A Polymeric Implant for Application in Post-Surgical Resection of Osteosarcoma Ayesha Suleman A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree Master of Pharmacy. Supervisors: Prof. Yahya E. Choonara University of the Witwatersrand, Department of Pharmacy and Pharmacology, Johannesburg, South Africa Associate Prof. Pierre P. D. Kondiah University of the Witwatersrand, Department of Pharmacy and Pharmacology, Johannesburg, South Africa 2023, Johannesburg, South Africa ii DECLARATION I, Ayesha Suleman, declare that this dissertation is my own, unaided work. It is being submitted for the Degree Master of Pharmacy at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at any other University. _______________________________________ (Signature of candidate) _______________day of_____________________20________________in_____________ 27th June 23 Pretoria iii DEDICATION For my Grandparents, Sultan Salahuddin Al Ayyubi (R.A.) and the Ottomans, especially Sultan Abdul Hamid Han Hz (Jannat Makkan). Their life stories have inspired me to always do my best and be my best. May the Almighty grant them the Highest Stages in Paradise. Ameen. iv RESEARCH OUTPUTS Presentations: 1. Ayesha Suleman, Pierre Kondiah and Yahya E Choonara. BRICS Summit 2021: 04 November 2021. Oral presentation. 2. Suleman A, Kondiah PPD, Mabrouk M and Choonara YE (2021) The Application of 3D- Printing and Nanotechnology for the Targeted Treatment of Osteosarcoma. Front. Mater. 8:668834. doi: 10.3389/fmats.2021.668834. 3. Suleman A, Kondiah PPD, Mabrouk M and Choonara YE . The Influence of Incorporating Bioglass Within a Composite Hydrogel for Application in Osteosarcoma. To be submitted to a suitable journal. v ABSTRACT Osteosarcoma is a malignant neoplasm of which there is osteoid formation by tumour cells. According to the American Cancer Society, osteosarcoma is prevalent in patients between the ages of 10 and 30 and those diagnosed with osteosarcoma over the age of 60, consist of 10% of the population afflicted. Surgery is a critical component in the treatment of osteosarcoma. Wide margin resections are standard in surgical treatment of osteosarcoma, therefore an osteotomy occurs, inducing a severe bone defect. Adjuvant chemotherapy is administered by IV after surgical removal of osteosarcoma to mitigate the risk of tumour micro metastases. The chemotherapeutic drugs are accompanied by severe side effects. To address the issue of a critical bone defect, the use of bone grafts and prosthetic devices (endoprosthesis or megaprosthesis), have been utilised. However, Osteosarcoma is mainly prevalent in adolescents, their growth poses a constant challenge to prosthetics and bone grafts have their own drawbacks. There is a need for customising implants for individual patients. To this end, this study focused on the development of a mouldable implants. The materials of these implants may have the ability to regenerate bone and provide localised drug delivery which may bypass the severe side effects of IV administered chemotherapeutics. Two hydrogel implants (one containing bioglass and the other without bioglass) were synthesised and compared. The polymers used were Alginate and Polyacrylamide. The hydrogel implants displayed great moulding ability when cast into different shapes and characterisation studies such as FTIR, XRD, TGA and DSC was conducted. Scaffolds containing bioglass initially possessed enough strength to match cancellous bone. Biomineralisation studies proved the formation of hydroxyapatite on the scaffold surface. Both hydrogel implants displayed ~20% of Doxorubicin released over 8 weeks. At 6 weeks, ALG-PAAM hydrogel implants degraded to 87,674% ± 5,042 of their original weight, while BG-ALG-PAAM hydrogel implants degraded to 86,528% ± 0,0987 of their original weight. Cell studies were conducted using MG-63 cells and proved that both scaffolds were non-cytotoxic and could facilitate cell adhesion. However, these hydrogel implants lacked the presence of pores from formation. Therefore, the same polymers were used to fabricate implantable scaffolds. These scaffolds were formed by lyophilisation and contained pores from formation. However, mechanical strength of these scaffold implants did not match the strength of bone and only two of the 5 criteria of the diamond concept were met in implantable scaffolds containing bioglass. Overall, it was concluded that BG-ALG-PAAM was better than the double network hydrogel without bioglass and had potential for application in Osteosarcoma as it met three of the five criteria mentioned in the diamond concept. Further research is needed to improve the scaffolds formed by lyophilization to meet at least three of the 5 criteria of the Diamond Concept. vi AKNOWLEDGEMENTS In the name of the Almighty, the most Beneficent, the most Merciful. All Praise and Thanks is due to the Almighty alone, Who, in His Infinite Mercy has allowed me to start and complete this M. Pharm journey. The biggest thank you goes to my Mum and brother for their never-ending support, affection and encouragement throughout my Master’s. I would like to thank my supervisors, Prof Yahya E Choonara and Prof Pierre P.D. Kondiah. Their supervision has helped me grow in leaps and bounds. A special thank you goes to Prof Thashree Marimuthu and Prof Pradeep Kumar for the encouraging words always passed to me in the hallways of 8th floor. A special mention goes to Prof Mostafa Mabrouk for his kind advice. A special mention goes to the technical team for the never-ending, always available assistance: Dr. Hillary Mndlovu, Mr. Kleinbooi Mohlabi, Mr. Bafana Themba, Miss Tshidi Mosete, Phumzile Madondo, and Siya Maphumulo. Your help and amazing personalities will continue to inspire me forever. A special thank you to Miss Petra Dinham for her expertise in SEM. To Yosra Sharfy, Iman Hoosen, Henna Cassimjee and Tayzuma Patel may the Almighty bless you for every little conversation and word of encouragement that you gave me. I will always treasure our friendship. To my other friends on 8th floor, you all deserve a mention. You all have acted as my friends, counsellors, personal cheerleaders and some of you, even comedians. This is list extensive, however, each of you deserve a mention for the special part you have played in my journey. Here goes: Ranya Ibrahim, Eeman Cohen, Taskeen Sarwan, Mashudu Mphaphuli, Leon Khoza, Lindokuhle Ngema, Variksha Singh, Divesha Essa, Onyinye Jennifer Uwaezuoke, Nombeko Sikhosana, Ayesha Wadee, Shazia Mansoor, Kruti Naik, Kate Da Silva, Dalton Brouwer, Kimaya Moodley, Magdi Abobaker, Ahmed Abdelgadir, Brian Karithi, Kara de La Harpe, Kundai Mazarura, Simisola Ayodele, Abu Bakr Nana, Lara Freidus, Vanessa Chivere, Fadzai Mutingwede, Alex Adekiya, Cuthbert Kibungu, Sarjan Patel, Lusanda and Mpume. To my fellow Texture Analyser custodians, Atang Motaung and Theresa Varughese, your friendship and work ethic deserve a special mention. To Dr. Gillian Mahumane: Thou art bae. I thank the following Postdocs for their very effective training and help with instruments and advice: Dr. Mershen Govender, Dr Sunaina Indermun, Dr Pavan Walvekar, Dr Sam, Dr Sifiso, vii Dr Mduduzi Sithole. Dr Philemon Ubanako, I thank you for the help and training in the cell lab and cell-work techniques. The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged (and thanked). Opinions expressed and conclusions arrived at, are those of the author and are not necessarily to be attributed to the NRF. viii TABLE OF CONTENTS Title: A Polymeric Implant for Application in Post-Surgical Resection of Osteosarcoma ………………………………………………………………………………………..i DECLARATION .....................................................................................................................ii DEDICATION ……………………………………………………………………………………….iii RESEARCH OUTPUTS ........................................................................................................ iv ABSTRACT ……………………………………………………………………………………….v AKNOWLEDGEMENTS ........................................................................................................ vi TABLE OF CONTENTS ...................................................................................................... viii LIST OF ABBREVIATIONS ................................................................................................. xiv LIST OF TABLES ................................................................................................................ xvi LIST OF FIGURES ............................................................................................................ xvii LIST OF EQUATIONS ....................................................................................................... xxii CHAPTER.1.INTRODUCTION AND OVERVIEW OF THE STUDY………. ……………………………………………………………………………………….1 1.1 Introduction and background to the study ....................................................... 1 1.2 Rationale and motivation ................................................................................ 4 1.3 Aims and objectives ....................................................................................... 7 1.4 Potential benefits of undertaking this research ............................................... 8 1.6 References ................................................................................................... 10 CHAPTER.2.LITERATURE REVIEW: THE APPLICATION OF 3D-PRINTING AND NANOTECHNOLOGY FOR THE TARGETED TREATMENT OF OSTEOSARCOMA ...................................................................................... 13 2.1 Introduction .................................................................................................. 13 2.2 Fabrication of 3D-Printed Scaffolds for Enhanced Bone Regeneration ......... 14 2.2.1 Natural polymers used for 3D-printing of scaffolds in bone regeneration ........ 18 2.2.1.1 3D-printed chitosan scaffolds ....................................................................... 18 2.2.1.2 3D- printed alginate scaffolds ....................................................................... 19 2.2.1.3 3D printed collagen scaffolds ....................................................................... 20 2.2.1.4 3D printed gelatin scaffolds .......................................................................... 21 2.2.1.5 3D printed silk fibroin scaffolds ..................................................................... 22 2.2.2 Synthetic polymers used for 3D-printing of scaffolds in bone regeneration .... 23 2.2.2.1 3D printed polycaprolactone scaffolds .......................................................... 23 2.2.2.2 3D printed polyurethane scaffolds ................................................................ 24 ix 2.2.2.3 3D printed poly (lactic acid) scaffolds ........................................................... 25 2.2.2.4 3D printed polyvinyl alcohol scaffolds ........................................................... 26 2.2.3 Bioceramics used for 3D-printing of scaffolds in bone regeneration ............... 27 2.2.3.1 3D printed calcium phosphate scaffolds ....................................................... 27 2.2.3.2 3D printed calcium silicates .......................................................................... 28 2.2.3.3 3D printed bioactive glasses in bone regeneration ....................................... 29 2.2.4 Comparison Between Different Scaffold Compositions .................................. 30 2.3 Stimuli-Responsive Scaffolds in Bone Regeneration .................................... 31 2.4 3D-Printing of Bioactive-Loaded Scaffolds for Bone Regeneration ............... 34 2.5 Nano-Enabled 3D-Printed Scaffolds for Bone Regeneration ........................ 35 2.5.1 Nano-liposomes and 3D-printed scaffolds ...................................................... 36 2.5.2 Polymeric nanoparticles and 3D printed scaffolds .......................................... 38 2.5.3 Iron oxide nanoparticles and 3D printed scaffolds .......................................... 