Abstract
To move towards clinical applications, tissue engineering (TE) should be
validated with human primary cells and offer easy connection to the
native vascularisation. Based on a sheet-like bone substitute
developed previously, we investigated a mesenchymal stem cells /
endothelial cells (MSCs/ECs) coculture to enhance pre-vascularisation.
Using MSCs from 6 independent donors, we focused on donor variability
and cell crosstalk.
Coculture was performed on calcium phosphate granules in a specific
chamber (one month). MSCs were seeded first then ECs were added after
two weeks, with control monocultures. Cell viability and organisation
(fluorescence, electronic microscopy), differentiation (ALP
staining/activity, RT-qPCR) and mechanical cohesion were analysed.
Adaptation of the protocol to coculture was validated (high cell
viability and proliferation). Activity and differentiation showed strong
trends towards synergistic effects between cell types. MSCs reached
early mineralization stage of maturation. The delayed ECs addition ECs
allowed for their attachment on developed MSCs. The main impact of donor
variability could be the lack of cell proliferation potential with some
donors, leading to low differentiation and mechanical cohesion and
therefore absence of sheet-like shape successfully obtained with others.
We suggest adapting protocols to cell proliferation potentials from one
batch of cells to the other in a patient-specific approach.
Introduction
Bone tissue engineering, among other regenerative medicine approaches,
aimed at developing new solutions to treat injured bone tissues and fill
critical size defects that cannot healed
naturally[1,2]. Cells of interests, namely
osteoblasts and/or mesenchymal stem cells (MSCs), are cultured in
vitro on a scaffold to form hybrid implantable substitutes with same
organisation and functions as the native
tissue[3]. Such substitutes could be an
alternative to allografts (suffering from risks of rejects and
transmission of infection)[3], autografts (limited
stock, donor site morbidity)[4,5] and biomaterials
used alone (limited bioactivity)[6,7]. However, a
current drawback in tissue-engineered substitutes preventing strong
clinical outcomes is their poor connexion to the existing vascular
network after implantation[8,9]. Native bone is
indeed a composite tissue containing a high density of blood vessels
allowing for the diffusion of interstitial liquid, waste and nutrients
from and to the cells[6,8]. If there is no blood
capillaries in the close environment of cells (150 – 200 μm), death of
the implanted rebuilt tissue can occur at mid or short
term[6,10]. To achieve translational progresses,
it is therefore mandatory to enhance the vascularisation potential of
bone substitutes to allow for easy connection in vivo .
One of the main approaches to perform pre-vascularisation is the
combination of endothelial cells (ECs) with the primary cells of
interest during the development of the implantable substitutes in
vitro through coculture protocols[11–15].
Important cross-talks have been reported between ECs and MSCs including
mutual beneficial effects and synergies on their respective growth and
differentiation[16–19]. Although this method has
been widely investigated, consensus are still lacking for many
parameters of the coculture protocols, such as ECs:MSCs cell ratio,
medium composition over time, seeding density or scaffold structure and
composition[13,20–22]. It is also still discussed
if cells should be cultured together at day 1 or the addition of one
cell lineage delayed, as suggested by some
studies[13,23]. Optimising these aspects would be
very promising to benefit from the mutual synergetic effects that could
appear between ECs and MSCs, in particular promoting the MSC
differentiation into osteoblasts, resulting in faster and more reliable
processes. This would in turn decrease the overall costs of such
cell-based therapies as less consumables and work force would be
required.
In a previous study, we developed a novel biohybrid bone substitute
specifically designed to meet clinicians’ expectations for the
regeneration of the maxillo-facial area[24]. After
culturing pre-osteoblastic cells on a monolayer of calcium phosphate
granules in a parallelepiped culture chamber, we obtained a sheet-like
tissue formed by both the newly formed cell matrix and the scaffold with
good mechanical cohesion[24]. This allowed the
substitute to be folded or stretched with easy handling. This original
shape would be beneficial for maxillo-facial surgeries as clinicians
could adapt it manually to the specific geometry of each patient’s face
but from a single standard fabrication
protocol[24]. Both biological and mechanical
properties of this substitute were characterized, optimised and
validated with a pre-osteoblastic cell line.
