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Transcript of To evaluate the cytotoxicity of a prototype 3D-printed titanium scaffold on L929 mouse fibroblasts ...
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