Cytotoxicity Screening of 3D-Printed Porous Titanium Scaffold using

Fibroblastats derived from Human Embryonic Stem Cells

Presenter: Tan Shao YongGroup Members: Ang Chui Noy Michelle

Lai Hiu fong SarahLim Li Zhen

Quek San Oon ShaunWoo Sing Yi Joanne

Yee Ruixiang

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

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)

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

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)

Applications of Titanium Scaffold

• Dental implants• Orthopedic surgery

– Spinal surgery– Joint replacement surgery– Other orthopedic surgery

• Cranio-facial reconstructionhttp://www.patriotdentalcenter.com/shared/images/implant-animation.gif

Why use Human Embryonic Stem Cells and their

Fibroblastic Derivatives

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-10993-5) of biomedical devices and materials

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)

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

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)

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

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

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

Materials & Methods

Sterilization of Titanium Scaffold

• Washing under double distilled water

• Autoclaving @ 121oC (20mins)

• Drying @ 37oC in an oven until use

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

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.

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)

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

Differentiation from hESC - Animation

Cytotoxicity test of Titanium 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

L929 cells PH 9 cells

37°C, 5%CO2

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.

Cytotoxicity test of Titanium Scaffold by Direct Contact Method

Cytotoxicity test of Titanium 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)

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.

Results

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

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 mag L929 cells at 20x mag

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 mag L929 cells at 20x mag

Cell Morphology of L929

• With Titanium 3D-printed scaffold– Yielded similar results as compared to the

negative control

L929 cells at 4x mag L929 cells at 20x mag

Cell Morphology of PH9

• With negative control– PH9 cells retained their spindle-shaped

morphology resembling normal healthy human fibroblasts

PH9 cells at 20x magPH9 cells at 4x mag

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

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 mag PH9 cells at 20x mag

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

Comparing Sensitivity of PH9 & L929 in MTT Assay

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10

30

50

70

90

110

IC50=0.00008708

log [concentration] of phenol

perc

enta

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cells

(L92

9)

-6 -5 -4 -3 -2-10

10

30

50

70

90

110

IC50=0.00001648

log [concentration] of phenolpe

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tage

of v

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H9)

Hence, fibroblasts derived from the hESC line are more sensitive to cytotoxic stimulus than L929.

Cytotoxicity of Titanium Scaffold on L929

Negative control Titanium Scaffold Positive Control0%

20%

40%

60%

80%

100%

120%

140%

Perce

ntage

of vi

able

cells

(L92

9)

Cytotoxicity of Titanium Scaffold on PH9

Negative control Titanium Scaffold Positive Control0%

20%

40%

60%

80%

100%

120%

140%

Pe

rce

nta

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of

viab

le c

ells

(P

H9)

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

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

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

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

Analysis & Discussion

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

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

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

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

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

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