38 2.5.4 Gold nanoparticles and 3D printed scaffolds .................................................. 39 2.5.5 Silver Nanoparticles and 3D printed scaffolds ................................................ 40 2.6 Nanocarriers for Potential Incorporation into 3D-Printed Scaffolds ............... 40 2.6.1 Mesoporous silica nanoparticles as anti-osteosarcoma nanocarriers ............. 41 2.6.2 Micelles as anti-osteosarcoma nanocarriers .................................................. 42 2.6.3 Targeted nanocarriers and stimuli-responsive nanocarriers ........................... 43 2.7 Concluding Remarks .................................................................................... 48 2.8 References ................................................................................................... 51 CHAPTER.3.DEVELOPMENT OF A COMPOSITE HYDROGEL IMPLANT FOR APPLICATION IN OSTEOSARCOMA ......................................................... 69 3.1 Introduction .................................................................................................. 69 3.2 Materials ...................................................................................................... 70 3.3 Methods ....................................................................................................... 71 3.3.1 Hydrogel Fabrication to determine the best cross linker ................................. 71 3.3.1.1 Hydrogel Fabrication .................................................................................... 71 3.3.1.2 Assessment of mechanical strength to determine best crosslinker: .............. 71 3.3.2 The Fabrication of hydrogel implants to establish the optimal concentration of the best crosslinker. ..................................................................................... 72 3.3.2.1 Synthesis of hydrogel implants with different crosslinker concentrations ...... 72 3.3.2.2 Assessment of hydrogel implants synthesised with different cross-linker concentrations to establish the optimal concentration of the best crosslinker 72 x 3.3.2.3 Assessment of mechanical strength of the hydrogel implants to establish the optimal concentration of best crosslinker ...................................................... 72 3.3.2.4 Assessment of swelling of the hydrogel implants to establish the optimal concentration of best crosslinker .................................................................. 72 3.3.2.5 Assessment of morphology of the hydrogel implant with the best crosslinker concentration ................................................................................................ 73 3.3.2.6 Assessment of mouldability .......................................................................... 73 3.3.3 Hydrogel Fabrication to induce macro-pores after determining the optimal cross- linking concentration: .................................................................................... 73 3.4 Results and discussion ................................................................................. 74 3.4.1 Hydrogel Fabrication to assess the best cross linker ...................................... 74 3.4.2 Hydrogel Fabrication to establish the optimal concentration of the best crosslinker: ................................................................................................... 75 3.4.3 Hydrogel Fabrication to induce to induce macro-pores after determining the optimal cross-linking concentration: .............................................................. 78 3.5 Concluding Remarks .................................................................................... 81 3.6 References ................................................................................................... 82 CHAPTER.4.THE INFLUENCE OF INCORPORATING BIOGLASS WITHIN A COMPOSITE HYDROGEL FOR APPLICATION IN OSTEOSARCOMA .. 85 4.1 Introduction .................................................................................................. 85 4.2 Materials ...................................................................................................... 86 4.3 Methods ....................................................................................................... 87 4.3.1 Fabrication of ALG-PAAM hydrogel implant: .................................................. 87 4.3.2 Bioglass incorporation into ALG-PAAM hydrogel implants to form BG-ALG- PAAM hydrogel implants .............................................................................. 87 4.3.3 Characterisation studies of ALG-PAAM and BG-ALG-PAAM hydrogel implants. . ……………………………………………………………………………………...88 4.3.3.1 Moulding capability of the ALG-PAAM and BG-ALG-PAAM hydrogel implants……………………………………………………………………………..88 4.3.3.2 Physicochemical properties of ALG-PAAM and BG-ALG-PAAM hydrogel implants…. ................................................................................................... 88 4.3.3.3 Thermal properties of ALG-PAAM and BG-ALG-PAAM hydrogel implants…..88 4.3.3.4 Biomineralization ALG-PAAM and BG-ALG-PAAM hydrogel implants .......... 88 4.3.3.5 Mechanical analysis of ALG-PAAM and BG-ALG-PAAM hydrogel implants……………………………………………………………………………..88 xi 4.3.3.6 Swelling and shape retention of ALG-PAAM and BG-ALG-PAAM hydrogel implants….. .................................................................................................. 89 4.3.3.7 Degradation, morphology and porosity of ALG-PAAM and BG-ALG-PAAM hydrogel implants ......................................................................................... 89 4.3.3.8 Doxorubicin Loading and release of ALG-PAAM and BG-ALG-PAAM hydrogel implants ........................................................................................................ 90 4.3.3.9 Invitro cytotoxicity and cell proliferation of ALG-PAAM and BG-ALG-PAAM hydrogel implants ......................................................................................... 90 4.3.3.10 Statistical analysis ........................................................................................ 91 4.4 Results ......................................................................................................... 92 4.4.1 Moulding capabilities of ALG-PAAM and BG-ALG-PAAM hydrogel implants.. 92 4.4.2 Physicochemical properties of ALG-PAAM and BG-ALG-PAAM hydrogel implants.. ...................................................................................................... 93 4.4.3 Thermal Characteristics of ALG-PAAM and BG-ALG-PAAM hydrogel implants…. ................................................................................................... 95 4.4.4 Biomineralization of ALG-PAAM and BG-ALG-PAAM hydrogel implants ....... 97 4.4.5 Mechanical analysis of ALG-PAAM and BG-ALG-PAAM hydrogel implants . 102 4.4.6 Swelling and shape retention of ALG-PAAM and BG-ALG-PAAM hydrogel implants.. .................................................................................................... 103 4.4.7 Degradation of ALG-PAAM and BG-ALG-PAAM hydrogel implants ............. 106 4.4.8 Morphology, Porosity and Pore size of ALG-PAAM and BG-ALG-PAAM hydrogel implants... ................................................................................................... 107 4.4.9 Drug Loading and Drug release of ALG-PAAM and BG-ALG-PAAM hydrogel implants………………………………………………………………………….113 4.4.10 In Vitro cell compatibility of ALG-PAAM and BG-ALG-PAAM hydrogel implants…. ................................................................................................. 115 4.5 Discussion .................................................................................................. 120 4.5.1 Moulding Capability of ALG-PAAM and BG-ALG-PAAM hydrogel implants . 120 4.5.2 Mechanical Strength of ALG-PAAM and BG-ALG-PAAM hydrogel implants 120 4.5.3 Swelling capabilities of ALG-PAAM and BG-ALG-PAAM hydrogel implants . 121 4.5.4 Biomineralisation of ALG-PAAM and BG-ALG-PAAM hydrogel implants ..... 122 4.5.5 Degradation of ALG-PAAM and BG-ALG-PAAM hydrogel implants ............. 122 4.5.6 Morphology of ALG-PAAM and BG-ALG-PAAM hydrogel implants .............. 122 xii 4.5.7 Drug Loading and Drug Release of ALG-PAAM and BG-ALG-PAAM hydrogel implants.. .................................................................................................... 123 4.5.8 In vitro cytocompatibility of ALG-PAAM and BG-ALG-PAAM hydrogel implants…. ................................................................................................. 123 4.6 Concluding Remarks .................................................................................. 123 4.7 References ................................................................................................. 125 CHAPTER.5.THE INFLUENCE OF INCORPORATING BIOGLASS WITHIN A COMPOSITE IMPLANT FOR APPLICATION IN OSTEOSARCOMA .................................................................................... 131 5.1 Introduction ................................................................................................ 131 5.2 Materials: ................................................................................................... 131 5.3 Methods: .................................................................................................... 132 5.3.1 Fabrication of APDL scaffolds: ..................................................................... 132 5.3.2 Fabrication of BGAPDL scaffolds ................................................................. 132 5.3.3 Characterisation studies of the implantable scaffolds. .................................. 132 5.3.3.1 Physicochemical properties of the implantable scaffolds. ........................... 132 5.3.3.2 Thermal properties of the implantable scaffolds ......................................... 132 5.3.3.3 Mechanical analysis of the implantable scaffolds ....................................... 133 5.3.3.4 Degradation of the implantable scaffolds .................................................... 133 5.3.3.5 Swelling analysis of the implantable scaffolds ............................................ 133 5.3.3.6 Morphology of the implantable scaffolds ..................................................... 133 5.3.3.7 Porosity of the implantable scaffolds .......................................................... 134 5.3.3.8 Biomineralisation of the implantable scaffolds ............................................ 134 5.3.3.9 Doxorubicin Loading and release of the implantable scaffolds ................... 134 5.4 Results ....................................................................................................... 135 5.4.1 Physicochemical Characterisation of the implantable scaffolds: ................... 135 5.4.2 Thermal Analysis of the implantable scaffolds.............................................. 137 5.4.3 Mechanical Strength of the implantable scaffolds: ....................................... 138 5.4.4 Degradation of the implantable scaffolds: .................................................... 139 5.4.5 Swelling of the implantable scaffolds: .......................................................... 140 5.4.6 Morphology of the implantable scaffolds ...................................................... 141 5.4.7 Porosity of the implantable scaffolds ............................................................ 143 5.4.8 Biomineralisation of the implantable scaffolds .............................................. 143 xiii 5.4.9 Doxorubicin loading and release of the implantable scaffolds ...................... 145 5.5 Discussion: ................................................................................................. 147 5.5.1 Mechanical Strength of the implantable scaffolds: ....................................... 148 5.5.2 Swelling of the implantable scaffolds: .......................................................... 148 5.5.3 Biomineralisation of the implantable scaffolds: ............................................. 