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Effect of mechanical loading
on osteogenesis of human
embryonic stem cell-derived
mesenchymal progenitors
within collagen microspheres
This item was submitted to Loughborough University's Institutional Repository
by the/an author.
Citation:
SHARIATZADEH, M. ... et al, 2018. Effect of mechanical loading
on osteogenesis of human embryonic stem cell-derived mesenchymal progenitors
within collagen microspheres. Journal of Cell Science & Therapy, 9 (3), 1000282.
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Please cite the published version.
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
Open Access
Research Article
Journal of Cell
Science & Therapy
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ISSN: 2157-7013
Shariatzadeh et al., J Cell Sci Ther 2018, 9:3
DOI: 10.4172/2157-7013.1000282
Keywords: Stem cell; Osteogenic dierentiation; Mechanical
stimulation; Collagen microspheres; Regenerative medicine
Introduction
Many cell types including osteoblasts, chondrocytes, broblasts
and endothelial cells are load sensitive and are subjected to daily
mechanical loading in vivo. Dense connective tissues like tendon and
ligament are stretched frequently through muscle contraction caused
by movement whereas bone is under dynamic loading to resist and
adapt to the experienced forces by maintaining homoeostasis through
tissue remodelling. Most of the forces applied in vivo are dynamic and
cyclic which means oen the tissue is under loading and resting cycles.
For example, femur and tibia undergo cyclic compression and tensile
forces during locomotion. erefore, it is possible that cells respond
more to cyclic loading as opposed to constant loading. Constant loading
may increase the risk of the cells being overloaded and becoming
unresponsive to the applied load [1-3]. A key area of research in tissue
engineering is concerned with nding the answers to how mechanical
loading transfers to the cells, how cells sense mechanical forces and
how and when cells respond to the applied external stimuli. Both 2D
and 3D cultures have been used to apply mechanical loading onto
cells [2], although 2D experiments such as gelatine coated plastic and
glass are somewhat limited since they do not accurately mimic the
complex 3D in vivo architecture. erefore, these surfaces do not full
the necessary requirements for culture and regeneration of functional
tissues. us, 3D in vitro models may provide more physiologically
relevant environments for mechanotransduction studies [4].
Studies have shown that cell response in 2D cultures in response to
dynamic stimulation is predominantly due to the deformation of the
substrate with additional minor uid ow eects, since the minimal
movement of uid ow has subsequent minimal eect on the cell [5-
7]. In contrast, cell response to load in 3D environments is related to
both the mechanical stimulation initiated within the system and the
nutrient transport mechanism generated by uid movement through
*Corresponding author: Shariatzadeh M, INSIGNEO Institute for In-silico
Medicine, Department of Mechanical Engineering, University of Shefeld, UK, Tel:
+44 (0)1509564826; E-mail: m.m.shariatzadeh@lboro.ac.uk
Received: May 21, 2018; Accepted: June 11, 2018; Published: June 18, 2018
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of
Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-Derived
Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282.
doi: 10.4172/2157-7013.1000282
Copyright: © 2018 Shariatzadeh M, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited.
Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem
Cell-Derived Mesenchymal Progenitors within Collagen Microspheres
Shariatzadeh M1*, Baldit A1,2, Perrault CM1 and Lacroix D1
1INSIGNEO-Institute for In-silico Medicine, Department of Mechanical Engineering, University of Shefeld, UK
2Université Lorraine - ENIM, CNRS, Arts et Métiers Paris Tech, LEM3, F-57000 Metz, France
Abstract
Mechanical forces and 3D topological environment can be used to control differentiation of mesenchymal stem
cells. However, mesenchymal stem cell fate determined by the effect of physical and mechanical cues is not yet
fully understood. Understanding how mechanical cues in the microenvironment orchestrate stem cell differentiation
provides valuable insight that can be used to improve current techniques in cell therapy. This study investigates the
osteogenic effect of mechanical stimulations on soft cellular microspheres loaded with human embryonic stem cell-
derived mesenchymal progenitors (hES-MPs) when subjected to dynamic loading and in the absence of chemical
stimulation. Microspheres were produced by gelation of bovine collagen type I with 1000 to 2000 hES-MP cells
seeded per droplet. Four loading conditions were studied: (1) 10% constant strain was applied by a Bose biodynamic
bioreactor for 15 min/day or 40 min/day for 5 or 10 days respectively; (2) 10% adjusted strain was applied (subtraction
of polydimethylsiloxane (PDMS) plastic elongation from global strain) using Bose biodynamic bioreactor for the same
4 duration/conditions as in the constant strain protocol. The results indicate that applying mechanical stimulation to
hES-MPs/collagen microspheres induced osteogenic differentiation of cells when the loading protocol was adjusted.