To bring this innovative process a step further towards a clinically
relevant solution, our objective here was therefore to adapt the
sheet-like substitute to a ECs/MSCs coculture to validate its in
vitro pre-vascularisation potential and to confirm assumptions on ECs
and MSCs interactions. As a translational research approach, we used
human primary mesenchymal stem cells obtained from bone marrow of 6
different donors and assessed bone differentiation within our system.
The comparison of variations between donor sources in a
tissue-engineering process previously validated was therefore also a
main objective of this study. Both lineages were cultured inside the
specific culture chamber on calcium phosphate granules over one month,
following optimisation of the technical parameters in our previous
study[24]. We used a delayed coculture approach,
seeding first MSCs before addition of ECs. We focused on the evaluation
of cell viability, morphology and organisation of the rebuilt tissue
through microscopy as well as early (alkaline phosphatase staining and
activity) and late (gene expression of specific markers) differentiation
of MSCs to osteoblasts, with a special care to the comparison of the
substitute’s cohesion between donors. Cross-analysis was then performed
in regard of the donor variability, to highlight the most affected
parameters and suggest ways of optimisation towards patient-specific
protocols.
Materials and Methods
Isolation and expansion of cells
Proliferation medium consisted in αMEM (Sigma, USA) supplemented with
10% foetal bovine serum (FBS, Eurobio, France), 2 mM L-glutamine
(Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA).
Differentiation medium consisted in DMEM high glucose (Sigma, USA) with
10% FBS (Eurobio, France), 2 mM L-glutamine (Gibco, USA), 1%
penicillin/streptomycin (Gibco, USA), 10 mM β-glycerophosphate (Sigma,
USA), 50 µg/mL ascorbic acid (Sigma, USA), 0.1 µM dexamethasone (Sigma,
USA). The endothelial medium consisted in EGM-2 medium (Lonza,
Switzerland).
MSCs: Bone marrow aspirates were obtained by standard puncture and
aspiration from the iliac crest of healthy human donors after receiving
informed consent. The protocol was approved by the Amiens University
Hospital Ethics Committee. Bone marrow aspirates were obtained from 6
donors (three males and three females, aged 35 – 56 years). After
centrifugation, the buffy coat layer (mononuclear cells) was isolated
and plated in T175 culture flasks with proliferation medium supplemented
with 2 ng/ml basic fibroblast growth factor (TebuBio, France). Cells were
seeded at 1x105 mononuclear
cells/cm2 and cultured in 5% CO2atmosphere humidified at 37°C. Medium was refreshed every 4 days, 3
times and then every weeks. Then, adherent cells were enzymatically
removed with 0.25% trypsin – EDTA (Sigma, USA) at 37°C and seeded at
1500 cells/cm² in T175 culture flasks. Cells cultured in proliferation
medium were used between passage 4 and 5.
ECs: Human umbilical cord bloods were collected from full-term births
with informed consent. The protocol was approved by the Amiens
University Hospital Ethics Committee. Umbilical cord bloods were diluted
at a ratio of 1/1 (v/v) in PBS. The cells suspensions were added to
Ficoll Plus solution (Sigma, USA) followed by centrifugation at 1800 rpm
for 30 min. Peripheral blood mononuclear cells were washed twice with
PBS, and then suspended in endothelial medium. Cell suspensions were
seeded at a density of 1.25x106 cells/well into
24-well plates. Medium was refreshed every day during the first week and
then every 2 days.