148 5.5.4 Degradation of the implantable scaffolds ..................................................... 149 5.5.5 Morphology and Porosity of the implantable scaffolds: ................................. 149 5.5.6 Drug Loading and Drug Release of the implantable scaffolds: ..................... 149 5.6 Concluding Remarks .................................................................................. 150 5.7 References ................................................................................................. 151 CHAPTER.6.: CONCLUSIONS, RECOMMENDATIONS, AND FUTURE PROSPECTS 155 6.1 Conclusions................................................................................................ 155 6.2 Future prospects ........................................................................................ 156 6.3 References ................................................................................................. 158 Appendix A: International Published Review Paper ................................................. 160 Appendix B: Research output: Oral presentation: BRICS Summit 2021: 04 November 2021… ....................................................................................................... 161 xiv LIST OF ABBREVIATIONS 3D- Three-dimensional 3DP Three-dimensional printing AgNP- Silver Nanoparticles ALG-PAAM- Alginate-polyacrylamide APDL- Alginate-polyacrylamide double lyophilised AuNP- Gold Nanoparticles β-TCP- β-Tricalcium phosphate BG- Bioglass BG-ALG-PAAM- Bioglass- Alginate-polyacrylamide BGAPDL- Bioglass-Alginate-polyacrylamide- double lyophilised BMP- Bone Morphogenic Proteins CAD- Computer-Aided Design CaCl2- Calcium Chloride DMEM- Dulbecco’s Modified Eagle Medium DN- Double Network Hydrogel FBS- Fetal bovine serum FeCl3- Iron (III) Chloride FTIR- Fourier transform infrared spectroscopy MBIS- N,N’-Methylenebisacrylamide Mw- Molecular Weight MRI- Magnetic resonance imaging MSN- Mesoporous silica nanoparticles OS- Osteosarcoma PBS- Phosphate Buffer Solution PCL Polycaprolactone PDLA- Poly (D-lactic acid) PLA- Poly (lactic acid) PLLA- Poly (L-lactic acid) P-S- Penicillin-Streptomycin PVA- Polyvinyl Alcohol SBF- Simulated Body Fluid SEM- Scanning Electron Microscopy TEMED- Tetramethylethylenediamine TEOS- Tetraethyl orthosilicate xv TGA- Thermogravimetric Analysis VEGF- Vascular Endothelial Growth Factor XRD- X-ray diffraction xvi LIST OF TABLES Table 1.1: The types of Osteosarcomas and their relevant treatments (Moore and Luu, 2014) (van der Spuy and Vlok, 2009) .............................................................................. 3 Table 1.2: Chemotherapeutic regimen for osteosarcoma (OS99). D- Doxorubicin, C- Carboplatin, I- Ifosfamide. Figure from (Union for International Cancer Control, 2014) Table licenced under Creative Commons (http://creativecommons.org/licenses/by-nc-sa/2 .............................................. 4 Table 1.3: Chemotherapeutic regimen for osteosarcoma (6 cycle MAP). A- Doxorubicin, P- Cisplatin, M- Methotrexate. (Union for International Cancer Control, 2014).Table licenced under Creative Commons (http://creativecommons.org/licenses/by-nc- sa/2.5/deed ....................................................................................................... 4 Table 2.1: Combinations of polymers and bioceramic composite scaffolds and their properties ....................................................................................................................... 17 Table 2.2: A summary of the different studies comparing different scaffold compositions and their bone regeneration outcomes .................................................................. 31 Table 2.3: Information on strategies to confer targeting properties to nanoparticles ........... 45 Table 2.4: Information on strategies utilized to confer intrinsic stimuli properties to nanoparticles as well as the anti-osteosarcoma drugs and cell lines utilized. .. 47 Table 3.1: Different alginate concentrations used to form the double network hydrogel ...... 75 Table 3.2: The strength capabilities of the double network hydrogel at different FeCl3 concentrations. ............................................................................................... 75 Table 3.3: The different concentrations of sodium bicarbonate and its resultant effects. ..... 79 Table 4.1: Porosity and range of Pore sizes present in ALG-PAAM and BG-ALG-PAAM hydrogel implants ......................................................................................... 113 xvii LIST OF FIGURES Figure 1.1: Schematic illustration of the concept and application of the proposed polymeric implant for the regeneration of bone and treatment of residual osteosarcoma cells. ................................................................................................................. 7 Figure 2.1: Illustration of the hierarchical structure of bone. Reproduced from X. Chen et al. 2018 under Creative Commons Attribution (CC BY) license (CC BY 4.0) (https://creativecommons.org/licenses/by/4.0/) ............................................... 15 Figure 2.2: Schematic depicting the use of NIR laser to trigger photothermal effects in tumor cells. HSP- Heat shock protein. Reproduced from Andersson et al. 2014 under Creative Commons Attribution license (CC BY-NC-SA 3.0)) ((http://creativecommons.org/licenses/by/3.0/) ................................................ 32 Figure 2.3: A depiction of how the magnetic field is generated from the coil to affect tumor cells. A generator is connected to a coil and oscilloscope. The generator provides voltage and frequency to the coil while the oscilloscope displays the frequency and amplitude on a screen. Reproduced from Vegerhof et al. 2016 under Creative Commons Attribution (CC-BY) license (CC BY 4.0) (http://creativecommons.org/licenses/by/4.0/). ................................................ 34 Figure 2.4: The various types of nanocarriers that may be incorporated into a scaffold. The nanocarriers are included in a 3D printed scaffold. The scaffold is then implanted into the critical defect site present in the femur due to osteosarcoma resection. Chemotherapeutics are released from the scaffold to target residual cancer cells while the scaffold regenerates the bone. This figure was created with BioRender.com ............................................................................................... 36 Figure 2.5: Nanoparticles with ligands for targeted delivery bind to cell receptors to be internalized by receptor mediated endocytosis. Reproduced from G. J. Kim and Nie 2005 under Creative Commons Attribution (CC-BY) license (CC BY-NC-ND 3.0) (https://creativecommons.org/licenses/by-nc-nd/3.0/) .............................. 44 Figure 3.1: Illustration of the method used to impart pores to the double network hydrogel. Illustrated using biorender. ............................................................................. 74 Figure 3.2: Swelling profiles of DN hydrogels crosslinked in 0,2 M, 0,3 M and 0,4 M of FeCl3. ....................................................................................................................... 76 Figure 3.3: SEM images of the optimal implant. The first SEM picture on the left indicates a single pore with the diameter of 79.99 ɥm at a magnification of 50 ɥm. The SEM picture on the left displays the implant at a higher magnification of 10 ɥm and a xviii single pore of 19.95 ɥm can be seen. No interconnected pores could be identified. ........................................................................................................ 77 Figure 3.4: Mouldability of hydrogel. A- the needle in the hydrogel after 24 hours. B- after the removal of the capped needle, no hydrogel is messed on the needle cap. B also displays that the hydrogel maintained the void after removal as indicated by the white arrow. C and D are pictures of the hydrogel at different angles. C and D display that the hydrogel maintained the void in the shape of the capped needle. D displays that the hydrogel even maintained the shape of the needle cap where it narrows as indicated by blue arrows. ........................................................... 78 Figure 3.5: SEM images of hydrogel implants with larger pores imparted by sodium bicarbonate. The images were taken at different scales (image on the right is at 100 ɥm while image on the left is at 400 ɥm). 1% w/v Sodium bicarbonate was added to the hydrogel scaffolds cross-linked in 0,3 M FeCl3. .......................... 80 Figure 3.6: SEM image of hydrogel implants with larger pores imparted by sodium bicarbonate at 200 ɥm. % w/v Sodium bicarbonate was added to the hydrogel scaffolds cross- linked in 0,3 M FeCl3. A variety of pore sizes above 100 ɥm in size could be observed. ....................................................................................................... 80 Figure 4.1: Moulding capabilities of ALG-PAAM and BG-ALG-PAAM hydrogel implants The moulding capabilities indicate the potential for customisation per individual patient. A- The various moulds the hydrogel implants were cast in. B- ALG-PAAM hydrogel implants moulding capabilities. C- BG-ALG-PAAM hydrogel implants. ....................................................................................................................... 92 Figure 4.2: FTIR spectra of Pristine Alginate, ALG-PAAM before immersion in FeCl3, ALG- PAAM, BG, BG-ALG-PAAM ........................................................................... 94 Figure 4.3: XRD spectra of Pristine Alginate, ALG-PAAM before immersion in FeCl3, ALG- PAAM, BG, BG-ALG-PAAM ........................................................................... 95 Figure 4.4: TGA thermograms of Pristine Alginate, ALG-PAAM before immersion in FeCl3, ALG-PAAM, BG, BG-ALG-PAAM ................................................................... 96 Figure 4.5: Derivative TGA thermograms of Pristine ALG, ALG-PAAM before CL, ALG-PAAM, BG-ALG-PAAM and BG. ................................................................................. 97 Figure 4.6: XRD Spectra of ALG-PAAM Hydrogel implants after immersion in SBF (Weeks 0- 4). The hydrogel implants before immersion is completely amorphous while crystalline peaks appear with a maximum at around 32° as the weeks go on after immersion in SBF. .......................................................................................... 98 Figure 4.7: XRD spectra of BG-ALG-PAAM after immersion in SBF (weeks 0-4). The hydrogel implants before immersion is completely amorphous while crystalline peaks xix appear with a maximum at around 32° as the weeks go on after immersion in SBF. ............................................................................................................... 99 Figure 4.8: EDS of ALG-PAAM before immersion in SBF. Only the presence of Fe and Cl could be noted. ............................................................................................... 99 Figure 4.9: EDS of ALG-PAAM hydrogel implants after immersion in SBF at week 2 (right) and week 4 (left). The presence of Ca and P was noted at both time points. 100 Figure 4.10: EDS of BG-ALG-PAAM before immersion in SBF. Only the presence of Fe, Cl, Si and P was observed. ................................................................................ 100 Figure 4.11: EDS of BG-ALG-PAAM after immersion in SBF for 2 weeks (right) and 4 weeks (left). The presence of Ca and P was noted at both time points. ................... 100 Figure 4.12: SEM images of ALG-PAAM hydrogel implants after immersion in SBF. Hydroxyapatite crystals can be observed on its surface. ............................... 101 Figure 4.13: SEM of BG-ALG-PAAM after immersion in SBF. Hydroxyapatite crystals can be observed on its surface. ................................................................................ 101 Figure 4.14: Graph depicting the decrease in compressive strength of ALG-PAAM and BG- ALG-PAAM hydrogel implants over time when exposed to SBF. .................. 