Alkaline phosphatase activity of samples in the adjusted loading protocol increased signicantly on day 14 whilst, the
deposited minerals, matrix reorganisation and alignment of collagen bres enhanced from day 21 post encapsulation
onward. Application of cyclic loading to 3D culture of hES-MP cells can be used as a model to regulate mechano-
stimulation and linage differentiation in vitro.
the scaold [8]. Culturing cells on/in 3D environments provides a
more realistic and physiologically-relevant model for studying load
driven biochemical responses in cells since they more closely mimic in
vivo conditions [9].
Many studies have investigated the role of mechanical stimulation
in controlling cell fate, including mechanical conditioning of
mesenchymal stem cells (MSCs) to direct MSC behaviour and
dierentiation for tissue engineering applications. Delaine-Smith et
al. reported that tensile loading favours osteogenic cell dierentiation
through initiation of a more brous matrix whilst, compression
loading encourages generation of a glycosaminoglycan (GAG) rich
matrix which facilitates chondrogenesis [10]. Other studies have
demonstrated the eect of longitudinal forces in up-regulating early
alkaline phosphatase (ALP) activity levels and mineralisation markers
both in the presence and absence of osteogenic media [11,12]. To
date there are limited studies on the eect of cyclic loading on the
osteogenesis of stem cells.
e aim of this study was to investigate the eects of cyclic
mechanical-induced osteogenic dierentiation and long-term
proliferation of progenitor cells and assess the cellular mechanisms
involved in the mechanotransduction and osteogenesis of embryonic
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 2 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
stem cell-derived mesenchymal progenitors (hES-MPs). e main
purpose was to design a polydimethylsiloxane (PDMS)-made loading
chamber and develop a loading protocol to apply short bouts of
mechanical loading, predominantly tensile and compression, on hES-
MPs seeded collagen microspheres. Another goal was to investigate
the eect of mechanical stimulation on osteogenic dierentiation of
cells through quantication of ALP activity and deposited minerals
levels in hES-MPs seeded collagen microspheres. In addition, cellular
matrix production and remodelling along with alignment of collagen
bres were evaluated to conrm load driven dierentiation of cells.
A previously established applied loading regime [10,13] was used to
subject cells to both indirect dynamic compression and tensile forces
through a PDMS loading chamber.
Material and Methods
All reagents are from Sigma-Aldrich UK unless otherwise stated.
hES-MPs culture conditions
A hES-MP cell line (Cellartis, Sweden) were cultured on 0.1% (wt/
vol) porcine gelatine type A (Sigma, UK) coated surfaces. Expansion
and proliferation medium contained 4 nm broblast growth factor-
basic recombinant human (FGF-β) (Life technologies, USA; as
recommended by manufacturer) added to αMEM (Lonza, UK), 10%
(vol/vol) fetal bovine serum (FBS, Life technologies, UK), 1% (vol/vol)
penicillin and streptomycin (Sigma, UK) and 1% (vol/vol) L-glutamine
(Sigma, UK). Cells were incubated at 37°C in 5% CO2. e medium (12
ml) was replenished every 2 days.
Collagen gelation procedure and cell seeding
Bovine collagen type I (5 mg/ ml) solution in 0.02 N acetic acid (BD
Biosciences, UK) was gelated following the manufacturers protocol.
