Objectives

1. Cytotoxicity Screening of 3D-Printed Porous Titanium Scaffold using Fibroblastats derived from Human Embryonic Stem Cells

2. Objectives • To evaluate the cytotoxicity of a prototype 3D-printed titanium scaffold on • L929 mouse fibroblasts • PH9 human fibroblasts derived from embryonic stem cells • To suggest a future use of PH9 cells as a standardised platform for in-vitro cytotoxicty testing

3. Titanium • Widely used for production of dental / orthopedic implants • Inert • Biocompatible • Resistant and durable • Good mechanical strength • Easily prepared in many shapes and textures without affecting biocompatibility (Vasconcellos, et al., 2008)

4. Titanium • Limited ability of conventional Ti to bond to bone and a higher stiffness compared to bone can result in loosening of implants • Problem tackled with porous Ti scaffold

5. Porous Titanium Scaffold • Allows bone tissues to grow in it • Enhanced osseointegration • Improved implant-bone bond • Relatively lower elastic moduli(Cachinho, et al., 2008) • Prevents bone resorption and decrease stress shielding (Lefebvrem, et al., 2008)

6. Applications of Ti Scaffolds • Dental implants • Orthopedic surgery • Spinal surgery • Joint replacement surgery • Other orthopedic surgery • Cranio-facial reconstruction

7. Why use Human Embryonic Stem Cells and their Fibroblastic Derivatives

8. L929 Cell lines • Immortalised cell lines of human lung fibroblasts over primary cultures explanted directly from living tissues • Recommended by current ISO protocol for cytotoxicity screening (ISO-10993-5) of biomedical devices and materials

9. L929 Cell Lines • Cancerous/ tumourous origin • Highly accustomized to invitro culture conditions after countless passages • Contains chromosomal and genetic aberrations that render it immortal • Not representative of how the cell behaves in vivo (Hay, 1996, Phelps et al., 1996)

10. L929 Cell Lines • Immortalized cell lines that originate from cancer/tumour and primary explants of discarded human tissue (Cowan et al., 2004; Reubinoff et al., 2000; Thomson et al., 1998)

11. L929 Cell Lines • Much less interbatch variability compared to primary explanted cells • This would translate to more reproducible results in cytotoxicity

12. Differentiated fibroblastic progenies of hESC • hESCs are self-renewable pluripotent cells harvested from inner cell mass of blastocyst • Genetically and karyotopically normal (Cai et al., 2004; Cowan et al., 2004; Reubinoff et al., 2000; Thomson et al., 1998) • Not tainted by pathological origin • More representative of how a cell would behave in vivo (normal physiology)

13. Differentiated fibroblastic progenies of hESC • Ready availability of several established hESC lines • Virtually inexhuastible reservoirs of differentiated somatic progenies (Cao, et al., 2008) • Potential to generate derivatives from all 3 germ layers (Alder, et al., 2008) • Readily available source of human cells

14. Differentiated fibroblastic progenies of hESC • Karyotopic stability • Able to replicate indefinitely and still express high levels of telomerase (Amit, et al., 2000) • Less interbatch variability • Better reproducibility of cytotoxicity test results

15. Differentiated fibroblastic progenies of hESC • Cytotoxic response of differentiated hESC fibroblastic progenies (PH9) to mitomycin C was more sensitive than L929 (Cao et al, 2008) • PCR data showed that pluripotency gene markers (Oct-4, Nanog, and Sox-2) were downregulated by passage 5 of random spontaneous differentiation, • Making pH9 representative of normal somatic cell physiology in vivo

16. Materials&Methods

17. Sterilisation of Ti Scaffolds • Washing under double distilled water • Autoclaving @ 121oC (20mins) • Drying @ 37oC in an oven until use

18. Preparation of Reference Materials • Negative Control • Agarose gel cylinders of same dimension as Ti scaffolds • 1.5% (w/v) agarose melted at 120°C for 20 min

19. Preparation of Reference Materials • Positive Control • Addition of an ultra-pure equilibrated phenol stock solution to the liquid-form agarose when the temperature of agarose dropped to and maintained at 60°C • Phenol-agarose solution poured into a sterile 96-well multidish, allowed to solidify at room temperature for 1 hour • Agarose gel cylinders then harvested from the 96-well multidish by aseptic technique.

20. Differentiation from hESC • H9 hESCs (WiCell, Wisconsin, USA) were scraped down with 1mg/ml collagenase IV (GIBCO) and plated on 0.1% gelatin pre-coated 75cm2 flask • Differentiation media - of DMEM, 1mM L-glutamine and 10% fetal bovine serum (FBS; Hyclone, UT, USA) • H9 hESCs were kept differentiating for around 3 weeks at first passage and then subsequently sub-cultured for another 3 passages until the fibroblastic morphology became pronounce and homogenous

21. Cytotoxicity test of Ti Scaffold by Direct Contact Method • L-929 seeded at 5×104 cell/cm2 in a 6-well plate and incubated overnight for 12 hours at 37°C, 5%CO2 • PH9 cells, were also seeded at 2×104 cell/cm2 into a similar 6-well plate and incubated under the same conditions

22. Cytotoxicity test of Ti Scaffold by Direct Contact Method • After cells reach 80% confluency, either the sterilized Titanium scaffold, the negative control cylinder or the positive control cylinder was added into the centre of the well using sterile forceps • The two six-well plates were then further incubated for another period of 48 hours with 1ml of fresh media to observe cellular response to the foreign object.

