Multi-material poly(lactic acid) scaffold fabricated via fused deposition modeling and direct hydroxyapatite injection as spacers in laminoplasty
Ghifari Syuhada, Ghiska Ramahdita, A. J. Rahyussalim, and Yudan Whulanza
Citation: AIP Conference Proceedings 1933, 020008 (2018); doi: 10.1063/1.5023942 View online: https://doi.org/10.1063/1.5023942
View Table of Contents: http://aip.scitation.org/toc/apc/1933/1 Published by the American Institute of Physics
Articles you may be interested in
Understanding the properties of chitosan aryl substituted thioureas in their role and potential as antibacterial agents
AIP Conference Proceedings 1933, 020002 (2018); 10.1063/1.5023936
Optimization growth of Spirulina platensis in bean sprouts extract medium with urea fertilizer for phycocyanin production as antioxidant
AIP Conference Proceedings 1933, 030014 (2018); 10.1063/1.5023961
Optimization of phycocyanin extraction from microalgae Spirulina platensis by sonication as antioxidant AIP Conference Proceedings 1933, 030013 (2018); 10.1063/1.5023960
Column chromatography isolation of nicotine from tobacco leaf extract (Nicotiana tabaccum L.) AIP Conference Proceedings 1933, 030011 (2018); 10.1063/1.5023958
Inertial focusing and passive micro-mixing techniques for rare cells capturing microfluidic platform AIP Conference Proceedings 1933, 040001 (2018); 10.1063/1.5023971
Sperm Na+, K+-ATPase and Ca2+-ATPase activity: A preliminary study of comparison of swim up and density gradient centrifugation methods for sperm preparation
AIP Conference Proceedings 1933, 030016 (2018); 10.1063/1.5023963
Multi-Material Poly(Lactic Acid) Scaffold Fabricated Via Fused Deposition Modeling and Direct Hydroxyapatite
Injection as Spacers in Laminoplasty
Ghifari Syuhada
1, Ghiska Ramahdita
2,4, Rahyussalim, A.J.
3, and Yudan Whulanza
1,4,a)1Department of Mechanical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok 16424, Indonesia
2Department of Metallurgy and Material Engineering, Faculty of Engineering, Universitas Indonesia, Kampus UI Depok 16424, Indonesia
3Department of Orthopedic and Traumatology, Faculty of Medicine, Universitas Indonesia-Cipto Mangunkusumo Hospital, Jl Salemba Raya No 6 Jakarta 10430, Indonesia
4Research Center for Biomedical Engineering Universitas Indonesia, Kampus UI Depok 16424, Indonesia
a)Corresponding author: yudan@eng.ui.ac.id
Abstract. Nowadays, additive manufacturing method has been used extensively to realize any product with specific attributes rather than the conventional subtractive manufacturing method. For instance, the additive manufacturing has enable us to construct a product layer-by-layer by successively depositing several materials in one session and one platform. This paper studied the properties of a 3D printed scaffold fabricated through Poly(Lactic-acid) (PLA) deposition modelling in combination with injectable hydroxyapatite (HA)/alginate as cell carrier. The scaffold was designed to serve as a spacer in cervical laminoplasty. Therefore, a series of test were conducted to elaborate the mechanical property, porosity and in-vitro toxicity testing. The results showed that the method is reliable to fabricate the scaffold as desired although the toxicity test needs more confirmation.
Keywords: additive manufacturing, Polylactic acid, hydroxyapatite/alginate, cervical laminoplasty
INTRODUCTION
Bone disorder such as in the backbone is a complication that can affect any people in many countries. In the United States, this problem has a growth rate of almost 13% per year and reaching one million bone grafting procedures done each year [1]. Moreover, musculoskeletal disease which can be caused by disorders of bones which also defines the pain in the musculoskeletal system have been reported that each year these conditions such as trauma, back pain, and arthritis are the three most common conditions [2]. Bone disorder such as spinal stenosis mostly found in men and women over 50 years old. However, younger people who were born with a narrow spinal canal or who have a history of injuries to their back may also get spinal stenosis. Other factor such as arthritis and inherited conditions may also cause this disease [3]. Spinal stenosis is a degenerative condition that happens gradually by time and certain conditions could occur such as narrowing of the spinal and nerve root canals, enlargement of the facet joints, stiffening of the ligaments, and overgrowth of bone and bone spurs [4].