Briey, collagen I was neutralized using 1 N NaOH and 10X phosphate
buered saline (PBS, Sigma, UK) and diluted to nal concentrations
of 2 mg/ ml and 3 mg/ml on ice. e cells were suspended throughout
the collagen solution at a density of 5 × 105 cells/ml. e volume
of each substance and the number of cells required per drop was
calculated according to the manufacturer’s protocol (BD Biosciences,
UK). Collagen microspheres of 2 and 5 μl were dispensed in a 90 mm
diameter Petri dish covered with UV-irradiated paralm and were
incubated for 45 min at 37°C and 5% CO2 to induce gelation. Gelated
collagen/hES-MPs microspheres were then gently ushed with medium
to remove them from the paralm and were transferred to a separate
Petri dish. Cell-seeded microspheres were maintained free-oating and
the medium was replaced with fresh complete media every 48 h.
Design of a polydimethylsiloxane (PDMS) made loading
chamber
Hypodermic needles (Sigma, UK) with dierent outer diameter’s
ranging from 0.6 mm to 0.9 mm were used as a template to create a
compression chamber. PDMS (PDMS SILGARD® 184) with the ratio
of 10:1 of base to initiator was mixed, poured on top of the template
and was baked at 60°C for 1.5 h. e needle was removed from the set
PDMS and the PDMS mould was cut into a rectangular shape (length:
44 mm, width: 28 mm, thickness: 6 mm). e same procedure was
followed using a 3 ml syringe as a template to make a cylindrical
chamber.
Characterisation of PDMS mechanical properties
Displacement and strain on the PDMS sample surface was
calculated using a high resolution digital imaging technique and digital
image correlation (DIC) soware Kelkins (University of Montpellier)
[14]. e Poisson’s ratio was obtained computing the transverse-
longitudinal strain ratio.
Finite element model
Using Abaqus (Dassault Systèmes, France) a nite element analysis
was performed within an elastic framework to estimate the link between
the macroscopic load applied on the PDMS and the local loading
applied on collagen microspheres. A frictionless surface contact law
has been used to manage the relative motion between PDMS and beads
bodies
Experimental boundary conditions were applied, i.e., bottom side
xed and top side displaced up to 10% of strain. anks to the geometry
of the PDMS chamber only a quarter was simulated taking into account
symmetry boundary conditions.
Cyclic loading protocol
Samples of 10-12 hES-MPs seeded collagen microspheres were
transferred to the loading chamber on day 6 post encapsulation (pc).
e loading chamber and embedded cell seeded microspheres were
subjected to dynamic mechanical stimulation of 10% strain, 1 Hz
for 15 min/day to 40 min/day for 5 and 10 days respectively using
ElectroForce® 5100 BioDynamic® test instrument (ElectroForce System
Group, BOSE, USA) and soware WinTest 7 as illustrated in Figure 1.
Assessment of collagen bre alignment
Alignment of collagen bres in the control and loaded samples
was compared using second harmonic generation (SHG) confocal
microscopy (type and brand) on day 21 (pc).
Evaluation of collagen microsphere surface micro structure
Cell attachment, elongation and new extracellular matrix (ECM)
collagen production was examined by scanning electron microscopy
(SEM) (Philips XL-20 SEM) on days 6, 16 and 28 pc. e microstructure
of the collagen beads and the reorganisation of collagen bres was
assessed in the loaded samples versus free oating controls on day 21 pc.
DNA count and cell viability
Cell viability and total DNA measurement of hES-MP cells of
free oating control versus loaded samples were determined using
uorescent Quant-iT dsDNA High-Sensitivity Assay Kit (Invitrogen,
UK) following the manufacturer’s protocol at 24 h, day 6, 21 and 28 pc.
Seeded cells were lysed with a cocktail of cell digestion buer consisting
of: 10% of cell assay buer that contained 1.5 m Tris-HCl, 1 mm ZnCl2
and MgCl2 in ddH2O mixed with 1% Triton X-100 and incubated at
4°C overnight. Cell lysate samples were then stored at 80°C for 10 min
before being incubated at 37°C for 45 min (two freeze thaw cycles)
and centrifuged at 10,000 rpm for 5 min. e resulting supernatant
was used for DNA quantication. 200 μl of diluted cell lysate reagent
(provided in the kit) and 20 μl of sample volumes were added to the
wells of a 96-well microplate, the uorescence signal intensity was
detected and recorded at λex 485 nm; λem 520 nm using Tecan innite
F200 PRO micro plate reader (Labtech, UK).
