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Cytotoxicity Screening of 3D-Printed Porous Titanium Scaffold using Fibroblasts derived from Human Embryonic Stem Cells Presenter: Lai Hiu Fong Sarah Group Members: Ang Chui Noy Michelle Lim Li Zhen Quek San Oon Shaun Tan Shao Yong Woo Sing Yi Joanne Yee Ruixiang
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Objectives To evaluate the cytotoxicity of a prototype 3D-printed titanium scaffold on L929 mouse fibroblasts PH9 derived from hESCs To validate the future use of PH9 cells as a standardized platform for in-vitro cytotoxicity testing
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Properties of Titanium
Inert Biocompatible Good mechanical strength Can be prepared in many shapes and textures (Vasconcellos, et al., 2008) Limitation: Higher stiffness compared to bone
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Porous Titanium Scaffold
Allows bone tissues to grow within it Enhanced osseointegration Improved implant-bone bond Relatively lower elastic moduli (Cachinho, et al., 2008) Prevents bone resorption and decreases stress shielding (Lefebvrem, et al., 2008)
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Printable Titanium scaffold
Design software
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Applications of Titanium Scaffold
Ti Scaffold Orthopedic surgery Spinal surgery Joint replacement surgery Dental Implants Cranio-facial reconstruction – hips - implant
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Why use Fibroblastic Derivatives of Human Embryonic Stem Cells
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L929 Cell Lines - Introduction
Immortalised cell lines of murine lung fibroblasts Recommended by current ISO protocol for cytotoxicity screening More reproducible cytotoxicity response Less interbatch variability
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L929 Cell Lines - Limitations
Not representative of how the human tissues behaves in vivo (Hay, 1996, Phelps et al., 1996) Contains chromosomal and genetic abberations
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Human embryonic stem cells
Self-renewable Karyotypically and genetically normal (Cao et al., 2004; Cowan et al., 2004; Reubinoff et al., 2000; Thomson et al., 1998) Potential derivatives from all 3 germ layers (Alder, et al., 2008) Not tainted by pathological origin Represents normal human physiology
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Differentiation from hESC - Animation
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Differentiated Fibroblastic Progenies of hESC - Advantages
Readily available source Inexhaustible reservoir (Cao, et al., 2008) Karyotypic stability Less interbatch variability Better reproducibility of cytotoxicity response
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Differences in Morphologies between PH9 and L929
PH9 cells at 20x magnification L929 cells at 20x magnification
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Materials & Methods
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L929 cells PH9 cells
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Cytotoxicity test of Titanium Scaffold by Direct Contact Method
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Results
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Results: Cell Morphology (at 20x mag.)
Positive control Negative control Titanium scaffold
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Comparing Sensitivity of PH9 & L929 in MTT Assay
Percentage of viable cells
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Cytotoxicity of Titanium Scaffold on L929 and PH9
Biocompatibility of titanium
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Cytotoxicity of Titanium Scaffold on L929 and PH9
Biocompatibility of titanium
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Discussion
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Biocompatibility of 3D Printed Porous Titanium Scaffold
Almost no cytotoxic effect Stable oxide layer Increased corrosion resistance
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Comparing L929 & PH9 PH9 more sensitive to cytotoxic stimuli than L929
Comparable to a previous cytotoxicity study (Cao, et al., 2008) L929 has disruptions in its cell cycle control
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PH9: A Potential Platform For Cytotoxicity Testing
Good reliability Using 3D titanium scaffold as a test material PH9 and L929 results showed no significant difference No false positive results
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Conclusions
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Use of 3D Printed Porous Titanium Scaffold
Future applications Dental implants Cranio-facial reconstructions Orthopedics
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Use of hESCs in Cytotoxicity Screening
More representative Reliable biological platform More sensitive cellular response Alternative to animal models
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Acknowledgements A/Prof Yeo Jin Fei A/Prof Cao Tong Lu Kai
NUS Faculty of Dentistry
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