Improving the mechanical and functional performance of extrusion based additive manufactured scaffolds for bone tissue engineering

IMPROVING THE MECHANICAL AND FUNCTIONAL PERFORMANCE OF EXTRUSION-BASED ADDITIVE MANUFACTURED SCAFFOLDS FOR BONE TISSUE ENGINEERING MUHAMMAD TARIK ARAFAT NATIONAL UNIVERSITY OF SINGAPORE 2011 IMPROVING THE MECHANICAL AND FUNCTIONAL PERFORMANCE OF EXTRUSION-BASED ADDITIVE MANUFACTURED SCAFFOLDS FOR BONE TISSUE ENGINEERING MUHAMMAD TARIK ARAFAT (B.Sc in Mechanical Engineering, BUET) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MECHANICAL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements Acknowledgements First and foremost, I would like to thank Associate Professor Ian Gibson. It is my pleasure to get him as my supervisor. His tender attention, patience, suggestions and prudence guidance encourage me throughout my candidature. I would like to express my gratitude to Dr Li Xu (Senior scientist I, IMRE) for supervising me during the last three years. His valuable guidance, continuous support and passion towards science encourage me throughout the difficult period of my research. I would like to thank National University of Singapore (NUS) for the NUS research scholarship and state of the art research facilities. I would also like to acknowledge the financial support for this research from the A*STAR Program under Grant No. R 397 000 038 305. I am grateful to all those colleagues, seniors and friends who have helped me in my PhD research when I was in need. My special thanks goes to Chris Lam for his guidance to develop my laboratory skills, Anand for his true support during the screw extrusion system development and maintenance, and Andrew for his valuable suggestions. I would also like to thank Liu Yuan and Dr. Monica for helping me a lot during the initial phase of my research. I would like to take this opportunity to convey my appreciation to Tanveer, Enamul, Pervej, Chandra Nath, Ahsan Habib, Afzal, Aravind, Asma, Anower, Zakaria, Abdul Hannan, Abu Taiyob and many more for i Acknowledgements being supportive. I would also like to acknowledge the effort of FYP student Daniel and Luo Hui. My heartfelt gratitude goes to my father Mr. Md. Shahjahan and Mrs. Monowara Begum for their blessings and mental support. I would also like to convey my gratitude to my younger brother Muhammad Tanvir Arafat for his inspiration. Last but not the least; I would like to convey my gratitude to my beloved wife Farhana Hoque for her prayer, inspiration and for always being with me. ii Table of contents Table of Contents Acknowledgements i Table of contents iii Summary xi Abbreviations xiv List of Figures xvii List of Tables xxiii Chapter Introduction 01 1.1 Background 01 1.2 Challenges in additive manufactured bone TE scaffolds 02 1.3 Research hypothesis and objectives 04 1.4 Significance of the research 05 1.5 Structure of thesis 06 Chapter Literature review 08 1.1 Background 08 2.2 Background of scaffold technology 09 2.3 Design of scaffolds 11 2.3.1 Porosity and pore size of scaffolds 11 2.3.2 Architecture of scaffolds 12 2.4 Materials of scaffolds 14 2.4.1 Bioactive ceramic phases 14 2.4.2 Biodegradable polymer matrices 15 iii Table of contents 2.4.3 Polymeric/ceramic composite scaffolds 2.5 Fabrication of scaffolds 18 19 2.5.1 Conventional fabrication techniques 19 2.5.2 Additive manufacturing techniques 19 2.5.2.1 Stereolithography apparatus (SLA) 20 2.5.2.2 Selective laser sintering (SLS) 22 2.5.2.3 Three-dimensional printing (3DP) 24 2.5.2.4 Extrusion based system 25 2.6 Modification of scaffolds 28 2.6.1 Through combinational approach 28 2.6.2 Through surface modification 30 2.7 Conclusions Chapter Screw extrusion system (SES) and its fabricated scaffolds 32 34 3.1 Introduction 34 3.2 Main features of in-house SES 35 3.2.1 Extruder screw 35 3.2.2 Extruder body part 36 3.2.3 Extruder nozzle 39 3.3 Evaluation of scaffolds fabricated via in-house SES 40 3.3.1 Scaffold Material 40 3.3.2 Scaffold design 40 3.3.3 Scaffold fabrication 41 3.3.4 Characterization of scaffolds 42 iv Table of contents 