Measurement of total alkaline phosphatase
ALP activity of loaded hES-MPs against free oating controls was
measured at 24 h, days 6, 14, 21 and 28 pc using an ALP uorometric
assay kit (Abcam, UK). Cells were transferred to a micro centrifuge
tube, washed with PBS and lysed with the same lysing cocktail as
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 3 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
1 (a)
1 (b)
1 (c)
Figure 1: Schematic diagram of the loading protocols, durations and mechanical conditioning of hES-MPs seeded collagen microspheres within 28 days post
en-capsulation. (a): Samples were subjected to either 15 min/day to 40 min/day, 10% strain, 1 Hz for 5 and 10 days. The DNA content, ALP activity level and
mineralisation of 12 samples were analysed for each time points. (b): Schematic of the applied constant strain on PDMS made loading chamber which resulted in
inconsistent load transfer to the microspheres. (c): Schematic of the applied adjusted strain which shows subtracting the calculated PDMS plastic elongation from
global strain may result in more consistent and uniform load transfer to the microspheres.
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 4 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
previously described for the DNA count. ALP activity was assessed
at λex 360 nm; λem 450 nm using a uorescence Nano drop-3300 2.8.0
(Labtech, UK) and normalised to total DNA content.
Quantication of minerals deposition
Quantication of deposited calcium, phosphorous and zinc was
measured by 1% Alizarin red S staining at 24 h, days 6, 14, 21 and 28
pc via Inductively Coupled Emission Spectrometry (ICP-ES), (Perkin
Elmer ELAN II DRC) according to the instrument’s protocol. Samples
were digested in 5% (w/v) perchloric acid and were diluted 10 times
prior to analysis. Samples then were mixed with 1% (w/v) nitric acid
with ratio of 1:1 in Eppendorf tubes. Mineralisation was also visualised
on 10 µm thick xed samples using uorescent microscope, Nikon Ti-
E, (Nikon, UK) by 1% Alizarin red staining of loaded and control cross
section samples over same period of time.
Statistical analysis
All experiments were performed three times in triplicate (n=9).
Cell viability at 24 h, days 6, 21 and 28 pc, ALP and mineralisation
comparison as well as statistical dierences between free oating,
unloaded controls and loaded samples were completed using one-way
ANOVA followed by Sidak’s or Tukey’s multiple comparisons test
using GraphPad Prism 6.
Results
Design of a PDMS made compression chamber
A loading chamber was made from PDMS and the central channel
diameter was optimised based on the measurement of average collagen
microsphere diameter (n=20) on day 6 pc as shown in Figure 2.
Based on the computational modelling, the chamber’s diameter was
calculated and made in such a way that it was approximately 50 μm-
80 μm bigger than the diameter of cell seeded collagen beads on day
6 pc [15]. Principal strain on the PDMS chamber was calculated by
computational modelling that revealed high stress appeared close to
the sample peripheries while local strain was quite constant and close
to 10% equivalent to the macroscopic load at the centre and along half
sample height (Figure 3). Also, the computational modelling indicated
that maximum of 5% strain would be transferred to the microspheres
inside the loading chamber. is is also consistent with the fact that the
chamber needs to be stretched with a 5% strain to reach the contact with
the beads. In addition, the contact sensitivity is related to the geometry
of PDMS sample as with the rectangular cross section, it appears that
the contact is rst reached in the direction corresponding to the shortest
geometry length compared with the square sample (Figure 4).
Characterisation of PDMS mechanical properties
e displacement/strain on the surface of the PDMS was calculated
using DIC soware Kelkins [14] and an average for the imposed 10%
global strain, 7.5% strain along the vertical direction of solicitation and
3.75% strain along the horizontal transverse direction was measured as
demonstrated in Figure 5. Poisson’s ratio was then calculated as 0.5 as
expected from polymer behaviour. Young’s modulus of the polymer
was calculated as 1000 kPa aer relaxation and plastic deformation was
calculated as 0.7 mm aer applying 10% strain and using cycle’s blocks
on the Bose biodynamic machine.