102 Figure 4.15: Graphs depicting matrix resilience (right) and deformation energy (left) of ALG- PAAM and BG-ALG-PAAM scaffolds after 24 hours and 4 weeks of exposure to SBF. Matrix resilience increased as exposure to SBF increased while deformation energy decreased as exposure to SBF increased. .................... 103 Figure 4.16: Swelling profiles of ALG-PAAM and BG-ALG-PAAM. ALG-PAAM displayed a slightly higher swelling capacity than BG-ALG-PAAM ................................... 104 Figure 4.17: ALG-PAAM shape maintenance over time. It can be seen that ALG-PAAM hydrogel implants can maintain its shape after swelling substantially. .......... 105 Figure 4.18: BG-ALG-PAAM shape maintenance over time. It can be seen that ALG-PAAM hydrogel implants can maintain its shape after swelling substantially. .......... 105 Figure 4.19: Degradation of ALG-PAAM hydrogel implants and BG-ALG-PAAM hydrogel implants over 6 weeks. ................................................................................. 106 Figure 4.20: Morphology at 24 hours of degradation. A- ALG-PAAM. B- BG-ALG-PAAM. No interconnecting pores could be observed. .................................................... 108 Figure 4.21: Morphology at 1 week degradation captured at different scales. A porous structure can be observed for both ALG-PAAM and BG-ALG-PAAM hydrogel implants. A- ALG-PAAM at 100 ɥm. B- ALG-PAAM at 30 ɥm. . C- BG-ALG-PAAM at 100 ɥm. D- BG-ALG-PAAM at 30 ɥm. ...................................................... 109 Figure 4.22: Morphology at 2 weeks degradation captured at different scales. A porous structure can be observed for both ALG-PAAM and BG-ALG-PAAM hydrogel xx implants. A- ALG-PAAM at 100 ɥm. B- ALG-PAAM at 30 ɥm. . C- BG-ALG-PAAM at 100 ɥm. D- BG-ALG-PAAM at 30 ɥm. ...................................................... 110 Figure 4.23: Morphology at 3 weeks degradation captured at different scales. Web like structures can be observed due to degradation implants as indicated by the white arrows. A porous structure can still be observed. A- ALG-PAAM at 100 ɥm. B- ALG-PAAM at 30 ɥm. . C- BG-ALG-PAAM at 100 ɥm. D- BG-ALG-PAAM at 30 ɥm. ............................................................................................................... 111 Figure 4.24: Morphology at 4 weeks degradation captured at different scales. . Web like structures can be observed due to degradation implants as indicated by the white arrows. A porous structure can still be observed A- ALG-PAAM at 100 ɥm. B- ALG-PAAM at 30 ɥm. . C- BG-ALG-PAAM at 100 ɥm. D- BG-ALG-PAAM at 30 ɥm. ............................................................................................................... 112 Figure 4.25: % drug loading of ALG-PAAM and BG-ALG-PAAM hydrogel implants. ALG- PAAM hydrogel implants displayed a slightly greater drug loading capability. ..................................................................................................................... 114 Figure 4.26: Cumulative drug release of Doxorubicin from ALG-PAAM and BG-ALG-PAAM hydrogels ...................................................................................................... 115 Figure 4.27: Cell viability of ALG-PAAM and BG-ALG-PAAM hydrogel implants treated with 3 days and 7 days conditioned media, conditioned media combined with the concentration of DOX released at that time point and Freed Doxorubicin drug. Data is presented as mean ± SD, n=3. * Indicates statistical difference (p < 0.05). ..................................................................................................................... 116 Figure 4.28:Cell adhesion onto ALG-PAAM hydrogel implants. Images show sparse cells. A- image of MG-63 cells on ALG-PAAM hydrogel implants at 200 ɥm. B,C- areas of A containing MG-63 cells at 100 ɥm. ............................................................ 118 Figure 4.29:Cell adhesion of BG-ALG-PAAM hydrogel implants. Images show cell clusters. A- image of MG-63 cells on ALG-PAAM hydrogel implants at 200 ɥm. B,C- areas of A containing MG-63 cells at 100 ɥm ......................................................... 119 Figure 5.1: FTIR spectra of APDL, BGAPDL, ALG-PAAM and BG-ALG-PAAM. The lines running through the spectra indicate the common bonds found throughout all four spectra. ........................................................................................................ 136 Figure 5.2: XRD spectra of APDL, BGAPDL, ALG-PAAM and BG-ALG-PAAM. The spectra indicate a lack of crystalline peaks proving the amorphous nature of the scaffolds. ..................................................................................................................... 136 Figure 5.3: TGA thermograms of APDL, BGAPDL, ALG-PAAM and BG-ALG-PAAM. ...... 137 xxi Figure 5.4: Derivatives of TGA thermograms for APDL,BGAPDL, ALG-PAAM and BG-ALG- PAAM. Derivative thermograms indicate that APDL and BGAPDL degrade at a lower temperature compared to ALG-PAAM and BG-ALG-PAAM ................ 138 Figure 5.5: Compressive strength, matrix resilience and deformation energy of APDL scaffolds and BGAPDL scaffolds. BGAPDL scaffolds have a higher compressive strength, matrix resilience and deformation energy than APDL scaffolds. ..... 139 Figure 5.6: Degradation profile of APDL and BGAPDL scaffolds over 8 weeks. BGAPDL scaffolds had a higher degradation than APDL scaffolds. ............................. 140 Figure 5.7: Swelling of APDL and BGAPDL scaffolds in SBF. APDL had a greater swelling capacity than BGAPDL scaffolds. ................................................................. 141 Figure 5.8: Cross-section of APDL scaffold under SEM. A variety of pore sizes can be observed. ..................................................................................................... 142 Figure 5.9: Cross-section of BGAPDL scaffold under SEM. Smaller pores can be seen in this image. .......................................................................................................... 142 Figure 5.10: FTIR and XRD Spectra of APDL and BGAPDL scaffolds displaying biomineralisation at various time points. A- FTIR spectra of APDL scaffolds. B- FTIR spectra of BGAPDL scaffolds. C- XRD spectra of APDL scaffolds. D- XRD spectra of APDL scaffolds ............................................................................ 143 Figure 5.11: EDX spectra of APDL and BGAPDL scaffolds before and after immersion in SBF. A- APDL scaffolds before immersion in SBF. B- BGAPDL scaffolds before immersion in SBF. C- APDL scaffolds after immersion in SBF. D- BGAPDL scaffolds after immersion in SBF. The EDX spectra display the presence of Ca and P for both APDL and BGAPDL scaffolds after immersion in SBF. ......... 144 Figure 5.12: Hydroxyapatite formation on APDL and BGAPDL scaffolds as seen at 500 nm. A- Hydroxyapatite crystals on the surface of APDL scaffolds that underwent exposure to SBF. B- Hydroxyapatite crystals on the surface of BGAPDL scaffolds that underwent exposure to SBF. ................................................................. 145 Figure 5.13: The doxorubicin loading profile of APDL and BGAPDL scaffolds. APDL scaffolds displayed a slightly higher loading capacity .................................................. 146 Figure 5.14: The doxorubicin release profiles of APDL and BGAPDL scaffolds................ 147 xxii LIST OF EQUATIONS Equation 3.1 Swelling ratio % .............................................................................................. 72 Equation 4.1 Swelling ratio % ............................................................................................. 89 Equation 4.2 Degradation % .............................................................................................. 89 Equation 4.3 % Cell viability .............................................................................................. 91 Equation 5.1 Degradation % ............................................................................................ 133 Equation 5.2 Swelling % .................................................................................................. 133 Equation 5.3 % Porosity .................................................................................................... 134 1 CHAPTER.1. INTRODUCTION AND OVERVIEW OF THE STUDY 1.1 Introduction and background to the study Osteosarcoma is a malignant neoplasm and is characterised by the production of osteoid by tumour cells (Heck and Toy, 2017). The American Cancer Society reports that people between the ages of 10 and 30 are most likely to develop osteosarcoma, and those over 60 make up 10% of those affected by the disease (American Cancer Society, 2020). Osteosarcoma can develop as a result of a number of predisposing factors, including Paget's disease, radiation exposure, chemotherapy, genetics, as well as when foreign bodies are inserted into bone such as orthopaedic implants. The majority of osteosarcomas develop in long bones, around the joint areas (proximal areas of the humerus and tibia, and the distal areas of the femur). The spine and flat bones are less likely to develop osteosarcomas (Reith, 2018). Surgery is a critical component in the treatment of osteosarcoma (Tiwari, 2012). Wide margin resections are standard in surgical treatment of osteosarcoma. A wide margin resection involves removing the tumour along with rim of healthy tissue. Thus an osteotomy occurs, inducing a severe bone defect (Tiwari, 2012). When treating Low-grade osteosarcoma, a wide margin resection of the tumour is the only treatment. Higher grade osteosarcomas, are treated with surgery as well as chemotherapy which me be administered before surgery to mitigate the risk ask of micrometastases (neoadjuvant chemotherapy) or after surgery (adjuvant chemotherapy) (Tiwari, 2012). Table 1.1 describes in detail the types of osteosarcoma and their treatments. Wide margin resections are limb-salvage procedures. A currently used modality to address defects caused by wide margin resections is the use of a prosthetic device (endoprosthesis or megaprosthesis). However, as Osteosarcoma is mainly prevalent in adolescents, their growth poses a constant challenge to the suitability of the fit of the prosthetic devices. Prosthetic enlargement to match the growth of affected adolescents has advanced from intermittent surgeries to non-invasive means (such as using magnetic devices). However, complications, such as loosening and wear and tear of the prosthetic device are frequent and the need for advancement is greatly needed (Tiwari, 2012; Bielack et al., 2016). The utilisation of bone grafts is another current approach to replacing bone. Bone grafts may be autogenous (from the patient’s own body) or homogenous (allografts) (from other humans). Each of these approaches have their own draw backs (autografts cause more pain and morbidity to patient and the quantity available is limited while allografts may cause an antigenic response and require processing that may cause loss of properties). Therefore research has 2 been focused on the use of safer, more cost effective synthetic grafts (Tiwari, 2012; Oryan et al., 2014; Martin and Bettencourt, 2018). Following the surgical resection of the osteosarcoma, adjuvant chemotherapy may be utilised to reduce the chance of tumour micrometastases. Methotrexate, doxorubicin, and cisplatin form part of the most common chemotherapeutic regimen for osteosarcoma. (Jones, 2020). The severe side effects of these chemotherapeutic include cisplatin's permanent ototoxic and nephrotoxic consequences, methotrexate's myelosuppression and mucositis, doxorubicin's cardiotoxicity, and tissue necrosis (Toy and Heck, 2017). As chemotherapeutics are systemically delivered and do not distinguish between healthy, normal cells and tumour cells, these side effects are common. While conventional chemotherapy has improved the survival rate of those patients with high-grade Osteosarcoma, no improvement has been made to the survival rate since the 1980’s, despite various attempts made at better chemotherapy regimens. Therefore, research into novel drug delivery systems is needed (Luetke et al., 2014). To address bone defects induced by surgery, osteo-regenerative platforms may be utilised. Bone regeneration is a complex process as molecular, biochemical, mechanical and cellular aspects have to be accounted for. Therefore, osteo-regenerative platforms, such as scaffolds and hydrogels, should be biocompatible and should have the appropriate porosity, degradability, compositional and mechanical properties(Wang et al., 2020). This project therefore proposes the development of an implant that will utilise localised delivery of chemotherapeutics, thereby bypassing the adverse effects of traditional chemotherapy administered systemically via intravenous infusion. The implant should have mouldable properties to allow for patient customisability. In addition, the implant will be fabricated with biomaterials with an osteo-regenerative potential to address the bone defects caused by osteosarcoma resection. 