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

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

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)

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 Ti Scaffolds Dental implants

Orthopedic surgery Spinal surgery Joint replacement surgery Other orthopedic surgery

Cranio-facial reconstruction

Why use Human Embryonic Stem Cells and their Fibroblastic

Derivatives

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

L929 Cell Lines

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

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)

L929 Cell Lines

Much less interbatch variability compared to primary explanted cells

This would translate to more reproducible results in cytotoxicity

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)

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

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

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

Materials&

Methods

Sterilisation of Ti Scaffolds Washing under double distilled water

Autoclaving @ 121oC (20mins)

Drying @ 37oC in an oven until use

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

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.

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

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

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.

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)

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.

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

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

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

Cell Morphology of L929

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

the negative control

Cell Morphology of PH9

With negative control PH9 cells retained their spindle-shaped

morphology resembling normal healthy human fibroblasts

Cell Morphology of PH9

With Titanium scaffolds Yielded no significant changes in cell

density and morphology

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

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

-6 -5 -4 -3 -2

10

30

50

70

90

110

IC50=0.00008708

log [concentration] of phenol

perc

enta

ge o

f via

ble

cells

(L92

9)

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

10

30

50

70

90

110

IC50=0.00001648

log [concentration] of phenolpe

rcen

tage

of v

iabl

e ce

lls (P

H9)

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

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

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

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 as the more sensitive hESC line.

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