Comparison of cell proliferation, ALP activity and
mineralisation between adjusted and constant loading
protocols
DNA pico green assay results showed that cells remained viable
and proliferated steadily well over 28 days pc in all conditions as shown
in Figure 6. In the constant loading regime, free oating controls had
a signicantly higher total DNA content than all other experimental
groups on days 14, 21 and 28 pc (p<0.0001). In addition, both 15 and
40 min/day loaded samples of 10 days regime showed slightly higher
cell number than 5 days regime conditions by the end of 28 days
experiment, but no statistical signicance was observed between the
loaded conditions of 5 and 10 days.
e results of ALP level revealed no substantial change in enzyme
activity in the loaded samples compared to unloaded and free-oating
controls following the application of constant loading over 28 days pc
(Figure 6). ALP activity in free oating controls increased to the highest
level on day 14 pc and reduced by half on days 21 and 28 pc (0.00186,
0.00121 and 0.0011 respectively). However, the enzyme activity did not
show any increase on days 14, 21 and 28 pc in the other experimental
groups. e 10 day loaded samples for both 15 min/day and 40 min/day
presented marginally more ALP activity level than 5 day loading regime
on day 21 (0.00074, 0.00082, 0.00071 and 0.00058 respectively). Still,
free oating controls reported higher enzyme activity level particularly
in comparison with 5 day loaded samples of 15 min/day and 40 min/
day and unloaded controls (p<0.001, 0.1 and 0.0001 respectively) on
day 21 pc.
ALP activity level in the adjusted loading protocol increased
substantially from day 6 to day 14 pc when the activity level reached
a peak, followed by a drop to nearly half the level on day 21, which
then remained stable up to day 28 pc. On day 14, the ALP level in 5
and 10 days loading regimes of 40 min/day loaded samples surged
signicantly up to 40% (p<0.0001) compared to both free oating
controls and 15 min/day samples. In addition, all 10 days loading
cycles samples showed slightly higher ALP activity levels compared to
other conditions on day 28 pc, but no statistical signicance was found
between these experimental groups on day 28 pc. Deposited calcium
was detected from day 21 and showed signicant increase on day 28
pc as shown in Figure 7. Loaded samples in adjusted loading protocol
presented the highest calcium level compared to other groups on day
28 pc which was nearly twice as much as detected deposited calcium
in free oating (27,000 μg/l, p<0.0001). In addition, the calcium level
of unloaded 10 days controls on day 28 pc was reported to be around
25% higher than free oating controls and 75% greater than its values
in loaded samples that were subjected to the constant loading protocol.
Loaded samples under the constant loading protocol presented
lowest calcium level among other experimental groups from day 21
onward. e calcium quantication results were further conrmed
by Alizarin red staining of loaded and control samples cross sections.
Both loaded samples under adjusted loading protocol and free-oating
controls presented more intense red colour in comparison with 10
days controls on day 28 pc (Figure 8). Phosphorous concentration of
adjusted protocol loaded samples increased up to 80% from day 21 to
28 pc and its concentration was reported 2.5 times higher than that of
loaded samples in constant protocol on day 28 pc (p<0.01) as indicated
in Figure 7. Also, free oating controls showed higher phosphorous
level compared to unloaded controls and loaded samples of constant
protocol (30%, p<0.01 and 67% respectively) on day 28 pc.
Zinc levels of loaded samples in the adjusted protocol was four
times higher than its value in free oating controls and nearly 10 times
greater than of that in loaded samples of the constant protocol on day
21 pc. Zinc concentration of loaded samples in the adjusted protocol
declined by 25% on day 28 pc whereas, all the other groups presented
higher level of zinc compared to the earlier time points.
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 5 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
Figure 2: PDMS made compression chamber, ID: 0.9 mm.
3(a) 3(b)
Figure 3: Beads strain eld corresponding to 10% of strain applied macroscopically on the PDMS sample (Abaqus). (a): Rectangular cross section, (b): Squared cross
section. Microspheres inside the loading chamber would sense the maximal strain of around 5% by applying 5% tensile strain to the loading chamber to reach the
contact with beads. Contact sensitivity is related the to the geometry of PDMS sample as with the rectangular cross section, it appears that the contact is rst reached
in the direction corresponding to the shortest geometry length compared with the square sample.
