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Cytotoxicity Screening of 3D-Printed Porous Titanium Scaffold using Fibroblastats derived from Human Embryonic Stem Cells Presenter: Tan Shao Yong Group.

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Presentation on theme: "Cytotoxicity Screening of 3D-Printed Porous Titanium Scaffold using Fibroblastats derived from Human Embryonic Stem Cells Presenter: Tan Shao Yong Group."— Presentation transcript:

1 Cytotoxicity Screening of 3D-Printed Porous Titanium Scaffold using Fibroblastats derived from Human Embryonic Stem Cells Presenter: Tan Shao Yong Group Members: Ang Chui Noy Michelle Lai Hiu fong Sarah Lim Li Zhen Quek San Oon Shaun Woo Sing Yi Joanne Yee Ruixiang

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 Properties of Titanium Inert Biocompatible Resistant and durable Good mechanical strength Easily prepared in many shapes and textures without affecting biocompatibility (Vasconcellos, et al., 2008)

4 Limitations of 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 Titanium Scaffold Dental implants Orthopedic surgery – Spinal surgery – Joint replacement surgery – Other orthopedic surgery Cranio-facial reconstruction animation.gif

7 Why use Human Embryonic Stem Cells and their Fibroblastic Derivatives

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

9 L929 Cell Lines - Limitations Cancerous/ tumourous origin Highly accustomized to in vitro 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 - Uses Immortalized cell lines that originate from murine (mouse) lung fibroblasts cancer/tumour and primary explants of discarded human tissue (Cowan et al., 2004; Reubinoff et al., 2000; Thomson et al., 1998) Much less interbatch variability compared to primary explanted cells This would translate to more reproducible results in cytotoxicity

11 Differentiated Fibroblastic Progenies of hESC - Introduction 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)

12 Differentiated Fibroblastic Progenies of hESC - Advantages 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

13 Differentiated Fibroblastic Progenies of hESC - Advantages 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

14 Differentiated Fibroblastic Progenies of hESC - Uses 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

15 Materials & Methods

16 Sterilization of Titanium Scaffold Washing under double distilled water 121 o C (20mins) 37 o C in an oven until use

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

18 Preparation of Reference Material 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.

19 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 75cm 2 flask Differentiation media - of DMEM, 1mM L- glutamine and 10% fetal bovine serum (FBS; Hyclone, UT, USA)

20 Differentiation from hESC 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 Differentiation from hESC - Animation

22 Cytotoxicity test of Titanium Scaffold by Direct Contact Method L-929 seeded at 5×10 4 cell/cm 2 in a 6-well plate and incubated overnight for 12 hours at 37°C, 5%CO 2 PH9 cells, were also seeded at 2×10 4 cell/cm 2 into a similar 6-well plate and incubated under the same conditions

23 L929 cellsPH 9 cells

24 37°C, 5%CO 2

25

26 Cytotoxicity test of Titanium 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.

27

28 Cytotoxicity test of Titanium Scaffold by Direct Contact Method

29 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)

30 Cytotoxicity test of Titanium 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.

31 Results

32 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 PH9 cells at 20x mag L929 cells at 20x mag

33 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 L929 cells at 4x magL929 cells at 20x mag

34 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 L929 cells at 4x magL929 cells at 20x mag

35 Cell Morphology of L929 With Titanium 3D-printed scaffold – Yielded similar results as compared to the negative control L929 cells at 4x magL929 cells at 20x mag

36 Cell Morphology of PH9 With negative control – PH9 cells retained their spindle-shaped morphology resembling normal healthy human fibroblasts PH9 cells at 20x mag PH9 cells at 4x mag

37 Cell Morphology of PH9 With Titanium scaffolds – Yielded no significant changes in cell density and morphology PH9 cells at 4x mag PH9 cells at 20x mag

38 Cell Morphology of PH9 With positive control – Displayed marked decrease in cell density more significant than that seen for L929 – Cell rounding and lack of typical spindle-cell morphology indicates a decrease in cell viability and metabolism PH9 cells at 4x magPH9 cells at 20x mag

39 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

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

41 Cytotoxicity of Titanium Scaffold on L929

42 Cytotoxicity of Titanium Scaffold on PH9

43 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

44 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

45 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

46 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

47 Analysis & Discussion

48 Biocompatibility of Titanium Biocompatibility - ability of a material to perform with an appropriate host response in a specific application Favourable biocompatibility response of Ti possibly due to excellent corrosion resistance – existence of a few nanometers thick native oxide film

49 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

50 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

51 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

52 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

53 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 L929 as well 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 as the more sensitive hESC line


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