23. Cytotoxicity test of Ti Scaffold by Direct Contact Method • At end of incubation, Ti scaffolds and control cylinders were removed • Cell viability quantitatively analyzed with CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS) kit • 200µl of MTS stock solution added to the 1ml media in both sets of cell cultures (L-929 and PH9) • Colorimetric analysis was subsequently performed by reading 490nm absorbance with an Infinite 200 microplate reader (Tecan Trading AG, Switzerland)

24. Cytotoxicity test of Ti Scaffold by Direct Contact Method • Data processed with Prism software version 5.01 (GraphPad Inc, USA) • Optical density readouts from control groups were used to plot the standard curve of phenol-induced cytotoxicity • Curve fitting performed with a non-linear regression model • Cytotoxicity of Titanium scaffold reported by percentage cell viability. • The cytotoxic level of scaffold also converted to equivalent dosage of phenol.

25. Results

26. Differences in Morphologies between PH9 and L929 • PH9 cells typically larger than L929 cells • Human cells are larger than murine cells • PH9 resemble the typical human fibroblast cells, with its more pronounced spindle shape morphology seen at higher magnification

27. Cell Morphology of L929 • With negative control • 90% confluency on a very dense cell monolayer • At higher magnification (20x), cell morphology clearly seen; cells appear viable

28. Cell Morphology of L929 • With positive control • Marked decreased cell density in the cell monolayer • Cell morphology has also changed by the loss of its typical fibroblastic spindle shape

29. Cell Morphology of L929 • With Titanium 3D-printed scaffold • Yielded similar results as compared to the negative control

30. Cell Morphology of PH9 • With negative control • PH9 cells retained their spindle-shaped morphology resembling normal healthy human fibroblasts

31. Cell Morphology of PH9 • With Titanium scaffolds • Yielded no significant changes in cell density and morphology

32. Cell Morphology of PH9 • With positive control • Displayed marked decrease in cell density • Decrease being more significant than that seen for L929 • Cell rounding and lack of typical spindle-cell morphology indicates a decrease in cell viability and metabolism

33. Comparing Sensitivity of PH9 & L929 in MTT Assay • Colorimetric readings reported the viability of L929 and PH9 cells by measuring mitochondrial activity of the cells • Dose-response curves of the viability of L929 and PH9 were constructed against increasing concentrations of phenol using GraphPad prism

34. Comparing Sensitivity of PH9 & L929 in MTT Assay Hence, fibroblasts derived from the hESC line are more sensitive to cytotoxic stimulus than L929

35. Cytotoxicity of Ti Scaffold on L929 • (To insert bar chart)

36. Cytotoxicity of Ti Scaffold on PH9 • (To insert bar chart)

37. Statistical Analysis • A series of t-tests comparing the cytotoxicity of the Titanium scaffold against the positive and negative controls when cultured in L929 cells and PH9 cells

38. Statistical Analysis • No significant difference in L929 cell viability between the negative control and Titanium scaffold treatment • Hence L929 cell viability was significantly higher with titanium scaffold treatment than with positive control treatment

39. Statistical Analysis • No significant difference in PH9 cell viability between negative control and titanium scaffold treatment • PH9 cell viability was significantly higher with titanium scaffold treatment than with positive control treatment

40. Statistical Analysis • Concluded that the Titanium scaffold is relatively biocompatible and non-cytotoxic • Comparing the cytotoxicity of the Titanium scaffold on L929 against that on PH9 cells • No significant difference between the cytotoxic effect of titanium on the L929 or PH9 cell lines

41. Analysis& Discussion

42. Biocompatibility of Titanium • Biocompatibility - ability of a material to perform with an appropriate host response in a specific application • Favourablebiocompatibility response of Ti possibly due to excellent corrosion resistance • existence of a few nanometers thick native oxide film

43. Biocompatibility of Titanium • Results demonstrate Ti exerts almost no cytotoxic effect on both L929 and PH9 cells • Cell viability at 98.9% and 99.9% respectively • T-tests conclude that the Titanium scaffold is relatively biocompatible and non-cytotoxic • No statistically significant difference in cytotoxicity of Ti scaffold on the 2 different cell lines

44. Comparing L929 & PH9 • Fibroblastic progenies derived from the hESC line are more sensitive to cytotoxic stimulus than L929 • Results comparable to a previous cytotoxicity study (Cao, et al., 2008) • Postulated explanation • L929 had disruptions in its cell cycle control due to genetic mutations, not unlike those found in cancerous cells

45. Comparing L929 & PH9 • Our findings demonstrated that the PH9 cell line can be a more reliable cell type to test for the cytotoxicity of materials • Titanium, a widely accepted biocompatible material, was used to compare the effects on PH9 and L929 • Results showed no significant difference • Proved that PH9 is reliable in that it did not produce false positive results

46. Comparing L929 & PH9 • Other factors in support of using hESC cell lines for cytotoxicity screening purposes • more representative of the behavior of somatic cells in vivo • reliable medium with which to test the cytotoxicity of drugs • more accurate cellular responses upon drug or chemical challenge • availability of hESC technology for in vitro studies makes it imperative to push the boundaries from animal models

47. Conclusions • Fibroblasts derived from hESC line is more sensitive to cytotoxic stimuli as compared to the ISO recommended L929 • 3D-printed Ti scaffolds non-cytotoxic to both the standard L929as well as the more sensitive hESC line.

48. Conclusions • hESC-derived fibroblasts, being genetically healthy human cells • Better representatives of normal human physiology • Hold potential to become the standardized platform for in vitro cytotoxicity test