There are various options commonly used for surgical method, such as anterior cervical corpectomy and fusion (ACCF), anterior cervical discectomy and fusion (ACDF), and posterior cervical laminoplasty. Technically speaking, posterior cervical laminoplasty has been considered to be the most effective approach for the treatment.
This treatment tends to have a simple operation procedure and could preserve cervical mobility with less post- operative complications compared to the other options [5]. There are two main laminoplasty techniques in order to relieve the pressure on the spinal cord: open-door and double open-door laminoplasty [6]. A technique of double open-door laminoplasty requires a spacer acted as bone graft in the spinal cord.
We investigated the usage a biomaterial scaffold as bone graft substitute for the open door laminoplasty. In a tissue engineering approach, several issues need to be addressed to define an ideal scaffold. Firstly, the scaffold able to degrade overtime as the scaffold gradually interact with the tissue host. At the same time, it allows bone cells to migrate and to form a new bone tissue or to be osteoconductive. Additionally, the geometrical design, mechanical properties and porosity of the spacer must be considered thoroughly before implanted. The material selection altogether with suitable fabrication strategy of the scaffold plays an important role in this approach [7,8,9].
It has been known that natural bone as a composite of collagen and bone mineral. The collagen acts as the matrix of the composite and the bone mineral acts as a filler to reinforce the collagen. Therefore, hydroxyapatite has been utilized as a bioactive material, which is osteoconductive and forms a strong bond to surrounding tissue [10]. Additionally, a scaffold for regeneration of missing or damaged bone must have a high strength and have porous internal structure. Therefore, a 3D polymer scaffold has been proposed since 2001 by Hutmacher using fused deposition modeling (FDM) technique to replicate the bone matrix [11]. Up to now, the usage of FDM or similar technique or similar is still being studied with an advancement in a composite material with bone mineral mostly hydroxyapatite [12-16]. The studies show that polymer such as polylactic acid or polycaprolactone mixed with hydroxyapatite has good structure, biocompatible and osteoconductive.
Several researches also used similar materials as multi material instead of mixing them in the first place. This approach allows the use of polymer as a construct to support the hydrogel embedded with cell. Schurmaan [17].
Moreover, Kundu has shown the realization of cartilage scaffold composed of polycaprolactone matrix together with viable cell in the alginate, whereas Kang successfully realized hydroxyapatite in gelatin inside the matrix for cartilage and skeletal muscle [18, 19]. This paper applied the realization of multi material bone scaffolds which consist of polylactic acid polymer to be filled with hydroxyapatite/alginate. Our previous study allowed us to create 3D printed polylactic acid with gelatin [20]. In this case, we used injectable hydroxyapatite/alginate as the candidate of cell carrier during deposition [21]. A characterization on the mechanical properties and vascularization of pores during the scaffold fabrication is the focal point of this research.
METHODS
The specific objective of this study is to design and to realize the 3D printed scaffolds via polylactic acid filament fused deposition modelling and filled with injected hydroxyapatite/alginate. The geometrical measurement and compression testing were conducted to obtain the properties of the scaffold. In two terms: the porosity and Young’s modulus.
FIGURE 1. Methodology Flow Chart
Scaffold Design
Based on the last study, it is known that our rapid prototyping machine were able to realize PLA strand 0.4- 0.8mm width and form an area 0.24±0.03; 0.41±0.01; and 0.53±0.08 mm2. Additionally, the scaffolds had 59% - 80% porosity corresponded to the pore’s area (whulanza2017). In this case, a pore area of 0.9mmx0.9mm or 0.81mm2 was aimed to be realized. This relatively large pore was assumed to be suitable to contain the Hydroxyapatite/alginate filler. Additionally, the laminoplasty operation was aimed to fill the gap between 3mm- 7mm. Therefore, scaffolds with 3 layers, 4 layers and 6 layers were designed to address those gaps as pictured in figure 2.
