Rajapakse CS, Wald MJ, Magland J, Sekhon KK, Adam MF, Sajda P, Wehrli FW, Guo XE. Accuracy of high-resolution in vivo micro magnetic resonance imaging for measurements of microstructural and mechanical properties of human distal tibial bone. J Bone Miner Res. 2010;25(9):2039-50. Markovic M, Van Hoorick J, Hölzl K, Tromayer M, Gruber P, Nürnberger S, Dubruel P, Van Vlierberghe S, Liska R, Ovsianikov A. Hybrid tissue engineering scaffolds by combination of three-dimensional
printing
and
cell
photoencapsulation.
J
Nanotechnol
Eng
Med.
2015;6(2):0210011-210017 Martin V, Ribeiro IA, Alves MM, Gonçalves L, Claudio RA, Grenho L, Fernandes MH, Gomes P, Santos CF, Bettencourt AF. Engineering a multifunctional 3D-printed PLA-collagenminocycline-nanoHydroxyapatite scaffold with combined antimicrobial and osteogenic effects for bone regeneration. Mater Sci Eng C Mater Biol Appl. 2019;101:15-26. Meskinfam M, Bertoldi S, Albanese N, Cerri A, Tanzi MC, Imani R, Baheiraei N, Farokhi M, Farè S. Polyurethane foam/nano hydroxyapatite composite as a suitable scaffold for bone tissue regeneration. Mater Sci Eng C Mater Biol Appl. 2018;82:130-140. Milazzo M, Contessi Negrini N, Scialla S, Marelli B, Farè S, Danti S, Buehler MJ. Additive manufacturing approaches for hydroxyapatite-reinforced composites. Adv Funct Mater. 2019, 1903055. Moreno Madrid AP, Vrech SM, Sanchez MA, Rodriguez AP. Advances in additive manufacturing for bone tissue engineering scaffolds. Mater Sci Eng C Mater Biol Appl. 2019;100:631-644.
17
Mota C, Wang SY, Puppi D, Gazzarri M, Migone C, Chiellini F, Chen GQ, Chiellini E. Additive manufacturing
of
poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate]
scaffolds
for
engineered bone development. J Tissue Eng Regen Med. 2017;11(1):175-186. Naghieh S, KaramoozRavari MR, Badrossamay M, Foroozmehr E, Kadkhodaei M.Numerical investigation of the mechanical properties of the additive manufactured bone scaffolds fabricated by FDM: The effect of layer penetration and post-heating. J Mech Behav Biomed Mater. 2016;59:241-250. Nakashima D, Ishii K, Nishiwaki Y, Kawana H, Jinzaki M, Matsumoto M, Nakamura M, Nagura T. Quantitative CT-based bone strength parameters for the prediction of novel spinal implant stability using resonance frequency analysis: a cadaveric study involving experimental micro-CT and clinical multislice CT. Eur Radiol Exp. 2019;3(1):1. Narayanan G, Vernekar VN, Kuyinu EL, Laurencin CT.Poly (lactic acid)-based biomaterials for orthopaedic regenerative engineering. Adv Drug Deliv Rev. 2016;107:247-276. Niaza K,Senatov F, Anisimova N, Kiselevskiy M, Kaloshkin S. Effect of co-incubation with mesenchymal stromal cells in cultural medium on structure and mechanical properties of polylactide-based scaffolds. BioNanoSci. 2017;7:712. Pereira T, Prendergast ME, Solorzano R. State of the art biofabrication technologies and materials for bone tissue engineering. J 3D Print Med.2018;2:103-13. Rakovsky A, Gotman I, Rabkin E, Gutmanas EY.β-TCP-polylactide composite scaffolds with high strength and enhanced permeability prepared by a modified salt leaching method. J Mech Behav Biomed Mater. 2014;32:89-98. Renders GA, Mulder L, van Ruijven LJ, van Eijden TM. Porosity of human mandibular condylar bone. J Anat. 2007;210(3):239-48. Rincón-Kohli L, Zysset PK. Multi-axial mechanical properties of human trabecular bone. Biomech Model Mechanobiol. 2009;8(3):195-208.