3.3.4.1 Porosity measurement 42 3.3.4.2 Morphology 44 3.3.4.3 Mechanical experiments 44 3.3.5 In vitro cell culture study 45 3.3.5.1 Cell seeding on scaffolds 45 3.3.5.2 Morphology of the cell-scaffolds constructs 45 3.3.5.3 AlamarBlue 46 3.3.6 Results and discussions 47 3.3.6.1 Porosity and dispensing speed 47 3.3.6.2 Modulus and dispensing speed 50 3.3.6.3 In vitro cell culture results 52 3.3.7 Conclusions 53 Chapter Improvement of mechanical properties of additive manufactured PCL/TCP scaffolds 4.1 Introduction 54 54 4.2 Part I – Silanized PCL/TCP(Si): composite synthesis, scaffolds fabrication and characterization 4.2.1 Materials and methods 57 57 4.2.1.1 Materials 57 4.2.1.2 Synthesis of PCL/TCP(Si) 57 4.2.1.3 Fabrication of the PCL/TCP(Si) scaffolds by SES 57 4.2.1.4 Characterization 58 4.2.1.5 Cell seeding on scaffolds 59 v Table of contents 4.2.1.6 Morphology of the cell-scaffold constructs 59 4.2.1.7 PicoGreen® Assay 60 4.2.1.8 Gene Expression (Real-time RT-PCR) 60 4.2.1.9 Western Blot Study (WB) 60 4.2.1.10 Statistical Analysis 61 4.2.2 Results and Discussions 62 4.2.2.1 PCL/TCP(Si) composite preparation and scaffolds fabrication 62 4.2.2.2 In vitro cells response 67 4.2.3 Conclusions 72 4.3 Part II – POSS modified PCL/TCP(Si): composite synthesis, scaffolds fabrication and characterization 4.3.1 Materials and methods 4.3.1.1 Materials 4.3.1.2 Synthesis of PCL/TCP(POSS) 73 73 73 73 4.3.1.3 Fabrication of the PCL/TCP(POSS) scaffolds by SES 73 4.3.1.4 Characterization 73 4.3.1.5 Cell seeding on scaffolds 74 4.3.1.6 Morphology of the cell-scaffold constructs 74 4.3.1.7 PicoGreen® Assay 74 4.3.1.8 Alkaline phosphate activity (ALP) 75 4.3.1.9 Statistical Analysis 75 4.3.2 Results and Discussions 75 vi Table of contents 4.3.2.1 PCL/TCP(POSS) composite preparation and scaffolds fabrication 75 4.3.2.2 In vitro cells response 80 4.3.3 Conclusions 83 Chapter Development of biomimetic composite coating on PCL/TCP(Si) scaffolds 85 5.1 Introduction 85 5.2 Materials and methods 87 5.2.1 Materials 87 5.2.2 Fabrication of PCL/TCP(Si) scaffolds by SES 87 5.2.3 Surface coating on PCL/TCP(Si) scaffolds 88 5.2.4 Scaffolds characterizations 89 5.2.5 Compression testing 89 5.2.6 Cell seeding on scaffolds 90 5.2.7 Morphology of cell-scaffolds constructs 90 5.2.8 PicoGreen® assay 90 5.2.9 Reverse transcription polymerase chain reaction (RT-PCR) 90 5.2.10 Western Blotting (WB) 90 5.2.11 Statistical analysis 91 5.3 Results and discussions 91 5.3.1 Scaffolds fabrication 91 5.3.2 Biomimetic CHA-gelatin composite coating 91 5.3.3 Scaffold characterization 93 vii Table of contents 5.3.4 In vitro cells response 5.4 Conclusions 98 105 Chapter Development of additive manufacturing-freeze drying integrated scaffolds with POSS modified PCL/TCP scaffolds 107 6.1 Introduction 107 6.2 Materials and methods 109 6.2.1 Materials 109 6.2.2 Fabrication of POSS modified PCL/TCP scaffolds by SES 109 6.2.3 Forming porous gelatin structure within the pores of the PCL/TCP(POSS) scaffolds and its characterization 110 6.2.4 Cell seeding on scaffolds 110 6.2.5 Morphology of the cell-scaffolds constructs 110 6.2.6 PicoGreen® assay 111 6.2.7 Alkaline phosphate (ALP) activity 111 6.2.8 Statistical analysis 111 6.3 Results and discussions 6.3.1 Fabrication of PCL/TCP(POSS) 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Rai B, Lin JL, Lim ZXH, Guldberg RE, Hutmacher DW, Cool SM. Differences between in vitro viability and differentiation and in vivo bone-forming efficacy of human mesenchymal stem cells cultured on PCL-TCP scaffolds. Biomaterials 2010;31(31):7960-7970. 160 List of publications List of publications Journals: 1. M. Tarik Arafat, Christopher X. F. Lam, Andrew K. Ekputra, Wong S. Yee, Xu Li, Ian Gibson. Biomimetic composite coating on rapid prototyped scaffolds for bone tissue engineering. Acta Biomaterialia; 2011, 7, 809-820. 2. M. Tarik Arafat, Christopher X. F. Lam, Andrew K. Ekputra, Wong S. Yee, Chaobin He, Xu Li, Ian Gibson. High performance additive manufactured scaffolds for bone tissue engineering. Soft Matter; 2012, 7(18), 8013-8022. 