START Equipment and
Material Preparation
Scaffold
Designing Scaffold
Fabrication
Testing Data Analysis Report and
Conclusion FINISH
FIGURE 2 (a) 3.6 mm scaffold (3 layers), (b) 4.8 mm scaffold (4 layers), (c) 7.2 mm scaffold (6 layers)
The second parameter designed was the interconnection of pores in the scaffold which involves spatial arrangement of HA/alginate filler in each layer. The HA/alginate was injected through a syringe which was connected with a piston pump mechanism to extrude the HA/alginate in semiliquid phase. Figure 3 shows the HA/alginate designed as such to fill specific area of 3D printed PLA layer. Ultimately, a multilayer of PLA matrix altogether with a HA/alginate was formed by sandwiching a set of layer as depicted in figure 3.
FIGURE 3The various HA/Alginate filler arrangement in a 3-layer-scaffold matrix (the scale does not represent the real size).
Scaffold Fabrication Technique:
Step 1: PLA Matrix Fused Deposition Modeling
A commercial RepRap 3D printer MendelTM using Fused Deposition Model (FDM) technique was used in realizing 3D matrix scaffold [7]. FDM is an additive manufacturing technology commonly used for modeling, prototyping, and production applications using a 3D printing technology. The principle of how FDM works is by laying down material in layers. The object was first designed from Computer Aided Design (CAD), when the FDM process run, a plastic filament or metal wire is unwounded from a coil to an extrusion nozzle. The nozzle is heated in order to melt the material and bring it to a thermoplastic state. Hence, it can be moved in both horizontal and vertical directions using a numerically controlled mechanism, directly controlled by Computer Aided Manufacturing (CAM). After extruded in its thermoplastic form and layers have been formed, the material hardens immediately when it cools down. In order to move the extrusion head, stepper motors or servo motors are typically installed.
(a) (b) (c)
Step 2: HA/Alginate Injection Method
The HA/alginate is prepared from brown algae with sodium chloride (NaCl) 0.9% and calcium chloride (CaCl2) 0.2 M (Merck). Alginate and hydroxyapatite powders were dissolved in 0.9% NaCl solution. The amount of liquid powder ratio (LPR) was kept fixed at 10 ml/g. The weight ratio given on alginate and HA were varied at 80:20 w/w. Later, the injection was done using syringe pump with a constant flow rate of 20 mL/hour during deposition. Based on the designed model, the hydroxyapatite/alginate were injected in the specific locations in the PLA matrix.
Mechanical Properties Testing
We also used the numerical and experimental methods for obtaining the Young’s modulus of the scaffold. The numerical method was by using finite element method performed using SolidWorks 2016 software. This method requires the design of the scaffold with fillers. Then, it should be imported to COMSOL to undergo simulation of mechanical solid. Some properties for each material such as Young’s modulus and Posison’s ratio should be inserted into COMSOL beforehand. Then, the simulation can be done by applying certain force on the model to obtain the stress and strain of the scaffold. This result can then be translated into graphs and processed to obtain the Young’s modulus by using the following formula, where the symbol of
V
is the stress andH
is the strain:ܧ ൌ ఘఌ (1)
The experimental was utilized Instron 5594 to do a compressive test with deformation rate of 2mm/minutes.
This equipment has the capacity of 2kN to obtain the mechanical properties from each sample of scaffold. The physical deformation can be seen and the value of Young’s modulus can also be obtained after the data from the experiment has been obtained.
Porosity Measurement
There were two methods for measuring porosity, first method was the analytical approach and the second was the experimental approach. The first method was conducted by designing the scaffold in SolidWorks 2016, applying the material properties, and measure the volume of the composite (PLA + void) and volume of void in SolidWorks 2016 software. We can then use the following equation:
(2) The second method uses water, glass beaker, and analytical balance. The variable being measured such as initial mass (MI), final mass (MF), initial volume (VI), final volume (VF), and volume of sample (VS = VI - VF).