Phenotypic and functional characterization of cells
Phenotypic characterization of MSCs was performed with antibody against
human CD44-PE, CD73-APC, CD90-FITC, CD105-PE, HLA DR-FITC, CD166-PE,
CD34-FITC, CD14-FITC, CD45-PE, CD146-PE (BD Biosciences, USA) and
analysed on a FACSAria II with the Diva software (BD Biosciences, USA)
with control IgG-FITC, -PE and -APC. Differentiation capacity of MSCs
was successfully tested prior to use (data not shown). For osteogenic
differentiation, the cells were cultured for 21 days in differentiation
medium as defined earlier. The osteogenic differentiation was evaluated
by ALP coloration with SIGMAFAST™ BCIP®/NBT (Sigma-Aldrich). For
adipogenic differentiation, the cells were culture for 21 days in a
defined adipogenic medium : DMEM low glucose (Sigma, USA), with 10% FBS
(Eurobio, France), 2 mM L-glutamine (Gibco, USA), 1%
penicillin/streptomycin (Gibco, USA), 0.5 mM isobutylmethyl xanthine
(IBMX), 60µM indomethacin, 1µM dexamethasone (Sigma, USA). Adipogenic
differentiation was confirmed by Oil Red O staining. For ECs phenotypic
characterization, ECs were incubated with antibody against human
CD31-FITC, CD184-PE and CD309-APC and analysed with the same device as
MSCs. Cells were used between passage 4 and 5.
Biomaterials
Sterile calcium phosphate granules (20% hydroxyapatite (HA)
Ca10 (PO4)6(OH)2, 80% b-tricalcium phosphate (TCP)
Ca3 (PO4)2) were kindly
provided by Biomatlante, France.
Culture protocols
We used the Clinicell25® culture cassette (Mabio-International, Figure
1 A) as a specific flat cell culture chamber for the
development of the tissue-engineered substitute. It is a gas-permeable,
plasma-treated parallelepiped chamber with a 24-cm2culture surface for a 10-mL volume filled with medium. After 1 month of
culture, the upper side of the chamber was opened to remove the tissue
samples and to perform analyses. Based on previous
optimisation[24], 400 mg of calcium phosphate
granules with a 80-200-mm diameter were inserted as scaffold material
inside the culture chamber, to obtain a single monolayer covering the
whole surface. To minimize ion release from the granules during cell
culture, they were incubated in culture chamber 48h in proliferation
medium before cell seeding.
The coculture protocol and feeding schedule are reported in Figure
1 B. In first phase, after gently elimination of chamber
medium, 2x106 MSC were injected in the culture
chamber. MSCs were cultured in proliferation medium over the first week
and in osteogenic differentiation medium over the second week. ECs were
then added (2.5x106 cells) with endothelial medium and
cells cocultured for 2 additional weeks. Medium was refreshed twice per
week. Three control groups were used in the same culture chamber with
scaffolds, (1) monoculture of MSCs in proliferation medium for 4 weeks
(“MSC” group), (2) monoculture of MSCs in proliferation medium for 1
week, differentiation medium for 1 week and endothelial medium for 2
weeks, i.e. the same feeding schedule as the coculture samples
(“MSC diff” group) and (3) monoculture of ECs in endothelial medium
for 2 weeks (Figure 1 B). For each independent donor, 3
chambers per group were seeded as technical replicates.
Cell viability and organisation
After one month in the chamber, cell viability was estimated by a
Live/Dead® kit (Invitrogen, USA) according to the manufacturer’s
protocol on sections of the tissue-engineered substitutes. Calcein AM (1
µM) and Ethidium homodimer-1 (EthD-1, 1 µM) fluorescent dyes were
respectively employed to stain viable and dead cells. The samples were
observed using fluorescence microscopy (Leica microsystems, Germany).
Samples were also analysed using immunofluorescence staining. Briefly,
after washing with PBS, sections of the tissue-engineered substitutes
were fixed with 4% paraformaldehyde for 10 minutes (Agar Scientific,
UK) then rinsed and permeabilised with 0.5% Triton X-100 (VWR, UK) for
10 minutes. They were then immersed in the same Triton solution
supplemented with bovine serum albumin (Sigma, USA) for 20 minutes.
Murine anti-connexin 43 (Cx43) primary antibodies (Invitrogen, USA) and
donkey anti-mouse 488 secondary antibodies (Invitrogen, USA) were used
to highlight gap junctions between cells. A rhodamine/phalloidin
solution (Invitrogen, USA) was used to visualise actin filaments.
Tissue morphology and density were observed using scanning electron
microscopy (Philips XL30 ESEM-FEG, the Netherlands). The samples were
immersed in Rembaum solution for 24h, rinsed with demineralized water
and then coated with gold before observations.
Cell differentiation
Section of the tissue-engineered substitutes were fixed with 2%
formalin after one month in the culture chamber and stained for alkaline
phosphatase (ALP) coloration using SIGMAFAST™ BCIP®/NBT (Sigma, USA).