3. M. Tarik Arafat, Andrew K. Ekputra, L. K. Anand, Chaobin He, Simon Cool, Dietmar W. Hutmacher, Xu Li, Ian Gibson. Hierarchical PCL/TCP(POSS)-foam scaffolds to improve the functional performance of additive manufactured scaffolds. [To be submitted]. 4. M. Tarik Arafat, Xu Li, Ian Gibson. Recent trends and challenges in additive manufactured scaffolds. [To be submitted]. Conferences: 1. M. Tarik Arafat, Christopher X. F. Lam, Wong S. Yee, Chaobin He, Xu Li, Ian Gibson. High performance rapid prototyped scaffolds for bone tissue engineering. MRS Fall meeting 2010. Boston, Massachusetts, USA. 2. M. Tarik Arafat, Christopher X. F. Lam, Andrew K. Ekaputra, Xu Li, Ian Gibson. Biomimetic composite coating on poly (ε-caprolactone) and silane modified tricalcium phosphate scaffolds for bone tissue engineering. TERMIS AP 2010 meeting. Sydney, Australia. 161 List of publications 3. M. Tarik Arafat, Christopher X. F. Lam, Xu Li, Chaobin He, Ian Gibson. Biomimetic Composite Coating on Rapid Prototyped Scaffolds for Bone Tissue Engineering. MacDiarmid Institute Student and Post‐Doc Symposium 2009. Auckland, New Zealand. 4. M. Tarik Arafat, Christopher X. F. Lam, Xu Li, Ian Gibson. Biomimetic Coating on Rapid Prototyped Poly (ε-caprolactone)-Tricalcium Phosphate Composite Scaffolds for Bone Tissue Engineering. TERMIS 2nd World Congress (TERMIS 2009), August 31 to September 3, 2009. Seoul, Korea. 5. M. T. Arafat, C.F.X. Lam, X. Li, I. Gibson. Evaluation of Nano-Carbonated Hydroxyapatite (CHA) Coating on Rapid Prototyped Scaffolds for Bone Tissue Engineering. 22nd European Society for Biomaterials Conference (ESB2009) 711 September, 2009. Lausanne, Switzerland. 6. M. Tarik Arafat , Monica M. Savalani, Christopher X. F. Lam, Xu Li , Ian Gibson. Control of Mechanical Properties of Rapid Prototyped Scaffolds by Varying Process Parameters. 2nd Asian Biomaterials Congress (2ABMC), 26th to 27th June, 2009, Singapore. 7. M. T. Arafat, M. M. Savalani, I. Gibson. Improving the mechanical properties in tissue engineered scaffolds. ASME International Mechanical Engineering Congress and Exposition 2008, Boston, USA. [Oral presentation] 8. Ian Gibson, M. M. Savalani, A. Tarik & Y. Liu. The use of multiple materials in Rapid Prototyping. 3rd International Conference on Advanced Research in Virtual and Rapid Prototyping 2007, Leiria, Portugal. [Key note]. 162 Appendix A Appendix A Improving the Mechanical Properties in Tissue Engineered Scaffolds A.1 Introduction Many patients who are in need of organ transplants suffer greatly and may even die from the lack of suitable donors with the demand for artificial organs far exceeding supply. In addition, bone grafting procedures are routinely carried out on a daily basis. Such bone grafts would not be required should there be satisfactory replacement bones. Scaffold-based tissue engineering aims to aid in the repair and regeneration of bone defects. Using this approach, scaffolds act as platforms that carry cells or therapeutic agents for regenerative therapies. To effectively achieve this, the scaffold should be osteoconductive, osteoinductive, biodegradable, highly porous and should have suitable mechanical properties and surface chemistry. To date, scaffold structures have only been used with some success in low-load bearing applications, despite the large variety of biomaterials and fabrication techniques explored in the last two decades. Poly (ε-caprolactone) (PCL) is a semicrystalline, bioresorbable polymer belonging to the aliphatic polyester family. Its melting temperature is 60ºC [3]. PCL has been used to make three-dimensional porous scaffolds, with a fully interconnected pore network, using Fused Deposition Modeling (FDM). β-Tricalcium phosphate (β-TCP) is a bioactive and biocompatible ceramic that has the ability to bind directly to bones. It has been used widely in orthopedic and dental applications due to its solubility and bone A-1 Appendix A remodeling capabilities. Composites including PCL-TCP are presently being used extensively as they have the suitable properties of both the polymer