Then we can use the following equation to obtain porosity percentage:
(3) In-Vitro Testing
The biocompatibility testing was conducted via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) reduction assay. The fabricated scaffolds were seeded by mesenchymal stem cell (MSC) in a multiwell. Later, the cultured cells were tested with Vybrant® and the absorbance value was determined using ELISA reader at 570 nm wave length. Cell proliferation and cell adhesion to scaffolds were evaluated at day-2 and day-6.
RESULTS AND DISCUSSION
PLA Fused Deposition Modelling
A FDM machine based on Reprap 3D printer is used for realizing 3D polylactic acid (PLA) matrix. The three- geometrical variation of scaffolds were fabricated as design in figure 2 for 3-layer, 4-layer and 6-layer. Below, the result of each samples are shown in figure 4. Additionally, figure 4 also shows the magnification of 3D PLA strand that formed during the FDM process.
% Volume 100 Total
Volume
Void x
Porosity
% V 100 x
M M
S Water
I
F x
Porosity U
FIGURE 4(a) 3 Layers Scaffold (b) 4 Layers Scaffold (c) 6 Layers Scaffold
The images in figure 4 are the magnification of PLA strand using Dino-Lite digital microscope. The image observation results a width of 0.44±0.02mm. The strand was designed in CAD environmental to have a value of 0.5 mm. However, some limitations regarding the solidification phenomenon also contribute to the printing process. Additionally, the phenomenon of having the pore size to be smaller might be due to two factors. The first one is that this might be caused by the occurrence of collapsing, which is the event when the filament being extruded falls with a bit of deviation to its line of extrusion. Second factor might be due to the extrusion of line width itself. As it has been explained before that the nozzle might not be able to extrude to 0.5 mm because of only having a diameter (Ø) of 0.4 mm. Hence, it affects the pore size of fabricated scaffold to be smaller. Hence, the results of fabricated multi-layer scaffolds have smaller measured strand width compared to the one designed in CAD environment.
Correspondingly, the pore size formed would also be different with that design. It was designed that the pore size of scaffold is around 0.81mm2or 0.9mmx0.9mm. The figure 10 depicts the top view of scaffold that brought the pore size at axial direction to be around 0.90±0.09 mm2pore. On the other hand, the pore size at lateral direction of the scaffold is at 0.44±0.07 mm2. It shows a significant different between the lateral pore size of those 3-layers, 4-layers and 6-layers. The 6-layer-scaffold tends to have smallest pore size among those three variations.
This smaller size due to the weight of more PLA layer gravitationally collapsing during the deposition process.
TABLE11.Measurement of pore size area from 3D printed PLA scaffolds 3D Printed PLA
Scaffold
Pore Area/ Rectangular Form of Strands (mm2)
Axial direction (top view) Lateral direction (side view)
Average Std. Dev. Average Std. Dev.
3-layer 0.80 0.03 0.51 0.07
4-layer 0.99 0.06 0.44 0.04
6-layer 0.92 0.03 0.38 0.02
HA/Alginate Injection
A syringe pump was used to inject the hydroxyapatite/alginate with a consistent flow rate of 20 mL/hour. The tip of the injector was installed in the RepRap 3D printer to allow the injection in certain position as desired. The results of injected HA into the PLA scaffolds are depicted in figure 5.
FIGURE 5 Top view: Overview of scaffolds and the magnification of (a) 3-layer; (b) 4-layer and (c) 6-layer 3D printed PLA/HA/Alginate
Figure 5 shows the result of injected HA into the 3-layer PLA scaffold captured using Dino-Lite digital microscope. It can be seen that the injectable HA/alginate filled the position in the pore as desired. The consistency of injectable HA is found to be optimum in composition of 20% w/w in alginate. However, after being injected the fabricated results are contained in a calcium chloride solution to maintain the HA in the scaffold and prevent it from shrinking. The figure 5 also shows the HA/alginate in the second row which indicated by a darker color.