Assessment of collagen bre alignment
e reorientation and aligned organisation of collagen bres in
loaded samples of adjusted strain protocol was substantially more
evident compared to the control free oating samples and unloaded
controls which presented more brous structure of collagen bres with
no preferential orientation at all depths (Figure 9). e most intense
SHG signal was recorded at depth 10 μm to 20 μm with less coverage
area at 20 μm. e signal was very weak at 30 μm which indicated a very
Figure 4: PDMS strain eld corresponding to 10% of strain applied macroscopically (Abaqus). (a): Rectangular cross section. (b): Squared cross section. Strong stress
appears close the sample ends but locally in the centre and along half sample height the strain is quite constant and close to 10% equivalent to the macroscopic load.
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 6 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
5(a)5(b)
5(
c
)
Figure 5: Measurement of transversal and longitudinal strains on the PDMS surface when subjected to 10% global strain using Kelkins software. (a): Transverse strain,
(b): PDMS mesh, and (c): Longitudinal strain.
6(a) 6(b)
6(c) 6(d)
Figure 6: Evaluation of cell viability and ALP activity of loaded samples of adjusted loading protocol and constant protocol versus free oating controls. (a) and (c): Total
DNA content of loaded samples 15 min/day and 40 min/day for 5 and 10 days, compared with controls in the constant and adjusted protocol, (b) and (d): ALP activity
of constant and adjusted loading protocols were also compared over 28 days pc. *indicates statistical signicance, (****p<0.0001; ***p<0.001; **p<0.01; *p<0.1). Data
presented as mean ± SD n=9.
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 7 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
low presence of collagen bres at this point. Evaluation of collagen bre
organisation was assessed using SEM microscopy. e surface structure
of collagen microsphere, cellular elongation and ECM reorganisation
of collagen bres were further assessed by SEM microscopy (Figure 10).
All cell seeded microspheres were covered with cells and matrix at both
time points. Cells and matrix on both time points and conditions did
not present any preferential direction of orientation and no cellular or
collagen bre alignment was visible in loaded samples of the adjusted
loading protocol versus free oating controls. In addition, deposition
of mineral particles was apparent in both loaded samples and free-
oating controls on day 28 pc in comparison with day 6 pc. Higher
accumulation and larger sizes of deposited mineral particles were
7(a) 7(b)
7(c)
Figure 7: Evaluation of deposited extracellular matrix calcium, phosphorous and zinc in loaded samples of 40 min/day in 10 days loading regime of constant and
adjusted protocols versus controls over 28 days pc. (a): Calcium deposition, (b): Phosphorous deposition, (c): Zinc deposition. *indicates statistical signicance,
(****p<0.0001; ***p<0.001; **p<0.01). Data is mean ± SD n=9.
Figure 8: Alizarin red staining of 10 μm thick seeded hES-MPs collagen microspheres on day 28 pc in 10 days cyclic loading regime. ffc: Free oating control; cls:
Constant loading sample; uc; Unloaded control; als: Adjusted loading sample. All images taken by light microscope, 20x.
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 8 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
Figure 9: SHG microscopic images of reorientation and alignment of collagen bres on day 21 pc in 10 days cyclic loading experiment. SHG signal intensity was higher
in loaded samples compared to the controls. Ffc; free oating control. Cls; constant loading sample. Uc; unloaded control. Als; adjusted loading sample. Collagen
bres of loaded sample in adjusted protocol visibly reorganised and aligned in comparison with more random organisation of collagen bre in other conditions. Settings
for images taken at 50 μm to 75 μm from the surface of all samples were optimised to visualise collagen. All images were taken by confocal microscope, EC 40x/1.3
plan-Neouar oil DIC.
Figure 10: SEM microscopy of hES-MPs seeded collagen microspheres surface on day 28 pc. FFC: Free oating control; CLS: Constant loading sample; UC;
Unloaded control; ALS: Adjusted loading sample. Accumulation of mineral deposits is more evident in both loaded samples and free-oating controls compared to free
oating controls on day 6 pc.
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 9 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
visualised in the loaded samples than free oating controls. Collagen
bres of microspheres in both samples and controls were visibly
thinner compared to the produced extracellular matrix bres on day 28
pc and showed more distinct organisation of bres as opposed to free
oating controls on day 6 pc (Figure 10).