3 Table 1.1: The types of Osteosarcomas and their relevant treatments (Moore and Luu, 2014)(van der Spuy and Vlok, 2009) Osteosarcoma Type Osteosarcoma subtype Description Treatment Intermedullary OS Conventional OS High grade intermedullary tumour. Mainly affects the metaphyseal region of bone. (May be further subclassified into chondroblastic, fibroblastic or osteoblastic OS depending on the histological profiles.) Wide margin resection and chemotherapy. Telangiectatic OS Affects metaphysis of long bones. One of the most aggressive forms of OS. Treated similarly to conventional OS. Small cell OS One of the rare forms of OS. Mainly affects the metaphyseal region of long bones. And is similar to Ewing’s sarcoma. Wide margin resection, Chemotherapy. Low grade central OS Has painless progression. Usually in older patients. Wide margin resection surgery. This type of OS has a recurrence rate if it is not properly resected. Surface OS Usually treated with surgery alone. This OS sits on the surface of the bone and is not continuous with the medullary Parosteal OS Slow growing surface OS. Can be found on many different bones of appendicular skeleton but most commonly found on the posterior distal femur. Wide margin resection surgery and reconstruction. Periosteal OS Can be found along the diaphysis of long bones, Wide margin resection surgery, 4 canal. It is usually painless (indolent) unless it is a high-grade surface OS. most commonly on the Tibia. High grade surface OS. Likely to erode cortical surface of the bone Treatment is similar to conventional OS. Wide margin resection surgery and chemotherapy. 1.2 Rationale and motivation Treatment of Osteosarcoma in the developing countries, such as South Africa, still lag behind the osteosarcoma treatments made in first world countries. Difficulties in access to healthcare facilities, the lack resources within healthcare facilities (which result in referral to another healthcare facility) as well as delays in access to diagnostic equipment and procedures delay the osteosarcoma diagnosis. This may lead to higher stage osteosarcoma and worse patient prognoses (Lisenda et al., 2017). Thus, when considering advancement in treatment, the South African context must be taken into account. According to the Unioin for International Cancer Control’s 2014 Review of the WHO’s cacncer medicines on the list of Essential medicines (Union for International Cancer Control, 2014), the standard chemotherapy regimens for osteosarcoma include a 6 cycle MAP (Table 1.3) where chemotherapy is provided to the patient for 17 weeks after surgery or 12 cycle OS99 where chemotherapy is provided to the patient for 21 weeks after surgery. (Table 1.2). These chemotherapy regimens include the delivery of doxorubicin by intravenous infusion after surgery. Table 1.2: Chemotherapeutic regimen for osteosarcoma (OS99). D- Doxorubicin, C- Carboplatin, I- Ifosfamide. Figure from (Union for International Cancer Control, 2014) Table licenced under Creative Commons (http://creativecommons.org/licenses/by-nc-sa/2 Table 1.3: Chemotherapeutic regimen for osteosarcoma (6 cycle MAP). A- Doxorubicin, P- Cisplatin, M- Methotrexate. (Union for International Cancer Control, 2014).Table licenced under Creative Commons (http://creativecommons.org/licenses/by-nc-sa/2.5/deed 5 These chemotherapy regimens require multiple visits to the clinic to receive IV infusions of Chemotherapy. Therefore, methods of the implant fabrication need to preferably use minimal resources and equipment. In addition, if the drug is loaded within the implant for localised therapy, it provides the advantage of bypassing IV methods of chemotherapy administration, which does not require patients to make difficult journeys to the clinic Thus, the patient receives chemotherapy, with minimal side effects and healthcare workers are not overburdened. Therefore, a key approach in developing treatments for Osteosarcoma would be to utilise a dual therapy approach, whereby the loading of an anticancer drug into a biocompatible implant would prevent the recurrence of Osteosarcoma by mitigating the risk of residual cancer cells after surgery, while filling the resection site and providing aid to regenerate the bone (Palamà et al., 2017). The implant that fills the defect site should possess the appropriate bone regenerative properties. According to the diamond concept, there are five general therapeutic targets that bone regeneration platforms should aim for. The implant should possess a minimum of three of these five properties (Giannoudis, Einhorn and Marsh, 2007; Giannoudis et al., 2008; Jahan and Tabrizian, 2016): • Osteoconduction, • Growth factors (osteoinduction), • Mechanical properties, • Vascularization (angiogenesis) and, • Osteogenesis (synthesis of new bone). Osteo-regenerative implants synthesised from biomaterials such as bioceramics, have the potential to address bone defects caused by surgical resection of osteosarcoma, due to their osteo-regenerative properties. Implants derived from bioceramics are structurally, compositionally and mechanistically similar to the apatite present in bone tissue (Wang et al., 2020). High mechanical strength materials are important for application in bone tissue engineering applications (Sithole, 2019). Bioglass is osteoconductive and osteoproductive (stimulates growth of new bone) (Rainer et al., 2008). However, despite the many applications in bone tissue engineering, bioglass tends to have weaker mechanical properties when used 6 alone which restricts use of these materials in certain applications (Mabrouk et al., 2014). Therefore, it would be important incorporate bioglass into other polymers. There is a need for the development of patient-customised implants for the individual patient (Howard et al., 2008) This need is emphasised when approaching osteosarcoma, as the population affected are growing adolescents. A method of ensuring customisability of implants would be to ensure that the implant may be mouldable to fit the defect site perfectly. Therefore, in brief, the development of a mouldable, polymeric implant that will utilise localised delivery of chemotherapeutics, thereby bypassing the adverse effects of traditional IV administered chemotherapy and fabricated with biomaterials that can meet the standards set by the diamond concept, is required for application in Osteosarcoma. This concept is illustrated in Figure 1.1. 7 Figure 1.1: Schematic illustration of the concept and application of the proposed polymeric implant for the regeneration of bone and treatment of residual osteosarcoma cells. 1.3 Aims and objectives The aim of this study was to fabricate a polymeric implant for the treatment of residual osteosarcoma that will also possess bone regeneration properties, once implanted at the site of resected bone. 8 Objectives: 1) To formulate a polymeric implant with mouldable properties that will allow for patient customisation. 2) To incorporate bioglass (a bioceramic) into the polymeric implant. 3) To undertake physicochemical, physicomechanical, morphological and thermal characterisation studies on the polymeric implant for determination of key characteristics such as mechanical strength. 4) To assess the polymeric implant’s suitability for localised chemotherapeutic drug delivery. 5) To evaluate the cytocompatibility by evaluating cytotoxicity of the polymeric implant utilising MG-63 cells. 1.4 Potential benefits of undertaking this research • Patients’ quality of life may be improved due to a restoration of limb function without implants or prosthesis that possess the potential for loosening and require adjustment. • Reduced expenditure by patient and decreased health burden due to reduced hospital visits. • Patient specific polymeric implants may be designed by utilising X-ray or CAT scans to design the mould. • The polymeric implant may be used for application in other morbidity types that require bone regeneration. 1.5. Overview of dissertation Chapter One of this dissertation provides a brief background about Osteosarcoma, its current treatment approaches and subsequent disadvantages. It also details the rationale and motivation by highlighting the need for research into bone regeneration platforms in the South African context and explains the aim and objectives of the study. Chapter Two covers the approaches taken to 3D printing scaffolds for application in Bone regeneration. It details the natural and synthetic polymers utilised as well as the bioceramics researched in 3D printing scaffolds for bone tissue engineering and criticizes how applicable the current scaffolds are for application in Osteosarcoma. The need for mouldable scaffolds is also briefly mentioned. Chapter Three discusses the preliminary approaches that were made to establish an appropriate hydrogel implant. The use of two polymers (alginate and polyacrylamide) to form a double network hydrogel implant are explained in this chapter as well the efforts to impart 9 appropriate porosity. Its moulding ability is also briefly illustrated. Experiments conducted in this chapter include mechanical analysis, swelling ratio % and imaging by SEM. Chapter Four of this dissertation covers the comparison of two double network hydrogel implant formulations, one containing bioglass and the other without bioglass. The hydrogels consist of Polyacrylamide (crosslinked with MBis) and Alginate (crosslinked with FeCl3). Various properties of the hydrogel implant such as physicochemical properties, mechanical strength, morphology, mouldable capabilities and biomineralization properties will be discussed in this chapter. Analysis of these properties were undertaken by various characterisation methods such as FTIR, XRD, Texture analysis, TGA and SEM. Drug release of the chemotherapeutic, doxorubicin, from both implantable hydrogels is also covered in this chapter. Chapter four also details the cytocompatibility of the hydrogel implant with cytotoxicity and cell adhesion studies. Chapter Five of this dissertation covers the conversion of the hydrogel implant into a scaffold implant by applying lyophilisation twice. A comparison between scaffold implants with bioglass and scaffold implants without bioglass was made. Various properties of the scaffold implant such as physicochemical properties, mechanical strength, morphology is discussed in this chapter. Analysis of these properties were undertaken by various characterisation methods such as FTIR, XRD, Texture analysis, TGA and SEM. Drug release of doxorubicin, from both implantable scaffolds is also covered in this chapter. Chapter Six provides a summary of the previously obtained results and presents the future prospects and limitations of the hydrogel implants (ALG-PAAM and BG-ALG-PAAM) as well as the scaffold implants (APDL and BGAPDL). 