ALP activity was determined using the kit “Alkaline Phosphatase
Activity Colorimetric Assay” (Biovision, USA), according to the
manufacturer’s recommendations. The level of ALP activity was normalized
by the amounts of total protein in the cell lysates (nmol/min/mg
protein). Details of the total protein mass measurements are available
as supplementary information (Figure S1).
Differentiation was also assessed through the expression of genes of
interest quantified by reverse transcription-polymerase chain reaction
(RT-qPCR). Total RNA was isolated using Tri Reagent (Sigma, USA). RNA
concentration and OD 260/280 value were measured by NanoPhotometer®
Pearl (Implen, Germany). The reverse-transcription PCR reactions were
performed with 1 µg total RNA using High capacity cDNA reverse
Transcription kit (Applied Biosystem, USA) in a 20-μL reaction volume.
The reaction was performed for 10 min at 25°C, followed by 2h at 37°C,
and 5 min at 85°C. Real time-PCR was also performed using SYBR
PrimeScript RT-PCR kit, according to the manufacturer’s recommendations.
All amplifications were normalized by GAPDH expression. Results were
analysed using the comparison Ct (2-DDCt) method, and expressed as
fold-change compared with MSC control group. Primers sequences used in
this study are reported in Table S1 (supplementary information).
Significance and donor variability
All results were obtained on samples seeded with MSCs from 6 independent
donors, as stated earlier. Cells obtained from 1 specific donor were
used in several technical replicates for each analysis method. The focus
was then given to the comparison between donors and variability
analysis. Statistical significance between groups (MSCs, MSCs diff, ECs
or coculture) was estimated after calculating the mean of all donors
using 2-way ANOVA with Tukey’s test, however no significance was found
due to this variability. Mean of donors and statistical analysis are
therefore not reported on figures and the detailed results of each donor
are instead plotted independently with error bars as standard deviation
of technical replicates.
Results
Cell viability and organisation
The cocultured tissue-engineered substitutes were first evaluated with
fluorescence microscopy and SEM at the end of the one-month culture.
Samples were also taken out of the culture chambers and handled to
assess stability and cohesion (i.e. handling with forceps was
possible without disrupting the sample). Observations of Live/Dead
staining (Figure 2A) showed a very good overall viability in all samples
regardless of the donor source. Only a few dead cells were found (red
dots) locally in the cell tissue covering the phosphate calcium
granules. No difference of viability was observed between the 6 donors
of MSCs. These observations revealed that cells were able to attach to
the granules and to overlay them with a 3D organization. The cells
organized together and formed a tissue that encompassed the granules,
although cell density could slightly differ from one donor to the other.
To confirm this, we investigated further the developed cell tissue with
other microscopy modalities. Indeed, slight differences were noticed
regarding cell density after analysis of the actin filament staining and
SEM observations (Figure 2B and C). On the one hand, the samples
obtained with cells from donors 1 to 4 showed a continuous cell tissue
penetrating the porous structure of the granules and filling the gaps
between them with many bridges formed by the actin filaments. Cells were
spread with standard morphology, similar to MSCs monocultures on the
same biomaterial although cell density could be slightly lower (Figure
2C), an expected result as the MSC group was maintained in proliferation
culture medium. Cx43 expression (presence of gap junctions) was
frequently noticed, highlighting early cell organisation, although no
pattern or specific location could be noticed. This resulted in
intermediate (donors 1 and 2) to good (donors 3 and 4) mechanical
cohesion when substitutes were handled. It has to be mentioned that
donor 4 showed higher heterogeneity of the cell tissue, and granules in
some areas were not covered by cells. The mechanical stability was
therefore very strong at some locations while granules were still moving
freely at others. Cx43 was however clearly present where the tissue
successfully developed. On the other hand, substitutes obtained with
cells from donors 5 and 6 showed mitigated results regarding density and
homogeneity of the cell tissue. Although viability was good as mentioned
earlier (Figure 2A), cells poorly proliferated in some areas, resulting
in holes between the granules, as shown in particular for donor 5
(Figure 2B), some granules are not covered by cells as showed by the
dark smooth red autofluorescence instead of bright actin filaments)
which led to low mechanical cohesion (immediate disruption upon
handling). This sample also showed very low expression of Cx43, barely
noticeable (Figure 2B).