This confirms the CAD design as pictured in figure 3.
Young’s Modulus Result
As we compare the Young’s modulus of simulation vs experimental of the scaffold with no fillers, we found that the value of compared Young’s modulus between simulation and experimental have similar results. The 3- layer- scaffold has a 56.68 MPa for its simulation results and an experimental result of 46.68 MPa. The difference for 4-layers-scaffold is much smaller with 58.01 MPa for its experimental and 59.45 MPa for the simulation result.
Whereas for 6-layer-scaffold, the experimental result is 84.42 MPa which is higher compared to the simulation result with 62.02 MPa (figure 6a).
Overall, comparing the results of Young’s modulus between simulation and experimental, it can be concluded that our simulation has a relatively small deviation with the experimental results. The result also shows that the incorporation of hydroxyapatite does not affect the elastic modulus of PLA matrix since the composition of HA relatively low. Additionally, as major part of injectable substance (80% w/w), alginate has a low elastic modulus compare to that the PLA matrix.
FIGURE 6 Measurement results of scaffolds: (a) elastic modulus and (b) porosity Porosity Result
After the process of injection, the porosity of injected scaffolds is measured using the same method used before. As the results are depicted in figure 6b, the measured porosity of scaffold with and without HA fillers shows a significantly different result. The value of simulated porosity percentage in all different type of layers is 61.5% in average, whereas the measured porosity is 55.3%, 51.7%, and 62.3% for 3-layer, 4-layer, and 6-layer scaffolds, respectively. In the HA/Alginate injected scaffolds, the overall trend of the actual porosity shows a lower value compared to the PLA scaffolds. The porosity ranges from 54.3%, 42.8%, and 48.7% for 3-layer, 4- layer, and 6-layer scaffolds, respectively.
Figuratively, the incorporation of HA/alginate decreases the porosity of PLA matrix since the HA compensate the bulk volume (figure 6b). However, this decreasing is not significantly for the case of 3-layer-scaffolds. The composition of injectable HA/alginate is mainly 80% of alginate which adsorb water during porosity measurement. In the case of 4-layer and 6-layer the different is significant. Additionally, the numerical computation for porosity shows a difference of porosity due to the collapsing PLA strands as described previously.
Biocompatibility: In-Vitro Testing
Table 2 shows the inhibition results of the fabricated scaffolds both PLA and PLA after injected with HA (hydroxyapatite)/alginate. A positive result shall be shown after 7 days of cells seeding in the scaffold. On contrary, it would be considered toxic if the inhibition value of the cells is more than 50%. Therefore, we can see that the PLA scaffolds is proven to be a non-cytotoxic substrate to living cells. PLA which is a scaffold fabricated from PLA shows the most promising result with 27.75% of cells adhering to it. Whereas, HA/alginate with a negative value indicates that the result was compromised during the sterilization process. However, the results show that the PLA matrix has less toxicity effect.
TABLE 2 Measurement of pore size area from 3D printed PLA scaffolds
Scaffolds Inhibition
Day-1 Day-7
PLA 40.25 27.75
HA/Alginate -44.3 -51.2
Control 100 100
CONCLUSION
Overall, a method of using additive manufacturing combined with injection of hydroxyapatite to realize an interconnected porous scaffold is successful. The method was proven to give a repeatable porous of PLA that combined using hydroxyapatite material. However, the osteoconductivity of hydroxyapatite was not yet established during this work.
REFERENCES
1. K. Hing, The Roy. Soc. 362(1825), 2821-2841 (2004).
2. Sylvia I. W-Castillo, PhD., Stuart L. Weinstein, MD, Edward H. Yelin, PhD., The Burden of Musculoskeletal Diseases in The United States 3rd Edition, (Bone and Join Initiative, Rosemont, IL, 2014)
3. U.S Department of Health and Human Services Public Health Service, "What is Spinal Stenosis?"
[Online] Available: https://www.niams.nih.gov/sites/default/files/catalog/files/spinal_stenosis_ff.pdf [Accessed: 18 February 2017]
4. North American Spine Society, "Diagnosis and Treatment of Degenerative Lumbar Spinal Stenosis"
[Online] Available:
https://www.spine.org/Documents/ResearchClinicalCare/Guidelines/LumbarStenosis.pdf [Accessed:
12 February 2017]
5. B. Yue, B. Chen, Xue-Xiao Ma, Yong-Ming Xi, Hong-Fei Xiang, You-Gu Hu, G. Zhang, Int. J. Clin.
Exp. Med. 8(6), 9565-9569 (2015).