and ceramic. Recent studies show the suitability of PCL-20% β-TCP scaffolds fabricated by FDM for lowload bearing bone tissue engineering applications. In this study, we demonstrate the possibility of increasing the mechanical properties of such scaffolds by introducing a through-hole. A number of scaffolds with through-holes of various sizes were fabricated in order to study the effect of the throughhole diameter on the stiffness of the complete scaffold. As well as possibly improving the mechanical properties of a scaffold, it is conjectured that through-holes may also become useful for the channeling or storage of nutrients. A.2 Materials and methods A.2.1 Scaffold processing A.2.1.1 Scaffold Material: For experiments we used PCL-20% β-TCP biocomposite. The composite was prepared by combining the PCL polymer and β-TCP in methylene chloride (JT Baker). The distribution of β-TCP within the composite was assessed and ensured to be homogeneous by visual inspection and analyzed using a microcomputed tomography (μCT) machine (Skyscan1076, Belgium). A-2 Appendix A A.2.1.2 Scaffold Fabrication: In house screw-extrusion system (SES) has been used to fabricate the scaffolds. Once the material is fed into the SES, the material melts to a semi-liquid state. The semiliquid material is then extruded through the nozzle due to pressure created by screw rotation. The extruded material is deposited in layers in a similar way to the commercial FDM process. The material solidifies and bonds to the preceding layer. After a layer is completed, the height of the extrusion head is increased and the subsequent layers are built to construct the entire scaffold. The SES was used to fabricate scaffolds with 0/90o lay down pattern and bulk dimension of 30 mm * 30 mm * mm. During the fabrication the processing temperature was set at 85 oC, the filament separation distance (centre to centre horizontal distance between adjacent filaments) was set to 1.5 mm and the nozzle diameter 0.5 mm. After the scaffolds were fabricated, through holes were drilled and subsequently scaffolds were cut into mm *6 mm * mm dimensions for analysis. The through holes were drilled because this technique was considered to be the simplest method for incorporating a sealed through hole into this type of scaffold. To study the relationship between hole size and strength, drill bits of 1.3 mm, 1.5 mm, 1.8 mm, 2.0 mm, 2.3 mm sizes were used. A.2.2 Morphology study by SEM Scaffold morphology and pore size were studied by using Scanning Electron Microscopy (SEM). Scaffolds were gold sputter coated by using a JEOL fine sputter A-3 Appendix A coater (JFC-1200) for 90 seconds at 10 mA. The SME images were taken by Philips XL30 FEG at a beam intensity of 10 kV. A.2.3 Mechanical characterization Compression tests were conducted on scaffolds by using an Instron 4502 Uniaxial testing system and 5kN load cell (Canton, MA, USA). From every batch five samples were tested adopting the guidelines for compression testing of acrylic bone cement set in ASTM F451-99a. The scaffolds were compressed at a rate of mm/min up to a strain level of approximately 80%. The stress-strain (σ-ε) curves were obtained to evaluate the compressive modulus. The modulus was calculated from the stress-strain curve as the slope of the initial linear portion of the curve. A.3 Results A.3.1 Scaffold morphology Figure shows SEM images of scaffolds with different sizes of drilled hole. It can be seen from the figure that, at the wall of the larger holes, a greater amount of material has been melted by the drill bit during drilling. A.3.2 Mechanical properties of the scaffolds The stiffness of scaffolds with different hole size is given in Table 1. The effect of the diameter of the holes on the stiffness of the scaffolds is given in Figure 3. Table A-4 Appendix A and figure show an increase of about 37% in stiffness for scaffolds with hole diameter 2.3 mm compared with that of scaffolds without a drilled hole. Fig. A-1: SEM images of the scaffolds. Table A-1: Calculated compressive stiffness for the scaffolds with