Discussion
Mechanical stimulation can enhance osteogenic dierentiation
of hES-MPs seeded collagen microspheres. Our results support other
studies that presented that mechanical and physical cues in the stem cell
microenvironment controls cell behaviour and promotes osteogenesis
of so 3D progenitor cells by supporting mineral deposition and
collagen bre realignment [16,17].
e adjusted strain regime was superior in stimulating the
osteogenesis of hES-MPs compared with the constant strain protocol.
e unloaded controls supported up-regulation of ALP activity on day
14 pc by presenting increased calcium and phosphorous deposition
on days 21 and 28 pc [18,19]. ese nding were further conrmed
by SHG images of reorganised and reoriented collagen bres near the
periphery of collagen beads, in addition to SEM images of more visible
deposited minerals on the surface of collagen microspheres in the
adjusted loading samples on day 28 pc [20,21].
Zinc levels have been shown to have a stimulatory eect on
mineralization, production of collagen and ALP activity level both in
vivo and in vitro [20]. Our results indicated that zinc accumulation
was signicantly up-regulated in loaded samples of adjusted loading
protocol on day 21 pc and declined to half of its value on day 28 pc.
However, other conditions and controls showed the opposite trend
for zinc at the same time points, with higher zinc expression level on
day 28 pc. Up-regulation of zinc on day 21 pc and prior to increase
in the deposited calcium and phosphorous could be explained by an
accumulation of zinc in the matrix vesicles that initiates the assembly
of hydroxyapatite crystals and mineralisation and was reported on day
28 pc [22]. Higher concentrations of zinc may indicate mineralisation
and osteogenic dierentiation of hES-MPs through higher MMPs
expression level in the ECM, but it also could indicate chondrogenesis
of progenitor cells [23,24]. erefore, more studies on the role of zinc in
calcication via matrix vesicles and expression levels of osteogenic and
chondrogenic MMPs are needed to fully link the up-regulation of zinc
with matrix vesicles and expression level of matrix metalloproteinase
family members.
Although various studies have shown that tensile and compression
loading enhances osteogenesis of primary cells, experimental
models still require meticulous planning and design to allow for the
transmission of appropriate and eective global strains to the cell
local micro environment. Dierent studies have reported the eect
of longitudinal stretching and compression on the osteogenesis and
chondrogenic dierentiation of stem cells [25,26]. Yet, it is still not
clear whether compression can induce osteogenesis of stem cells on
a so scaold [27]. In real physical activity mechanical strain modes
are complex and combined mechanical forces act together to inuence
stem cell fate in the body [28]. us, it is more relevant to study the
synergistic eect of external mechanical forces on osteogenesis of
progenitor cells [29]. erefore, analysing the combined eects of
tensile and compression loading on the cell seeded microspheres was an
important focus of this study. e results of the mechanical stimulation
of progenitor cells indicated the eectiveness of the adjusted loading
protocol in enhancing osteogenesis of the cells, which is in agreement
with other studies [10,11]. However, there are multiple factors that
should be considered for obtaining more signicant outcome in future
studies [4]. Controlling these factors increase accuracy whilst reducing
variability in measuring osteogenic markers and increase the validity of
the eect of mechanical loading on osteogenic dierentiation of hES-
MPs in so 3D cultures [5,6].
One of the most important factors is the geometry of the collagen
microspheres that determine the transmission of loads (compression
and tensile) through collagen bres and into the cells. e geometry
of collagen beads was assumed spherical and as it was calculated by
computational modelling applying 10% global strain which results in
a transfer of around 5% compression to the centre and more tensile
loads to the periphery of the spheres. Due to the nonhomogeneous
distribution of the cells in the microspheres and cells tendency to
migrate to the surface of collagen beads, in theory, hES-MPS would
sense more tensile loads compared to compression. In addition,
applying tensile load to the PDMS and compression of the chamber
will cause collagen microspheres to be pulled in a longitudinal direction
(top to bottom) that would change the geometry of the beads during
the experiment. is alteration of bead geometry in opposite direction
of being conned and compressed by the PDMS under the loads would
also add to the complexity of load transfer. In reality, microsphere
geometry varies considerably depending on the number of seeded
cells from being close to spherical, oval or three quarter of a sphere
that once again increase the variability between samples, experimental
conditions and repeats [29,30].