6. T. Kaito, K. Yonenobu, Atlas of Neurosurgical Techniques spine and peripheral nerves, Edition: 2nd, Chapter: Cervical Laminoplasty, (Thieme, pp.228-233, Osaka, 2016)
7. F. Gattazzo, C. De Maria, Y. Whulanza, G. Taverni, A. Ahluwalia, and G. Vozzi, J. Biomed. Mater.
Res. B. Appl. Biomater. 103(5), pp.1107-1119 (2015)
8. C. De Maria, Y. Whulanza, A. Ahluwalia, and G. Vozzi, Smart Sensing Scaffolds, in Smart Membranes and Sensors: Synthesis, Characterization, and Applications (ed A. Gugliuzza), (John Wiley & Sons, Inc., Hoboken, NJ, USA., 2014)
9. AJ, Rahyussalim; Kurniawati, T.; D., Aprilya; Anggraini, R.; Ramahdita, Ghiska; Whulanza, Yudan, AIP Pub. 1817(1), 020004-1 - 020004-6 (2017)
10. Hench LL., Current Opinion Solid State Mater. Sci. 2(5), 604–610 (1997)
11. Hutmacher DW, Schantz T, Zein I, Ng KW, Teoh SH, Tan KC, J. Biomed. Mater. Res. 55(2), 203- 206 (2001)
12. Danoux, C.B., Barbieri, D., Yuan, H., de Bruijn, J.D., van Blitterswijk, C.A. and Habibovic, P., Biomatter 4(1), p.e27664 (2014).
13. Lee, J.B., Kim, J.E., Bae, M.S., Park, S.A., Balikov, D.A., Sung, H.J., Jeon, H.B., Park, H.K., Um, S.H., Lee, K.S. and Kwon, I.K., Nanomed.: Nanotech., Bio., and Med. 12(2), 496-497(2016) 14. Senatov, F.S., Niaza, K.V., Zadorozhnyy, M.Y., Maksimkin, A.V., Kaloshkin, S.D. and Estrin, Y.Z.,
Mech. Behav. of Biomed. Mater. 57, pp.139-148 (2016)
15. Nyberg, E., Rindone, A., Dorafshar, A. and Grayson, W.L., Tissue Eng. Part A 23(11-12), pp.503-514 (2017)
16. Jo, H.H., Lee, S.J., Park, J.S., Lee, J.H., Kim, W.D., Kwon, S.K., Lee, J.H., Lim, J.Y. and Park, S.A., J.
of Nanosci. and Nanotech. 16(11), pp.11943-11946(2016)
17. Schuurman, W., Khristov, V., Pot, M.W., van Weeren, P.R., Dhert, W.J. and Malda, J., Biofabri. 3(2), p.021001 (2011)
18. Kundu, J., Shim, J.H., Jang, J., Kim, S.W. and Cho, D.W., J. Of Tiss. Eng. And Regen. Med. 9(11), pp.1286-1297 (2015)
19. Kang, H.W., Lee, S.J., Ko, I.K., Kengla, C., Yoo, J.J. and Atala, A., Nature biotech. 34(3), pp.312- 319 (2016)
20. Whulanza, Yudan; Hidayaturrahmi, Pretty; Kurniawati, Tri; AJ, Rahyussalim, AIP Pub. 1817, 040001 (2017)
21. Lestari, Gusti Ruri; Yuwono, Akhmad Hermand; Sofyan, Nofrijon; Ramahdita, Ghiska, AIP Pub.
1817, 020006 (2017).