different hold diameter. Diameter of hole (mm) Without hole 1.3 1.5 1.8 2.0 2.3 Stiffness (MPa) 25.86 ± 1.09 22.74 ± 1.3 27.61 ± 1.14 27.65 ± 1.56 34.29 ± 0.55 35.54 ± 2.64 A-5 Stiffness (MPa ) Appendix A 45 40 35 30 25 20 15 10 1.2 1.4 1.6 1.8 Hole size (mm) 2.2 2.4 Fig. A-2: Effect of the hold size diameter on the stiffness of the scaffold. A.4 Discussions Table A-1 and Figure A-2 show that for the smaller diameter hole the effect on stiffness is negative. After this we appear to get a threshold value for the hole diameter. Finally, there is noteworthy increment in the stiffness for the larger hole diameter. The stiffness of the scaffold with hole diameter 1.3mm is lower than that of the scaffolds without hole. For the 1.5 mm hole diameter the difference in stiffness is also not remarkable. Therefore, it can be said that hole diameter smaller than the filament separation distance has negative impact on the stiffness of the scaffolds. This may be because, for a smaller diameter hole, the structure is damaged during the hole making process. Evidence of this can be observed in the developed micro cracks as shown in Figure A-3. It should be noted that for 1.5 mm hole and for 1.8 mm hole we observed only a 6.9% increment in stiffness. However, a large change in stiffness was found for 2.0mm hole diameter. For 2.0 mm and 2.3 mm hole diameters, the stiffness increases by 32.6% and 37.4% respectively than that of scaffolds without drilled holes. Hence, it can be said A-6 Appendix A that 1.8mm diameter hole acts as a threshold value for 1.5 mm filament distance and hole diameters larger than the filament distance have significant effect on the stiffness of the scaffold. From Figure A-4, it can be seen that larger diameter drilled holes involve more material in the column making process. As the amount of fused material is more for the larger hole diameter, the strength of the scaffold has increased correspondingly. It can be seen clearly that the holes mask off the interconnected pores in the remainder of the scaffold. Such columns can act as a means for storing nutrients or for channeling flow through the scaffold. The number and distribution of the columns must therefore be limited in relation to the remainder of the scaffold, but nonetheless can be an important contributor to the overall effectiveness of the scaffold for tissue regenerative purposes. Micro cracks at lower diameter hole Fig. A-3: Micro cracks at the wall of the hold with 1.5 mm diameter. This molten material acts as a column and thus increases the strength Fig. A-4: Molten material acts as a column and increases the strength of the scaffold. This image is for 2.3 mm hole. A-7 Appendix A A.5 Conclusions This experiment demonstrated the possibility of increasing the mechanical properties of the PCL-20% β-TCP scaffolds by making a through hole. From the experiment we can conclude that, for a specific filament distance there is a threshold value of the hole diameter. A hole larger than that of the threshold value has a significant impact on increasing the strength of the scaffold. In addition it is believed, this column type hole can also be useful for channeling or storage of nutrients. By using this through hole for nutrient storage we can make scaffolds more suitable for load bearing application without decreasing the effectiveness for tissue regeneration. Future study may investigate how to incorporate through-hole into the direct fabrication process. A-8 [...]