18
Saska S, Pires LC, Cominotte MA, Mendes LS, de Oliveira MF, Maia IA, da Silva JVL, Ribeiro SJL, Cirelli JA. Three-dimensional printing and in vitro evaluation of poly(3-hydroxybutyrate) scaffolds functionalized with osteogenic growth peptide for tissue engineering. Mater Sci Eng C Mater Biol Appl. 2018;89:265-273. Senatov FS, Niaza KV, Zadorozhnyy MY, Maksimkin AV, Kaloshkin SD, Estrin YZ. Mechanical properties and shape memory effect of 3D-printed PLA-based porous scaffolds. J Mech Behav Biomed Mater. 2016;57:139-48. Singh D, Rana A, Jhajhria SK, Garg B, Pandey PM, Kalyanasundaram D. Experimental assessment of biomechanical properties in human male elbow bone subjected to bending and compression loads. J Appl Biomater Funct Mater. 2019;17(2):2280800018793816. Wang G, Shen L, Zhao J, Liang H, Xie D, Tian Z, Wang C. Design and compressive behavior of controllable irregular porous scaffolds: based on Voronoi-tessellation and for additive manufacturing. ACS Biomater Sci Eng. 2018;4:719−27. Wüst S, Müller R, Hofmann S. Controlled positioning of cells in biomaterials-approaches towards 3d 3D tissue printing. J Funct Biomater. 2011;2(3):119-54. Xia H, Zhao D, Zhu H, Hua Y, Xiao K, Xu Y, Liu Y, Chen W, Liu Y, Zhang W, Liu W, Tang S, Cao Y, Wang X, Chen HH, Zhou G. Lyophilized scaffolds fabricated from 3d3D-printed photocurable natural hydrogel for cartilage regeneration. ACS Appl Mater Interfaces. 2018;10(37):31704-31715. Zhang L, Yang G, Johnson BN, Jia X. Three-dimensional (3D) printed scaffold and material selection for bone repair. Acta Biomater. 2019;84:16-33.
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FIGURES
Fig. 1. 3D printed PLA scaffolds. Virtual porogen size: 500 µm (A), 400 µm (B), 600 µm (C), 400 – 600 µm (D)
20
Fig. 2. Calorimetric curves of PLA before and after 3D printing
21
Fig. 3. Slices of the 3D printed PLA scaffolds collected from the micro-CT analysis
22
Fig. 4. Slice of a specimen from human iliac crest bone and the related morphometric parameters as computed from the micro-CT analysis
Fig. 5. Compression curves for the scaffolds fabricated setting a height layer of 0.25 and 0.4 mm
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Supplementary Information
An example of the STL file representative of a random pore distribution of 500 µm diameter is shown in Fig. S1, while a picture of the sliced version to be 3D printed is reported in Fig. S2. A random PLA scaffold was obtained, even if a close match with the model cannot be observed by visual inspection.
Fig. S1 – The CAD model of the scaffold characterized by a random pore distribution of 500 µm diameter
Fig. S2 – The sliced PLA scaffold starting from the model shown in Fig. S1
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Table I - Thermal properties of PLA before and after 3D printing. Tg: glass transition temperature, Tm: melting temperature, ∆H: meting enthalpy; χ: crystallinity degree
Tg [°C]
Tm [°C]
∆H [J/g]
χ [%]
PLA Filament
63.68
151.54
19.43
21.0
PLA Scaffold
65.38
156.36
17.82
19.2
Table II – micro-CT morphological evaluation of 3D printed scaffolds
400 µm
500 µm
600 µm
400 - 600 µm
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
Layer
0.1 mm
0.25 mm
0.4 mm
0.1 mm
0.25 mm
0.4 mm
0.1 mm
0.25 mm
0.4 mm
0.1 mm
0.25 mm
0.4 mm
76.75
75.86
71.83
55.83
61.73
58.35
40.02
39.65
39.77
70.76
67.00
67.66
0.43
0.46
0.51
0.28
0.41
0.44
0.27
0.35
0.41
0.34
0.36
0.40
1.77
1.64
1.40
1.97
1.50
1.33
1.46
1.12
0.98
2.06
1.87
1.67
6.15
5.77
5.24
12.54
8.25
7.81
14.41
10.77
10.77
8.37
10.22
8.45
23.25
24.14
28.17
44.16
38.26
41.65
60.00
60.34
60.23
29.23
33.00
32.34
Percent scaffold volume (%) Structure thickness (mm) Structure linear density (1/mm) Scaffold surface/volume ratio (1/mm) Total porosity (%)
Conflict of interest
The authors have no conflict of interest
Author statement
No competing financial interests