Cell differentiation
ALP is an early marker of osteoblast
differentiation[25]. ALP staining and activity
measurement on samples removed from the culture chamber after one month
can be seen in Figure 3. First, this confirmed the differences of
stability depending on the culture group (Figure 3A). As it could be
expected, ECs didn’t produce substantial extracellular matrix,
preventing the calcium phosphate granules to be bound together.
Mechanical cohesion was similar between differentiated and
non-differentiated MSCs, but the combination of both lineages in
coculture led to improved stability for some donors, in particular donor
3. We can also notice on these results the differences reported earlier
with microscopy analysis depending on the donor source, with donor 5
showing poor mechanical cohesion and donor 4 high heterogeneity.
Purple colour on tissue samples highlighted the presence of ALP (Figure
3A) and the activity of the produced enzyme was evaluated quantitatively
(Figure 3B). ALP was differently expressed depending on donor although
overall very high levels could be achieved in both MSC diff and
coculture groups for most of them. These results were shown by both
methods with good consistency, ALP activity highlighting quantitatively
the important increase in these two groups (up to 15-20 fold). As
expected, non-differentiated MSCs and ECs didn’t produce this bone
marker. Donor 6 showed the lowest expression in all groups.
The expression of genes of interest was then quantified with RT-qPCR to
assess late bone differentiation (Figure 4A, Runx2, ALP, Col1a2, OCN,
OPN and BSP) as well as presence and activity of endothelial cells at
the end of the coculture (Figure 4B, Vegfr2, Von Willebrand factor).
Runx2, a transcription factor, is considered as the first and most
specific marker of early osteoblast
differentiation[25], followed by increase in the
expression of ALP, OPN and the non-specific Col1a2. All of these three
markers reach a peak of production as osteoblast differentiation occurs,
followed by as slight decrease as cells start to
mineralize[25,26]. BSP and OCN are then the latest
markers to be expressed, highlighting the osteoblast
maturation[27,28]. OPN would be the only marker to
be still expressed by osteocytes[29]. Results were
normalized by the expression obtained with non-differentiated MSCs
cultured alone to highlight the variations through coculture. Vegfr2-
and Von Willebrand factor-related genes were selected as markers of the
presence of endothelial cells[30,31]. Results of
bone-related markers in monoculture of ECs are not reported in the
figure as they led to null expression or background noise only, as
expected.
Expression of Runx2 and ALP-related genes, both early markers, showed
different behaviours. Overall, a decrease in Runx2 expression was
noticed in MSC diff and coculture groups compared to MSC (for all donors
but donor 6) while ALP expression drastically increased. The expression
of Col1a2, non-specific matrix protein, showed a clear trend for
increase in coculture but with high variability. The same behaviour was
noticed with the late differentiation markers OCN and BSP, showing high
over-expression for some donors only (donor 6 in particular). The
expression of OPN gene was never increased compared to MSC control.
However, overall results showed a strong example of synergistic effects
that can happen thanks to cell types crosstalk, as gene expressions in
most cocultures samples were far higher than the sum of respective
monocultures.
Regarding endothelial-related genes (Figure 4B), over-expression of
Vegfr2 and Von Willebrand factor genes was obtained in the cocultured
chambers after one month for all donors, except 4 and 5 regarding
Vegfr2. The other samples led to high or very high expression of this
markers compared to non-differentiated MSCs, validating the presence of
active ECs in coculture at the end of the culture process but also
confirming a high variability in adhesion and MSCs/ECs crosstalk. As
expected, monocultures of ECs led to extremely high expression for these
two genes (not plotted in the figure for readability reasons),
respectively 103 to 104 and
104 to 106-fold increases for Vegfr2
and Von Willebrand factor. Populations of ECs were observed attached to
the tissue developed by the MSCs as seen on Live/Dead images (Figure
4C), highlighting cell-cell contact and mixing between both cell types
on the scaffold.
