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 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 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 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) Printable Titanium scaffold
Design software Applications of Titanium Scaffold
Ti Scaffold Orthopedic surgery Spinal surgery Joint replacement surgery Dental Implants Cranio-facial reconstruction hips - implant Why use Fibroblastic Derivatives of Human Embryonic Stem Cells 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 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 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 Differentiation from hESC - Animation 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 Differences in Morphologies between PH9 and L929
PH9 cells at 20x magnification L929 cells at 20x magnification Materials & Methods L929 cells PH9 cells Cytotoxicity test of Titanium Scaffold by Direct Contact Method Results Results: Cell Morphology (at 20x mag.)
Positive control Negative control Titanium scaffold Comparing Sensitivity of PH9 & L929 in MTT Assay
Percentage of viable cells Cytotoxicity of Titanium Scaffold on L929 and PH9
Biocompatibility of titanium Cytotoxicity of Titanium Scaffold on L929 and PH9
Biocompatibility of titanium Discussion Biocompatibility of 3D Printed Porous Titanium Scaffold
Almost no cytotoxic effect Stable oxide layer Increased corrosion resistance 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 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 Conclusions Use of 3D Printed Porous Titanium Scaffold
Future applications Dental implants Cranio-facial reconstructions Orthopedics Use of hESCs in Cytotoxicity Screening
More representative Reliable biological platform More sensitive cellular response Alternative to animal models Acknowledgements A/Prof Yeo Jin Fei A/Prof Cao Tong Lu Kai
NUS Faculty of Dentistry