Expansion, at 37 ºC in a humidified atmosphere

Expansion,
Seeding, and Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem
Cells

hBM-MSCs were obtained from a 95-year-old female donor undergoing knee
arthroplasty at the Hospital Center of Alto Ave, Guimarães, in accordance with
a protocol established between the 3B’s Research Group, University of Minho and
the Hospital Center of Alto Ave and approved by the ethics committee of this
hospital. hBM-MSCs were obtained from the patient after signing an informed
consent. hBM-MSCs were characterized and cultured using our standard protocols.(9) hBM-MSCs were
expanded in basal medium consisting of a-MEM (Life Technologies) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) and 1% antibiotic/antimycotic solution and cultured at 37 ºC
in a humidified atmosphere of 5% CO2. Cells were expanded in the
basal medium until passage 4 and then were harvested, counted and a cell
suspension of 200 000 cells/50 mL was seeded over
each NFM. The conditions assessed were nanofibrous substrates biofunctionalized
with antibodies against IGF-I and TGF-b3 in a mixed or
single fashion. The corresponding growth factors were bound from recombinant or
PL -origin. The hBM-MSCs seeded on the biofunctionalized nanofibrous substrate
were cultured under basal medium without further supplementation. The
experimental positive control comprises hBM-MSCs cultured on functionalized
NFM, without antibodies immobilization under standard chondrogenic
differentiation medium (basal medium supplemented with Insulin-Transferrin-Selenium-G
Supplement (ITS; Invitrogen), 1 mM dexamethasone (Sigma-Aldrich), 0.1 M sodium
pyruvate (Invitrogen), 17 mM ascorbic acid-2-phosphate (Wako Pure Chemical
Industries, Ltd.), 35 mM L-proline (Sigma-Aldrich), 10 ng mL-1 TGF-b3 and 100 ng mL-1
IGF-I). The hBM-MSCs cultured under basal medium on functionalized NFM, without
antibodies immobilization were used as negative control. The nanofibrous
substrates were retrieved at predefined culturing times, namely 7, 14, and 28
days. All experiments were performed in triplicates and repeated at least three
times independently.

Cellular
Biochemistry Analyses

Cell proliferation was evaluated by DNA quantification
(Quant-iTPicoGreen dsDNA assay, Invitrogen, Alfagene), metabolic activity by
the MTS assay (CellTiter 96 AQueous One Solution, Promega) and
cellular protein by Micro BCA assay (Micro BCATM Protein Assay Kit, Thermo
Fisher Scientific, USA), according to the manufacturer`s instructions; and glycosaminoglycans
quantification by a colorimetric assay.(9)

Scanning
Electron Microscopy (SEM)

The samples were collected at each defined time point and analyzed in a
scanning electron microscope (Model S360, Leica Cambridge, U.K.) as previously
reported.(9)

RNA
Isolation and Real-Time Quantitative Polymerase Chain

At each culturing time, the hBM-MSCs were washed with PBS, immersed in
Tri reagentÒ, and kept at -80 ºC
for later RNA extraction. The extraction was performed as described elsewhere.(9) RNA was
reversed-transcribed into cDNA according to the protocol from qScript cDNA
Synthesis Kit (Quanta BioSciences; VWR, USA). Afterwards, the obtained cDNA was
used as a template for the amplification of the target genes shown in Table 2,
according to manufacturer’s instructions of the PerfeCtaTM SYBR®
Green system (Quanta Biosciences, VWR, USA). Forty-five cycles of denaturation
(95 ºC, 10 s), annealing (temperature-dependent on the gene; Table 2; 30 s),
and extension (72 ºC, 30 s) were carried out in a MastercyclerÒ ep Gradient S realplexÒ thermocycler (Eppendorf;
Hamburg, Germany) for all genes. The transcript expression data were normalized
to the housekeeping gene glyceraldehydes-3-phosphate-dehygrogenase
(GAPDH) and the quantification performed according to the Livak method (2 -??CT
method), considering the basal medium condition (negative control) as
calibrator.

Histological
Analysis

Samples were collected at 28 days of culture, washed twice with sterile
PBS, transferred into a sterile 24-well plate, fixed in a 4% paraformaldehyde
solution in PBS, and kept at 4 ºC until further used for staining procedures. Alcian
blue staining and immunolocalization of type II collagen (mouse anti-human type
II collagen monoclonal antibody Millipore) were performed as described
previously.(3)  

Statistical
Analysis

Statistical analysis was performed using the SPSS statistic software
(realese 24.0.0.0 for Mac). First, a Shapiro-Wilk test was used to ascertain
the data normality and Levene test for test the homogeneity of variances. The data following a normal distribution, parametric tests were used,
namely one-way
ANOVA test followed by Tukey’s HSD test.  When the normality and variance homogeneity were
rejected, non-parametric tests were used, namely a Kruskal-Wallis test followed
by Tukey’s HSD test. A p < 0.01 was considered statistically significant in the analysis of the results.