10 1.6 References American Cancer Society (2020) Key Statistics for Osteosarcoma. Available at: https://www.cancer.org/cancer/osteosarcoma/about/key-statistics.html (Accessed: 10 February 2020). Bielack, S. S. et al. (2016) ‘Advances in the management of osteosarcoma’, F1000Research, 5(0), pp. 1–10. doi: 10.12688/f1000research.9465.1. Giannoudis, P. V. et al. (2008) ‘The diamond concept - open questions’, Injury, 39(SUPPL.2), p. S5. doi: 10.1016/S0020-1383(08)70010-X. Giannoudis, P. V., Einhorn, T. A. and Marsh, D. (2007) ‘Fracture healing: The diamond concept’, Injury, 38(S4), pp. S3–S6. 10.1016/s0020-1383(08)70003-2. Heck, R. K. and Toy, P. C. (2017) ‘Malignant Tumors of Bone’, in Campbell’s Operative Orthopaedics. 13th edn. Philadelphia: Elsevier, pp. 945–983. Available at: https://www.clinicalkey.com/#!/content/book/3-s2.0- B9780323374620000276?scrollTo=%23hl0000691. Howard, D. et al. (2008) ‘Tissue engineering: Strategies, stem cells and scaffolds’, Journal of Anatomy, 213(1), pp. 66–72. doi: 10.1111/j.1469-7580.2008.00878.x. Jahan, K. and Tabrizian, M. (2016) ‘Composite biopolymers for bone regeneration enhancement in bony defects’, Biomaterials Science. Royal Society of Chemistry, 4(1), pp. 25–39. doi: 10.1039/c5bm00163c. Jones, R. L. (2020) ‘Malignant Tumors of Bone, Sarcomas, and Other Soft Tissue Neoplasms’, in Goldman-Cecil Medicine. 26th edn. Elsevier, pp. 1341–1344. Available at: https://0-www- clinicalkey-com.innopac.wits.ac.za/#!/content/book/3-s2.0- B9780323532662001922?scrollTo=%23top. Lipson, H. and Kurman, L. (2013) Fabricated: The new world of 3D Printing. John Wiley & Sons, Inc,1st edition 1–5. Lisenda, L. et al. (2017) ‘Osteosarcoma outcomes at a South African tertiary hospital’, South African Medical Journal, 107(9), pp. 754–757. doi: 10.7196/SAMJ.2017.v107i9.11424. Luetke, A. et al. (2014) ‘Osteosarcoma treatment - Where do we stand? A state of the art review’, Cancer Treatment Reviews. Elsevier Ltd, 40(4), pp. 523–532. doi: 10.1016/j.ctrv.2013.11.006. Mabrouk, M. et al. (2014) ‘Effect of ciprofloxacin incorporation in PVA and PVA bioactive glass composite scaffolds’, Ceramics International. Elsevier, 40(3), pp. 4833–4845. doi: 11 10.1016/j.ceramint.2013.09.033. Martin, V. and Bettencourt, A. (2018) ‘Bone regeneration: Biomaterials as local delivery systems with improved osteoinductive properties’, Materials Science and Engineering C. Elsevier B.V., 82, pp. 363–371. doi: 10.1016/j.msec.2017.04.038. Moore, D. D. and Luu, H. H. (2014) ‘Osteosarcoma’, in Peabody, T. D. and Attar, S. (eds) Orthopaedic Oncology. Switzerland: Springer International Publishing, pp. 65–92. doi: DOI: 10.1007/978-3-319-07323-1_4,. Oryan, A. et al. (2014) ‘Bone regenerative medicine: Classic options, novel strategies, and future directions’, Journal of Orthopaedic Surgery and Research., 9(1), pp. 1–27. doi: 10.1186/1749-799X-9-18. Palamà, I. E. et al. (2017) ‘Therapeutic PCL scaffold for reparation of resected osteosarcoma defect’, Scientific Reports, 7(1), pp. 1–12. doi: 10.1038/s41598-017-12824-3. Rainer, A. et al. (2008) ‘Fabrication of bioactive glass-ceramic foams mimicking human bone portions for regenerative medicine’, Acta Biomaterialia, 4(2), pp. 362–369. doi: 10.1016/j.actbio.2007.08.007. Reith, J. D. (2018) ‘Bone and Joints’, in Rosai and Ackerman’s Surgical Pathology. 11th edn. Elsevier, pp. 1740–1809. Available at: https://www.clinicalkey.com/#!/content/book/3-s2.0- B9780323263399000408?scrollTo=%23top. Sithole, M. N. (2019) In situ conjugation-co-fabrication of bioarchetypes employing 3D print processing. University of the Witwatersrand, South Africa. van der Spuy, D. and Vlok, G. (2009) ‘Osteosarcoma: Pathology, staging and management’, SA Orthopaedic Journal, 8(3), pp. 69–78. Tiwari, A. (2012) ‘Current concepts in surgical treatment of osteosarcoma’, Journal of Clinical Orthopaedics and Trauma. Elsevier, 3(1), pp. 4–9. doi: 10.1016/j.jcot.2012.04.004. Toy, P. C. and Heck, R. K. (2017) ‘General Principles of Tumors’, in Campbell’s Operative Orthopaedics. 13th edn. Philadelphia: Elsevier, pp. 829–895. Available at: https://0-www- clinicalkey-com.innopac.wits.ac.za/#!/content/book/3-s2.0- B9780323374620000240?scrollTo=%23top. Union for International Cancer Control (2014) ‘Osteosarcoma: 2014 Review of Cancer Medicines on the WHO List of Essential Medicines’, (3), pp. 1–10. Available at: http://www.who.int/selection_medicines/committees/expert/20/applications/Osteosarcoma.pd f?ua=1. 12 Wang, C. et al. (2020) ‘3D printing of bone tissue engineering scaffolds’, 5, pp. 82–91. doi: 10.1016/j.bioactmat.2020.01.004. 13 CHAPTER.2. LITERATURE REVIEW: THE APPLICATION OF 3D-PRINTING AND NANOTECHNOLOGY FOR THE TARGETED TREATMENT OF OSTEOSARCOMA 2.1 Introduction Osteosarcoma is a malignant neoplasm of which there is osteoid formation by tumor cells (Heck and Toy, 2017). According to the American Cancer Society, osteosarcoma is prevalent in patients between the ages of 10 and 30. Those diagnosed with osteosarcoma over the age of 60, consist of 10% of the population afflicted (The American Cancer Society, 2020). Osteosarcoma may arise from certain predisposing factors such as Paget’s disease, exposure to radiation, chemotherapy, genetics as well as foreign bodies inserted into bone such as orthopaedic implants. The majority of osteosarcomas occur in long bones, close to the joint areas (proximal areas of the humerus and tibia, and the distal areas of the femur). Osteosarcomas are less prevalent in flat bones and the spine (Reith, 2018). Current therapy for osteosarcoma employs the administration of neoadjuvant chemotherapy followed by surgery and adjuvant chemotherapy. Adjuvant chemotherapy is utilized after surgical removal of osteosarcoma to mitigate the risk of tumor micro metastases. The most common chemotherapeutic regimen for osteosarcoma consists of methotrexate, doxorubicin and cisplatin (Chou, Geller and Gorlick, 2008; Luetke et al., 2014). These chemotherapeutic agents vary in severe adverse effects, including irreversible ototoxic and nephrotoxic complications (cisplatin), myelosuppression and mucositis (methotrexate), cardiotoxicity and tissue necrosis (doxorubicin) (Toy and Heck, 2017). These adverse effects are prevalent as chemotherapeutics do not differentiate between healthy, normal cells and tumor cells. Surgery is a critical component in the treatment of osteosarcoma. The current approach to replace bone after surgery is to employ bone grafts. Bone grafts may be autogenous (from the patient’s own body), homogenous (from other humans), xenografts (from other species). Each of these approaches has its own limitations, therefore research has been focused on the use of safer, more cost-effective synthetic grafts (Martin and Bettencourt, 2018). Synthetic osteo-regenerative scaffolds should be biocompatible and have the appropriate porosity, degradability, compositional and mechanical properties to be suitable for bone regeneration as bone regeneration is a complex process as molecular, biochemical, mechanical and cellular aspects have to be considered (C. Wang et al., 2020). 3D printing is an advantageous method to fabricate implantable scaffolds for bone regeneration. 3D printed scaffolds can be precisely designed to mimic bone tissue morphologically (C. Wang et al., 2020) and provides control over scaffold pore shape, size 14 (Ghorbani et al., 2020), and facilitates the incorporation of other functional agents within the scaffold (C. Wang et al., 2020). In addition, one of the strategies to target the delivery of chemotherapeutic compounds in osteosarcoma has been to employ nanocarriers. For example, targeted nanoparticles can increase the bioavailability and stability of chemotherapeutics while reducing the risks of side effects (Raj et al., 2019). Selective and precise release of chemotherapeutic compounds may also be achieved by the employment of stimuli-responsive nanoparticles. In addition, nanocarriers can be designed to release chemotherapeutic drugs according to triggers such as intrinsic stimuli (S. Y. Wang et al., 2020). This review details the 3D printing and nano-enabling of synthetic and natural polymers as well as bioceramics that have been researched as potential candidates for targeted bone regeneration in Osteosarcoma. In particular, magneto- and photo-responsive scaffolds for application in osteosarcoma are discussed as well as a concise incursion into nanoparticles that have been incorporated into scaffolds for targeted bone regeneration. Nanoparticles that have the potential to be incorporated into osteo-mimetic scaffolds for localized adjuvant therapy are also elaborated on. 2.2 Fabrication of 3D-Printed Scaffolds for Enhanced Bone Regeneration Among the techniques used to fabricate 3D printed scaffolds, extrusion printing is most popular for bone regeneration (Martínez-Vázquez et al., 2015; Bendtsen, Quinnell and Wei, 2017; Fahimipour et al., 2017; Pei et al., 2017; H. Kim et al., 2018; Luo et al., 2018; Du et al., 2019; Martin et al., 2019). Other techniques that have been employed include Fused Deposition Modeling (FDM) (Rajzer et al., 2018; Chen, Chen and Wang, 2019; Liu et al., 2020), Binder Jet Printing (Sarkar and Bose, 2019), Melt Electrohydrodynamic 3D Printing (Bai et al., 2020) and Selective Laser Sintering (Feng et al., 2020; Shuai et al., 2020). Bone is a dynamic and complex tissue consisting of a hard matrix. Two components contribute to the matrix of bone- mineral (majority in the form of hydroxyapatite) and matrix proteins (the main protein being collagen type I). Bone consists of two types of cells- the osteoblast family and osteoclasts. The osteoblast family includes osteoblasts (which form bone by depositing mineral). Once surrounded by bony matrix the osteoblasts are referred to as osteocytes (which maintain bone). Osteoclasts resorb (remove) bone tissue. Together, these cells continuously remodel and maintain bone (White, Black and Folkens, 2012). Structurally, human bone tissue consists of cancellous and cortical bone. Cancellous bone is spongy-like with a high porosity at 50-90% and accounts for 20% of the human skeleton. Cortical bone makes up 80% of the weight of the human skeleton with a porosity of 5-10%. Cortical bone has a greater Young’s modulus and compressive strength than cancellous bone (Zhang et al., 2014). 