Discussion
We aimed here at investigating the pre-vascularisation potential of a
sheet-like bone substitute previously
developed[24]. Thanks to a new step of coculture
between human primary MSCs and ECs, we also used this culture system to
assess the variability between donors. The coculture approach was
expected to lead to tissue-engineered substitutes mimicking better the
native structure of tissues towards easier connexions to the existing
vascular network after implantation[11,32].
However, cocultures could also be beneficial directly during thein vitro culture steps to improve and speed up cell growth and
differentiation. Indeed, growth factors produced by MSCs after
differentiation towards bone lineage can promote angiogenesis and ECs
proliferation, such as VEGF secreted by osteoblasts in the presence of
BMP-2[33]. Moreover, mutual beneficial effects
exist as the ECs population can significantly increase proliferation and
differentiation of MSCs[16,17]. Cell-cell direct
contact was here prioritized over paracrine signalling as MSCs and ECs
can both form gap junctions[11,34,35]. The
ECs/MSCs coculture could therefore allow for the development of
substitutes with better vascularisation potential than the respective
monoculture, even with synergetic
effects[8,16,33,36], as we noticed here for the
expression of some genes of interest, especially Col1a2. However, the
successful development of both cell types requires specific culture
conditions, still discussed nowadays (cell ratio, simultaneous or
delayed co-culture, medium composition,etc. [16,20,21,23,33,37]), and in particular
3D growth structures appear to be
mandatory[23,33,38]. We were thus expecting the
calcium phosphate scaffolds used in our system to promote coculture
growth and activity despite the absence of specific coating such as
collagen, commonly used for primary EC monoculture in vitro .
Moving from cell line to human primary cells is not a straightforward
step but requires adjustments and more complex protocols, in particular
for initial seeding density and culture medium
schedules[13,17,36], but the fundamental analysis
of the coculture parameters was not the initial purpose of this study.
We wanted to focus instead on the results variability with cells
obtained from 6 donors treated and cultured independently. Selection of
the optimal parameters for the tissue-engineering protocol presented
here was therefore based first on a previous study using murine
pre-osteoblastic cells, but also thanks to preliminary results and
literature background[13,24]. We decided then to
work with a delayed addition of ECs to allow for a first phase where
MSCs would be able to proliferate and generate extra-cellular matrix
prior to ECs anchorage. We used then endothelial medium to supply the
coculture with nutrients ensuring ECs survival, although osteogenic
medium could also promote vascularisation[39].
Coculture samples were compared to the respective monocultures to
highlight the effects of the crosstalk.
Microscopy observations showed that a continuous cell tissue was
successfully developed in coculture samples, with good viability and
early signs of cell organization (gap junctions, endothelial cell islets
attached on the MSCs matrix, Table 1). Cells spread and bonded the
calcium phosphate granules together, in a similar way as it was achieved
previously with a cell line monoculture[24]. This
absence of cytotoxicity induced by the calcium phosphate-based scaffold
and the coculture conditions was expected but this confirmation was a
first crucial result towards the use of human primary cells. Ultimately,
the goal of such hybrid tissue-engineered substitutes is the
implantation to strengthen injured or lacking tissues, and optimal cell
viability in contact with the biomaterial, as validated here, is
therefore mandatory. The newly formed cell tissue however showed
sometimes more cracks and non-covered areas in case of lower
proliferation of MSCs (donor 5), leading to limited mechanical
stability, but coculture didn’t induce drastic decrease in proliferation
potential of either cell types, in contrast with some literature
results[40,41]. As stated previously, one of the
main advantages of the tissue-engineered substitute reported here is its
sheet-like shape[24]. The mechanical cohesion of
the samples showed here a certain variability. Some coculture samples
offered very easy handling with forceps after being removed from the
culture chamber while other were immediately completely disrupted. The
mechanical cohesion level could be linked to a certain extent to the
degree of over-expression of late markers (Col1a2, BSP, Table 1), but it
could depend more likely on the proliferation potential of the MSCs
source. High cell density with good organisation seemed directly
correlated to high mechanical properties (Table 1).
