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 http://theodoregray.com/periodictable/Samples/022.46/s12s.JPG

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) http://picasaweb.google.com/askdoctorbone/PicturesForMediaUse?authkey=Gv1sRgCJfMmqzvhuSEvAE&feat=directlink#5381276157897710626 http://picasaweb.google.com/askdoctorbone/PicturesForMediaUse?authkey=Gv1sRgCJfMmqzvhuSEvAE&feat=directlink#5381276163683870338

Printable Titanium scaffold Design software

Applications of Titanium Scaffold Ti Scaffold Orthopedic surgery Spinal surgery Joint replacement surgery Dental Implants Cranio-facial reconstruction http://www.patriotdentalcenter.com/shared/images/implant-animation.gif http://www.rsabiomedical.com/umrsa/?PHPSESSID=c889f29db5ea1e16dadd402169fa4080 – hips http://www.patriotdentalcenter.com/shared/images/implant-animation.gif - 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