15 Cortical bone possesses the ultimate strength of 30- 211 MPa and an elastic modulus of 16- 20 GPa. Cancellous bone on the other hand has the ultimate strength of 51-193 MPa and an elastic modulus of 4.6-15 GPa (Huang, Wang and Wang, 2014). Figure 2.1 illustrates the hierarchical structure of bone tissue. Figure 2.1: Illustration of the hierarchical structure of bone. Reproduced from X. Chen et al. 2018 under Creative Commons Attribution (CC BY) license (CC BY 4.0) (https://creativecommons.org/licenses/by/4.0/) In general, there are five therapeutic targets that bone regeneration scaffolds aim for; 1) growth factors (osteoinduction), 2) vascularization (angiogenesis), 3) mechanical properties, 4) osteogenesis and 5) osteoconduction. These five targets form part of the “diamond concept” developed by Giannoudis et al., 2007, 2008. Scaffolds for bone regeneration should possess at least three of these five properties. (Giannoudis, Einhorn and Marsh, 2007; Giannoudis et al., 2008; Jahan and Tabrizian, 2016). When a scaffold is referred to as “osteoconductive” it facilitates the ingrowth of mesenchymal stem cells (MSCs), perivascular tissue and capillaries. When a scaffold is referred to as “osteoinductive”, it facilitates the recruitment and differentiation of MSCs into osteoblasts and chondroblasts which leads to new bone formation. Osteoinduction is modulated by growth factors such as bone morphogenetic proteins (BMP) - 2, -4 and -7 and vascular endothelial growth factor (VEGF). Osteogenesis is the synthesis of new bone by viable cells (Khan et al., 2005; Roberts and Rosenbaum, 2012). Several researchers have studied and reviewed the effects of porosity and pore size on angiogenesis, cell behaviour during ossification, mechanical and degradation properties and concluded that scaffold pore size and porosity may influence ossification, angiogenesis as https://creativecommons.org/licenses/by/4.0/ 16 well as mechanical and degradation properties. Scaffolds that are microporous (100-600 ɥm) displayed superior vascularization and integration with the bone tissue of the host. Increased pore size increased bone ingrowth (and therefore osteoconduction). Angiogenesis was supported by triangular, rectangular and elliptic pores while mechanical strength was improved by square pores. Smaller pores, staggered orientation of pores and a gradient porosity provided greater mechanical strength (compressive modulus). Larger pore size was associated with faster degradation rates (Abbasi et al., 2020). Zaharin and co-workers 3D printed titanium alloy scaffolds (Ti6Al4V) with cube and gyroid pore structures. The scaffold’s pore sizes ranged between 300 to 600 ɥm. Interestingly, in this study, pore size was the deciding factor of whether the scaffold displayed similar properties to bone. It was concluded that scaffolds of both pore geometries showed similarity to natural bone properties at a pore size of 300 ɥm (Zaharin et al., 2018). Pore structure and size impact the mechanical strength of scaffolds. Zhao and co-workers researched the effect of honeycomb structured pores on the mechanical strength of 3-D printed polylactic acid and photosensitive resin scaffolds. PLA scaffolds with honeycomb structure displayed a greater compressive modulus. It was also suggested that a smaller pore size be used when designing scaffolds to improve mechanical strength (Zhao et al., 2018). Hence, the geometry and physical characteristics of scaffolds such as curvature, pore size and shape need to be carefully controlled to enhance mechanical strength and cellular responses for effective bone tissue regeneration. Most polymers and bioceramics are therefore used in combination to form composite scaffolds due to individual polymers lacking the required properties for optimal bone tissue regeneration. Table 2.1 provides a list of selected combinations of polymer-ceramic composite scaffolds that have been explored. 17 Table 2.1: Combinations of polymers and bioceramic composite scaffolds and their properties Polymer/ s Bioceramic Drug Properties: Ref Chitosan/PVA Hydroxyapatite BMP-2 BMP enhanced proliferation and attachment of cells Mechanical Strength- Elastic modulus of 91.14 MPa. (Ergul et al., 2019) Alginate/Gelatin Hydroxyapatite - Nanoapatite increased proliferation and osteogenic differentiation. Nanohydroxyapatite increased mechanical strength of scaffolds. (Luo et al., 2018) Alginate - BFP-1 In vitro: Cell viability, migration, proliferation In vivo: accelerated bone regeneration (Heo et al., 2017) Collagen/ decellularized extracellular matrix (dECM)/ Silk Fibroin (SF) - - Collagen/dECM and Collagen/dECM/SF scaffolds displayed better cell proliferation and differentiation compared to pure collagen scaffold. Collagen/dECM/SF had better mechanical strength. (Lee et al., 2018) Gelatin/PVA - - Cell proliferation and differentiation. Mechanical Strength (H. Kim et al., 2018) Silk Fibroin/sodium alginate Hydroxyapatite Bovine Serum Albumin Cell attachment and migration. Increased SF/HA ed to increased cell proliferation. (Huang et al., 2019) 18 PCL β-tricalcium phosphate - β-tricalcium phosphate enhanced proliferation and differentiation. Mechanical strength (Bruyas et al., 2018) Piperazine based- polyurethane-urea - - Osteoconductive Sufficient mechanical strength. (Ma et al., 2019) PLLA/MgO/Halloysite nanotubes - - Cell adhesion, proliferation, migration facilitated. Mechanical Strength (Liu et al., 2020) PVA Biphasic Calcium Phosphate Platelet rich fibrin (PRF) In vitro: scaffolds with PRF promoted greater cell adhesion, proliferation and differentiation. In vivo: scaffolds with PRF stimulated greater bone formation (Song et al., 2018) 2.2.1 Natural polymers used for 3D-printing of scaffolds in bone regeneration 2.2.1.1 3D-printed chitosan scaffolds Chitosan is a natural biopolymer (Ahmed et al., 2015; Saravanan, Leena and Selvamurugan, 2016) that can be 3D-printed with appropriate binders . Chavanne and co-workers (2013) explored various binders such as citric acid, lactic acid and acetic acid on chitosan/hydroxyapatite composites. Due to high wettability, medium viscosity and short solidification time lactic acid (40% wt.) was found to be an optimal binder (Chavanne et al., 2013). Native chitosan poses some limitations for fabricating scaffolds in bone regeneration due to its limited potential to repair bone defects. These limitations include poor antimicrobial activity, quick depolymerization that occurs in vivo, poor water solubility and hemo-incompatibility. Therefore, chemical modifications such as phosphorylation, carboxyalkylation, hydroxylation, quaternization, copolymerization and sulfation may be necessary (Logithkumar et al., 2016) and should be considered when 3D printing chitosan scaffolds for application in bone tissue 19 engineering. Combining chitosan with other polymers (such as alginate and gelatin), ceramics (such as hydroxyapatite) and other materials (such as silicon dioxide) can assist with attaining the desired chemical and biological properties required for bone tissue regeneration (Saravanan, Leena and Selvamurugan, 2016). Caballero and colleagues (2019) studied the relationship between rheology and the composition of chitosan/calcium phosphate inks. It was shown that the higher the concentration of chitosan, the greater the 3D-printability of the ink. The polymer chain entanglement was influenced by chitosan. With higher chitosan concentrations, the ink had more structure (i.e., more viscous), less Newtonian in nature and displayed increased shear thinning behavior. The calcium phosphate morphed from dicalcium phosphate dihydrate into hydroxyapatite that was mineralized in the chitosan scaffold upon printing in basic water/ethanol baths. In addition to the chitosan concentration, the properties of the inks depended on the inorganic to organic ratio. The inorganic to organic ratio influenced the ionic strength and mineral content of the inks. While chain entanglement and mineral content resulted in the ink being less Newtonian, increasing the ionic strength made the ink more Newtonian. The chitosan/calcium phosphate inks were used to fabricate 3D printed scaffolds by robocasting. (Caballero et al., 2019). In another study, chitosan and chitosan/hydroxyapatite hydrogels laden with MC3T3-E1 pre- osteoblast cells were compared with alginate and alginate/hydroxyapatite hydrogels. In terms of cell differentiation and proliferation, chitosan was regarded to be superior compared with alginate. These hydrogels were printed by an extrusion based printer. (Demirtaş, Irmak and Gümüşderelioǧlu, 2017). 2.2.1.2 3D- printed alginate scaffolds Alginate is a biopolymer derived from brown algae (Venkatesan et al., 2015) and may require further purification for application in bone regeneration applications(Torres et al., 2019). Alginates form hydrogels by ionic crosslinking with divalent cations and can be used as bioinks to form 3D-printed scaffolds (Hernández-González, Téllez-Jurado and Rodríguez-Lorenzo, 2020). Typically, alginate is deficient in biological properties that can precipitate bone formation (Park et al., 2018), neither does it have sufficient mechanical strength (Venkatesan et al., 2015). However, alginates have been still used in bone regeneration as they are suitable vehicles for the delivery of peptides such as BFP-1 (Heo et al., 2017). Introducing sulfate groups to alginate in bio-inks for 3D printing has shown to prolong BMP-2 activity (Park et al., 2018). To overcome the lack of mechanical properties, alginate may be used in combination with other natural or synthetic osteo-compatible biopolymers used in bone regeneration (Venkatesan et al., 2015). 20 Bendsten and co-workers (2017) studied various concentrations of 3D printable hydrogels comprising alginate, polyvinyl alcohol (PVA) and hydroxyapatite. Two hydrogels had optimized printing quality. Among the two optimized hydrogels, the hydrogel constituting 2.5% alginate, 0.15% Na2HPO4, 0.20% CaSO4, 2.5% hydroxyapatite and 0.72% NaCl possessed the optimal 3D-printing quality when developed with cell culture media. The presence of PVA and hydroxyapatite within the alginate hydrogel contributed significantly towards the printability, viscosity and cell viability. These hydrogels were fabricated by extrusion printing. (Bendtsen, Quinnell and Wei, 2017). In addition, Luo and colleagues (2017) fabricated 13-93 bioactive glass/alginate scaffolds composed of different mass ratios by extrusion printing. The mass ratios of 13-93 bioactive glass: alginate were 4: 4, 2: 4, 1: 4 and 0: 4. When compared to the pure alginate scaffold, the scaffolds that contained 13-93 bioactive glass had increased apatite mineralization and improved mechanical strength. The presence of bioactive glass within the scaffold also improved scaffold porosity and pore size. Optimal mechanical strength, and the highest proliferation and attachment of rat bone mesenchymal stem cells (rBMSCs) was found in the scaffold consisting of mass ratio 2: 4 (Luo et al., 2017). These are classical examples of combining alginate with biomaterials having superior mechanical strength to render alginate suitable for the fabrication of 3D-printed bone regenerative scaffolds. However, based on the values provided above for cortical and cancellous bone regeneration, further improvements have to be made to enhance the mechanical strength of alginate scaffolds to facilitate implantation at load-bearing sites. 2.2.1.3 3D printed collagen scaffolds Collagen (type I) is secreted by osteoblasts and is the most abundant type of collagen found in the extracellular matrix (ECM) of bone (Boskey, 2003; Ferreira et al., 2012). Collagen is osteoconductive, has weak antigenicity (Ferreira et al., 2012; Zhang et al., 2018) and is the most abundant organic component of bone; therefore it serves as an excellent mineralization template. Collagen also provides a surface for cell adhesion (Zhang et al., 2018). However, native collagen is deficient in mechanical strength And therefore may be combined with other biopolymers to increase the mechanical strength and achieve enhanced cellular activity (Lee et al., 2018). In a study by Montalbano and co-workers (2018), 3D-printed scaffolds fabricated from collagen Type I (with bioactive components such as mesoporous bioactive glass containing strontium) were combined to form a hybrid system. Interestingly, the results of the bioactivity studies displayed that the bioactivity of the constructs may be enhanced due to the acidic groups of the collagen fibers providing increased sites for hydroxyapatite nucleation (Montalbano et al., 2018). 