Furthermore, numerical modelling indicated that when a 10%
strain is applied to the construct, the embedded cells receive a dierent
magnitude of strain that decreases exponentially with increasing
the distance from the construct surface [31]. ese ndings were
conrmed by Pfeiler’s et al. [32] model that evaluated the local loading
conditions on hMSCs seeded in 3D collagen scaold that was exposed
to cyclic tensile strain. eir study concluded that global strain applied
tensile strain of 10% resulted in 18.3% local strain because of geometric
variation in the gel shape.
Moreover, osteogenic marker assays were performed on a pool of
total extracted cells from the collagen microspheres without considering
the local cell number on the specic area (centre or periphery) of the
beads and dominance of load type. erefore, they cannot be a true
representative of the eectiveness of the applied mechanical strain
on the cells. As a result, cells in dierent areas of microspheres would
sense non-identical load transfer that may lead in inconsistent cellular
response and promotion of dierentiation. eses variabilities were
conrmed by assessing the histological staining of collagen beads
across sections that indicated random accumulation of osteogenic
markers stains with no signicant preference of the deposited stains
intensity in the periphery or centre of the cross sections and between
dierent cross sections of one sample. In addition, the intervaribility
between samples caused by very low initial seeded cells and proliferated
cells in random areas of beads periphery increased the inconsistency of
the results. e intensity of the deposited stain can be aected by very
low cell number in specic area of the beads despite assuming that the
optimum load transfer condition was carried out.
Better handling of collagen beads is required as hydrogel so
geometry can easily be aected by transferring microspheres to the
loading chamber. Due to the very small size and lightweight nature of
the microspheres, collagen microspheres may rotate through loading
chamber and may cause non-homogeneous transfer of forces to the
cells as shown in histological staining of microspheres. erefore,
cells in dierent areas of a microsphere may experience more random
mechanical forces and respond dierently to the applied load.
Citation: Shariatzadeh M, Baldit A, Perrault CM, Lacroix D (2018) Effect of Mechanical Loading on Osteogenesis of Human Embryonic Stem Cell-
Derived Mesenchymal Progenitors within Collagen Microspheres. J Cell Sci Ther 9: 282. doi: 10.4172/2157-7013.1000282
Page 10 of 11
Volume 9 • Issue 3 • 1000282
J Cell Sci Ther, an open access journal
ISSN: 2157-7013
Advanced computational modelling is required to dene a
more detailed and accurate map of load transfer for specic areas
of the collagen microspheres [33]. Such a map would improve our
understanding of the transmission of loads and load types to the cells
and therefore, would assist in developing an optimum protocol to study
the eect of mechanical loading on the cells and cellular response.
In addition, conventional in vitro osteogenic marker assays are not
sensitive enough for low cell number therefore, even with optimised
mechanical conditioning regime quantication and analysis of the
changes in osteogenic markers would not be valid. Although applying
mechanical stimulation can aect cell commitment, mechanical
loading alone may not provide all of the necessary signals for full
cell commitment and osteogenesis, thus, combining mechanical and
chemical stimulation is critical in osteogenic dierentiation of hES-
MPs as suggested in other studies [34,35].
Conclusion
is study has demonstrated that mechanical loading can
stimulate osteogenic dierentiation of 3D culture of self-assembly
hES-MPs seeded collagen microspheres by enhancing both early
stage bone markers and mineralisation. Combining computational
and experimental studies has provided precise and consistent load
transfer into microsphere’s bres and enabled mechanical forces to
eciently be transmitted to the cells. Understanding how mechanical
cues in the microenvironment orchestrate stem cell dierentiation can
provide valuable insight to improve current techniques in cell therapy
and organ repair. Hence, the eect of cyclic loading on 3D culture of
hES-MP cells could be employed as a model to achieve more in-depth
understanding of mechano-stimulation and lineage dierentiation
both in vitro and in silico. e result of this study can be used as a tool
to build more optimised constructs to transfer mechanically stimulated
stem cells to the specic area of a defected bone or as cells carriers and
implants in bone and cartilage regenerative therapies.
Acknowledgements
SHG microscopy of samples was carried out by Dr Nicola Green (University
of Shefeld) who is gratefully acknowledged. Financial support from the European
Research Council (258321) is acknowledged.
Conict of Interest
The authors declare that they have no competing interests.
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