... improve the mechanical and functional performance of extrusion- based additive manufactured scaffolds. ” The main objective of this study was therefore to develop additive manufactured scaffolds with improved mechanical and functional performance for bone TE applications In this context, a number of objectives that have been set to accomplish the principal aim are as follows: Evaluate the suitability of in-house... the cell seeding efficiency, and thereby to improve the functional performance of the scaffolds The hypothesis, objectives and significance of this research will be elaborated in the following sections 1.3 Research hypothesis and objectives Having the above mentioned challenges in mind the following hypothesis can be formed: “To further improve the range of bone TE applications we need to improve the. .. proliferative and osteoconductive properties of the additive manufactured scaffolds 139 8.1.3 Improvement of the functional performance of the additive manufactured scaffolds 140 8.1.4 Evaluation of the biomimetic composite coated additive manufactured scaffolds in vivo 8.2 Limitations and recommendations 141 142 References 144 List of publications 161 Appendix A-1 x Summary Summary Scaffold -based tissue engineering. .. particularly contribute to the better understanding of the effects of different coupling agents on polymer/ceramic composites for bone TE application Furthermore, the results may extend the understanding of the basic principles to give effective biomimetic coating on additive manufactured scaffolds and also different ways to improve functional performance of additive manufactured scaffolds for bone TE In this... approach to improve the cell seeding efficiency of the developed scaffolds Evaluate the biomimetic composite coated scaffolds in rat calvarial defect 1.4 Significance of the research The results of this present study may have significant impact on the application of scaffolds in bone TE application by providing additive manufactured scaffolds with improved mechanical and functional performance The results... improve the mechanical properties of the composite scaffolds in context of bone TE Moreover, different surface modification techniques to improve the proliferative and osteoconductive properties of the additive manufactured scaffolds are still in its infancy 1.2 Challenges in additive manufactured bone TE scaffolds In scaffold -based bone TE an ideal scaffold should be mechanically stable; that is the mechanical. .. appendice in this thesis This chapter started with the importance and present challenges of additive manufactured scaffolds for bone TE, and ends up with the hypothesis, aim and significance of this research work Chapter 2 presents a comprehensive review on additive manufactured scaffolds for bone TE by describing design, materials and fabrication technologies used for scaffolds Moreover, the recent challenges... native tissue at the time of implantation [6] Other highly desirable features concerning the scaffold are its controllable interconnected porosity and vascularization This chapter aims to identify the state -of -the art and future direction of additive manufactured bone TE scaffold The emphasis will be on the design, material and additive fabrication of the bone TE scaffolds 8 Chapter 2 2.2 Background of. .. disruption of donor site bone structure and considerable donor site morbidity associated with the harvest [3, 4] Therefore, the development of new synthetic bone substitutes or scaffolds that could be used instead of autogenous cancellous bone grafts has become a key priority in bone TE [5-7] In scaffold -based bone TE the scaffold acts as a platform to carry cells or therapeutic agents for regenerative therapies... PCL/TCP(POSS)-foam scaffolds compared to PCL/TCP(POSS) scaffolds In summary, it has been found that coupling agents improve mechanical properties of the polymer/ceramic scaffolds significantly Scaffolds with improved mechanical properties can be further modified to enhance functional performance of the scaffolds This study will make a significant contribution in the field of extrusion based AM scaffolds by improving . NATIONAL UNIVERSITY OF SINGAPORE 2011 IMPROVING THE MECHANICAL AND FUNCTIONAL PERFORMANCE OF EXTRUSION- BASED ADDITIVE MANUFACTURED SCAFFOLDS FOR BONE TISSUE ENGINEERING MUHAMMAD. IMPROVING THE MECHANICAL AND FUNCTIONAL PERFORMANCE OF EXTRUSION- BASED ADDITIVE MANUFACTURED SCAFFOLDS FOR BONE TISSUE ENGINEERING MUHAMMAD TARIK. manufactured scaffolds 138 8.1.2 Improvement of the proliferative and osteoconductive properties of the additive manufactured scaffolds 139 8.1.3 Improvement of the functional performance of the additive