21 Lin et al. (2016) fabricated chemical and solvent-free collagen/hydroxyapatite scaffolds by robocasting. The hydroxyapatite powder used measured <100nm in particle size. Optimal printing parameters were established as rods of 600 ɥm in diameter for moderate mechanical strength and bone regeneration outcomes. Non-printed scaffolds were compared to 3D printed scaffolds. Non-Printed scaffolds were prepared by filling molds with ink utilized for the 3D printed scaffolds. The molds matched the dimensions and shape of the 3D printed scaffolds. The ink filled molds were then lyophilized to form non-printed scaffolds. The results displayed that when compared in vitro, 3D printed scaffolds improved cell proliferation and differentiation. When compared in vivo the 3D printed scaffolds facilitated cell migration, osteogenesis and enhanced repair. However, despite possessing moderate mechanical strength, the scaffolds were still deemed as suitable for application only in bone defects with low load-bearing capacity or cancellous bone (Lin et al., 2016). This may not be suitable for application in post- surgical resection of osteosarcoma of the tibia or femur due to the load-bearing nature of the bone. A collagen-based scaffold by use of indirect 3D printing was developed by Sachlos and co- workers (2006). Nano-sized carbonate substituted hydroxyapatite crystals with dimensions of approximately 180x80x20 nm were precipitated within collagen fibers. This was achieved by enlisting a biomimetic precipitation technique. A calcium chloride solution served as the source of calcium while potassium dihydrogen phosphate served as the phosphate source. These two solutions were separated by a collagen membrane and precipitated within the membrane as carbonate substituted hydroxyapatite crystals. The collagen membranes were air-dried after precipitation was allowed to occur, then shredded into flakes and mixed in a collagen dispersion. This mixture was then cast into 3D printed molds that were fabricated by hot-melt inkjet printing. The mold could facilitate the formation of microchannels that would permit the perfusion of the scaffold. However, appropriate mechanical testing, in vitro cell studies and in vivo experiments were not conducted (Sachlos et al., 2006). Appropriate testing will have to be conducted to prove that this scaffold meets at least 3 of the 5 criteria mentioned in the diamond concept and will have to prove sufficient mechanical strength if scaffolds of this nature is to be considered for application of post-surgical resection of osteosarcoma. 2.2.1.4 3D printed gelatin scaffolds Gelatin is a derivative of collagen synthesized by partial acid (type A) or alkaline (type B) hydrolysis from animal collagen. (Djagny, Wang and Xu, 2001; Hoque et al., 2015). Type A gelatin can be employed as a vehicle for acidic proteins in vivo, while type B has been used for the prolonged release of basic molecules (Echave et al., 2017). To improve the thermal and mechanical properties of gelatin for in vivo applications, crosslinking of gelatin is 22 necessary (Yang et al., 2016). Compared to collagen, gelatin displays lower immunogenicity (Santoro, Tatara and Mikos, 2014; Echave et al., 2017) and has several positive traits such as elasticity and cell-adherence due to the RGD sequences present in its primary structure (Su and Wang, 2015). When gelatin is extracted at a reduced temperature, greater mechanical strength is obtained but not sufficient for application in bone tissue engineering or regeneration (Usta et al., 2003; Kuttappan, Mathew and Nair, 2016). To overcome this, Kim H. and co-workers (2018) fabricated combined gelatin/PVA scaffolds by extrusion printing using different gelatin: PVA ratios. The pure gelatin scaffold displayed exceptional protein and water absorption capabilities and the optimal gelatin: PVA ratio for cell differentiation, cell proliferation and mechanical strength was established to be 5:5 (H. Kim et al., 2018). Hydroxyapatite/gelatin scaffolds were also fabricated by extrusion printing by Martinez-Vazquez and colleagues (2015). In their study, hydroxyapatite was doped with silicone and the average size of the hydroxyapatite-silicone crystals was 35 ± 5 nm. The presence of gelatin within the scaffold resulted in an increase in cell differentiation when compared to pure ceramic scaffolds. The mechanical characteristics of the scaffolds were similar to trabecular bone tissue (Martínez-Vázquez et al., 2015) and thus may not be suitable for application in post-surgical resection of osteosarcoma in load-bearing bones. In another study by Celikkin and co-workers (2019), 3D-printed gelatin methacrylate hydrogel scaffolds containing gold nanoparticles (AuNP) were fabricated by extrusion printing. AuNPs were included to enhance Computed Tomography (CT) imaging. The study also researched the impact of different AuNP sizes and concentrations on scaffold cytocompatibility and mechanical properties. The optimal hydrogel formulation was determined to contain AuNPs of 60 nm in size and 0.16 mM concentration. This formulation was then utilized to fabricate 3D printed scaffolds to assess the behavior of Mesenchymal stem cells. Cell studies indicated that both gelatin methacrylate scaffolds with and without AuNPs successfully facilitated osteogenic differentiation. The scaffolds containing AuNPs also enhanced ɥCT imaging (Celikkin et al., 2019). The inclusion of AuNPs to enhance CT imaging may be of advantage in monitoring the bone regeneration and healing process after scaffold implantation into the resected tumor site. 2.2.1.5 3D printed silk fibroin scaffolds Silk is a proteinaceous biopolymer (Melke et al., 2016; Bhattacharjee et al., 2017; Ma, Wang and Dai, 2018) commercially available from two families of silk worms. Silk fibroin from the tropical tasar silkworm possesses the RGD peptide sequence that facilitates superior cell adhesion (Datta, Ghosh and Kundu, 2001; Bhattacharjee et al., 2017). In bone regeneration silk protein is advantageous in that it possesses mechanical strength and biodegradability. 23 Silk fibroin systems have slow degradation while maintaining their load-bearing capacity (Ma, Wang and Dai, 2018). Mulberry silk in particular has a Young’s Modulus of 12.4–17.9 GPa. (Pérez‐Rigueiro et al., 2001; Melke et al., 2016). However, these mechanical characteristics cannot be compared to silk fibroin processed into scaffolds as their mechanical characteristics will differ depending on the process parameters used to form the scaffold (e.g. matrix stiffness, processing techniques and composition) (Melke et al., 2016). A study by Huang and co-workers (2019) researched combined 3D printed silk fibroin/hydroxyapatite scaffolds. Silk fibroin/hydroxyapatite nanocomposites of <100 nm in width were prepared by co-precipitation. The nanocomposites were combined with sodium alginate to form a bio-ink and were 3D printed by extrusion. The mechanical strength of the 3D printed silk fibroin/hydroxyapatite scaffolds demonstrated that they were suitable for trabecular bone applications. However, when compared to hydroxyapatite/sodium alginate scaffolds, scaffolds containing increased amounts of silk fibroin/hydroxyapatite nanocomposites displayed poorer mechanical strength. The scaffolds with higher silk fibroin/hydroxyapatite facilitated greater cell proliferation and osteogenic differentiation. (Huang et al., 2019). In a study employing pure collagen, collagen/decellularized ECM and collagen/dCEM/silk fibroin scaffolds, the 3D printed scaffolds with silk fibroin displayed the most potential for bone regeneration due to superior mechanical properties, increased cell viability and increased preosteoblast cell deposition of calcium (Lee et al., 2018). However, the mechanical strength of these scaffolds may not be suitable for application in post-surgical resection of osteosarcoma at load-bearing sites. 2.2.2 Synthetic polymers used for 3D-printing of scaffolds in bone regeneration 2.2.2.1 3D printed polycaprolactone scaffolds Polycaprolactone (PCL) is a semi-crystalline polymer that is suitable in bone tissue engineering due to its prolonged biodegradation rate (2-3years)(Dwivedi et al., 2020). Although not osteoconductive, PCL may be modified to obtain osteoconductivity and osteoinductivity, for example, by incorporation of growth factors (Mantila Roosa et al., 2010). 3D printed PCL scaffolds are available as bioresorbable implants used in craniofacial surgery(Prasadh and Wong 2018). The mechanical properties and osteoinductivity of PCL can be improved by the addition of β-tricalcium phosphate. Bruyas et al. (2018) observed that the concentration of β-tricalcium phosphate influenced the mechanical properties of the scaffolds fabricated by FDM. PCL scaffolds containing 0, 20, 40, and 60 wt.% β-tricalcium phosphate displayed a Young’s Modulus of 264, 355, 495, and 1140 MPa, respectively. Furthermore, by adjusting the β-tricalcium phosphate concentration, the biodegradation ratio can be optimized 24 (Bruyas et al., 2018). The mechanical strength and osteoconductivity of these scaffolds make it a potential candidate for application in post-surgical resection of osteosarcoma, however, at least one other criteria of the diamond concept has to first be observed. Heo and co-workers (2019) developed 3D printed PCL scaffolds coated in fish bone extract. The scaffolds were first fabricated by extrusion of melted PCL pellets then soaked in 1 wt% and 3 wt% fish bone extract solutions. Native PCL scaffolds, as well as scaffolds coated with fish bone extract, exhibited similar mechanical properties suitable for bone regeneration. Scaffolds coated in fishbone extract had increased cell proliferation, calcium and phosphorous deposition and osteoblast differentiation (Heo et al., 2019). Kim J-Y and colleagues (2018) researched the fabrication of 3D printed PCL scaffolds incorporated with β-tricalcium phosphate and bone-derived decellularized ECM (bone dECM) derived from porcine. The PCL and PCL-β-tricalcium phosphate scaffolds were first fabricated by extrusion printing at 120°C and then immersed in bone dECM solution followed by incubation and lyophilization. In vitro studies conducted with the 3D printed scaffolds coated in bone dECM demonstrated excellent cell seeding, proliferation and differentiation. In vivo studies demonstrated that scaffolds decorated with bone dECM displayed bone tissue growth within the scaffold. 3D printed scaffolds without bone dECM only had bone tissue growth on the edges of the scaffolds whereas those with bone dECM displayed greater mineralization and osteoid formation. The scaffolds also mimicked the mechanical properties of human cancellous bone (J. Y. Kim et al., 2018). In another study, 3D printed PCL scaffolds were fabricated by FDM into different geometries namely, honeycomb, gyroid and mesh structures. These 3D printed scaffolds were then loaded with hydrogels formulated from alginate and gelatin and the hydrogel retention capabilities of the different scaffold structures were researched. The gyroid structured 3D printed PCL scaffold retained the most hydrogel and was selected for further research. Apatite formation increased within the hydrogel, whereas smaller apatite formation was noted on PCL surfaces, which reduced over time due to dissolution. The PCL-gel scaffold displayed cytocompatibility, adhesion and viability of cells (Hernandez, Kumar and Joddar, 2017). 2.2.2.2 3D printed polyurethane scaffolds Polyurethanes are biopolymers that comprise hard segments formed by the reaction between a chain extender and a diisocyanate; and the soft segments are formed from olygodiols and diisocyanates (Marzec et al., 2017). The properties of polyurethanes are influenced by its hard-soft segment ratio. One study indicated that as the ratio of hard segments increased, the hydrophilicity of the polyurethane surface increased as well as cell proliferation of human 25 bone-derived cells. However, an increase in hard segments of polyurethane decreased the osteogenic potential (Bil et al., 2009). The properties of polyurethanes can be tailored by manipulation of chemical composition and synthesis technology and parameters. Polyurethanes are non-toxic, promote calcification in vivo, possess mechanical and physicochemical flexibility and are biocompatible. Their properties can range from thermoplastic to thermosetting and can be hydrophilic or hydrophobic. Polyurethanes implanted in vivo have supported bone regeneration. Polyurethanes can serve as bone void fillers, shape memory scaffolds and drug carriers (Marzec et al., 2017). Ma et al. (2019) showed that piperazine can regulate the osteogenesis of osteoblasts in a dose-dependent manner, and may enhance the osteoconductivity of 3D printed piperazine- based polyurethane-urea scaffolds. These scaffolds were 3D printed by extrusion printing. The optimal concentration of piperazine was determined to be ∼0.5 mM (Ma et al., 2019). Wang and co-workers (2018) fabricated biodegradable shape memory polyurethane 3D printed scaffolds by low temperature fused deposition modeling. The soft segments of the