1. 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

2. 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

3. 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

4. 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)

5. Printable Titanium scaffold
Design software

6. Applications of Titanium Scaffold
Ti Scaffold
Orthopedic surgery
Spinal surgery
Joint replacement surgery
Dental Implants
Cranio-facial reconstruction
– hips
- implant

7. Why use Fibroblastic Derivatives of Human Embryonic Stem Cells

8. 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

9. 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

10. 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

11. Differentiation from hESC - Animation

12. 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

13. Differences in Morphologies between PH9 and L929
PH9 cells at 20x magnification
L929 cells at 20x magnification

14. Materials & Methods

15. L929 cells
PH9 cells

16. Cytotoxicity test of Titanium Scaffold by Direct Contact Method

17. Results

18. Results: Cell Morphology (at 20x mag.)
Positive control
Negative control
Titanium scaffold

19. Comparing Sensitivity of PH9 & L929 in MTT Assay
Percentage of viable cells

20. Cytotoxicity of Titanium Scaffold on L929 and PH9
Biocompatibility of titanium

21. Cytotoxicity of Titanium Scaffold on L929 and PH9
Biocompatibility of titanium

22. Discussion

23. Biocompatibility of 3D Printed Porous Titanium Scaffold
Almost no cytotoxic effect
Stable oxide layer
Increased corrosion resistance

24. 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

25. 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

26. Conclusions

27. Use of 3D Printed Porous Titanium Scaffold
Future applications
Dental implants
Cranio-facial reconstructions
Orthopedics

28. Use of hESCs in Cytotoxicity Screening
More representative
Reliable biological platform
More sensitive cellular response
Alternative to animal models

29. Acknowledgements A/Prof Yeo Jin Fei A/Prof Cao Tong Lu Kai
NUS Faculty of Dentistry