More importantly, overall, bone differentiation results in coculture
showed systematically similar or higher trends than the monocultured
groups of MSCs, with hints for strong synergistic effects (gene
over-expression levels). The presence of ECs and the specific coculture
feeding schedule didn’t jeopardize MSCs differentiation towards the bone
lineage and even increased it for some donors, as it was expected thanks
to the crosstalk between both cell types. Due to low Runx2 expression
along with high ALP, moderate Col1a2 and occasional BSP/OCN
over-expressions compared to the proliferating MSCs, we could assume
that cocultured MSCs were starting to maturate and produce mineralized
matrix[25–28]. This was also consistent with the
total absence of OPN. Moreover, even if the order of magnitude was
drastically different from the EC monoculture, Vegfr2 and Von Willebrand
factor expressions confirmed that a population of active ECs was still
present in the coculture samples after one month of tissue development.
Samples 4 and 5, which showed no over-expression of VEGFR2, also had the
lowest or most heterogeneous cell densities with no expression of late
bone-specific genes. Therefore, we hypothesized that important MSCs
growth and matrix production was mandatory to allow the ECs to attach on
the newly formed tissue and to survive in coculture without the need for
a specific coating. This was confirmed by the absence of cohesion in the
monoculture of ECs, with a very low number of cells after one month.
This behaviour strengthened the rationale behind using a delayed
coculture protocol and a specific feeding schedule, with a first step of
MSCs development on the scaffold before addition of ECs. Using
endothelial medium in cocultures for two weeks maintained the
development of both lineages without stopping MSCs differentiation.
Overall, our system allowed therefore for cell proliferation, viability
and activity of MSCs and ECs in coculture over the one-month culture
period required for the development of the sheet-like substitute, paving
the way to sheet-like bone substitute with enhanced vascularisation
potential, which should be validated through in vivo assays in
further studies..
Although our process was therefore successfully adapted from cell line
monoculture to primary cell coculture, the important variability between
donor sources prevented to guarantee a relevant mechanical cohesion for
all samples. As a solution, the culture time in the chamber could be
adjusted for further experiments in order to allow cells from each
source to reach confluence state on the calcium phosphate granules.
Indeed, variability in proliferation potential between donor sources was
noticed during the tissue-engineering process, but also during the
standard pre-amplification steps (expansion in T175 flasks) needed to
obtain the initial number of MSCs to be seeded in the specific culture
chamber. To allow for the development of a consistent cell tissue in the
chamber, the culture time could vary from batch to batch, in a
patient-specific approach, to be consistent with the pre-culture
proliferation rate. This could lead to better homogeneity in the gene
expression levels of late differentiation markers, and therefore
consistently high mechanical cohesion. It has to be noticed that this
can be however difficult to achieve due to more complex protocols and
regulations. Another solution would therefore be to use higher initial
cell densities. Even for cell batches with high differentiation and
proliferation potentials, one million cells per culture system could be
considered as low density[42]. This would imply
additional pre-culture expansion steps to reach the expected number of
cells. Finally, although no clear trend appeared with our results, the
donor age remains a parameter to consider[43].
In conclusion, we investigated in this paper how the tissue-engineering
protocol leading to the development of a bone sheet-like substitute with
easy handling for maxillo-facial regeneration could be adapted to the
coculture of human primary MSCs and ECs, in a translational approach.
From optimized technical parameters, both cell types were cultured
together in a specific chamber on a monolayer of calcium phosphate
granules to enhance pre-vascularization potential with samples obtained
from 6 independent donors. Activity and proliferation of both cell types
were confirmed after one month, without detrimental effect from one on
the other or from the feeding schedule, but rather synergistic effects
compared to the respective monocultures. Cell viability was always very
high, validating the absence of cytotoxicity from the scaffold and the
tissue-engineering protocol for the use of human primary cells.
Production of cell matrix by MSCs enhanced EC survival which, in turn,
supported bone differentiation. MSCs were found to be in early
mineralization stage at the end of the protocol. More importantly, we
highlighted that the important variability in proliferation potential
and, in turn, gene expression, could have prevented the development of
the sheet-like shape with enough mechanical stability. A
patient-specific approach would be to adapt culture time based on the
cell proliferation rate. Further studies will therefore investigate this
approach before first in vivo validation in a small animal model.