Expansion, at 37 ºC in a humidified atmosphere

Expansion,Seeding, and Chondrogenic Differentiation of Human Bone Marrow Mesenchymal StemCellshBM-MSCs were obtained from a 95-year-old female donor undergoing kneearthroplasty at the Hospital Center of Alto Ave, Guimarães, in accordance witha protocol established between the 3B’s Research Group, University of Minho andthe Hospital Center of Alto Ave and approved by the ethics committee of thishospital.

hBM-MSCs were obtained from the patient after signing an informedconsent. hBM-MSCs were characterized and cultured using our standard protocols.(9) hBM-MSCs wereexpanded in basal medium consisting of a-MEM (Life Technologies) supplemented with 10% heat-inactivated fetalbovine serum (FBS) and 1% antibiotic/antimycotic solution and cultured at 37 ºCin a humidified atmosphere of 5% CO2. Cells were expanded in thebasal medium until passage 4 and then were harvested, counted and a cellsuspension of 200 000 cells/50 mL was seeded overeach NFM. The conditions assessed were nanofibrous substrates biofunctionalizedwith antibodies against IGF-I and TGF-b3 in a mixed orsingle fashion.

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The corresponding growth factors were bound from recombinant orPL -origin. The hBM-MSCs seeded on the biofunctionalized nanofibrous substratewere cultured under basal medium without further supplementation. Theexperimental positive control comprises hBM-MSCs cultured on functionalizedNFM, without antibodies immobilization under standard chondrogenicdifferentiation medium (basal medium supplemented with Insulin-Transferrin-Selenium-GSupplement (ITS; Invitrogen), 1 mM dexamethasone (Sigma-Aldrich), 0.1 M sodiumpyruvate (Invitrogen), 17 mM ascorbic acid-2-phosphate (Wako Pure ChemicalIndustries, Ltd.

), 35 mM L-proline (Sigma-Aldrich), 10 ng mL-1 TGF-b3 and 100 ng mL-1IGF-I). The hBM-MSCs cultured under basal medium on functionalized NFM, withoutantibodies immobilization were used as negative control. The nanofibroussubstrates were retrieved at predefined culturing times, namely 7, 14, and 28days. All experiments were performed in triplicates and repeated at least threetimes independently.CellularBiochemistry AnalysesCell proliferation was evaluated by DNA quantification(Quant-iTPicoGreen dsDNA assay, Invitrogen, Alfagene), metabolic activity bythe MTS assay (CellTiter 96 AQueous One Solution, Promega) andcellular protein by Micro BCA assay (Micro BCATM Protein Assay Kit, ThermoFisher Scientific, USA), according to the manufacturer`s instructions; and glycosaminoglycansquantification by a colorimetric assay.(9)ScanningElectron Microscopy (SEM) The samples were collected at each defined time point and analyzed in ascanning electron microscope (Model S360, Leica Cambridge, U.K.

) as previouslyreported.(9) RNAIsolation and Real-Time Quantitative Polymerase ChainAt each culturing time, the hBM-MSCs were washed with PBS, immersed inTri reagentÒ, and kept at -80 ºCfor later RNA extraction. The extraction was performed as described elsewhere.(9) RNA wasreversed-transcribed into cDNA according to the protocol from qScript cDNASynthesis Kit (Quanta BioSciences; VWR, USA). Afterwards, the obtained cDNA wasused 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 normalizedto the housekeeping gene glyceraldehydes-3-phosphate-dehygrogenase(GAPDH) and the quantification performed according to the Livak method (2 -??CTmethod), considering the basal medium condition (negative control) ascalibrator.HistologicalAnalysisSamples were collected at 28 days of culture, washed twice with sterilePBS, transferred into a sterile 24-well plate, fixed in a 4% paraformaldehydesolution in PBS, and kept at 4 ºC until further used for staining procedures. Alcianblue staining and immunolocalization of type II collagen (mouse anti-human typeII collagen monoclonal antibody Millipore) were performed as describedpreviously.(3)  StatisticalAnalysisStatistical analysis was performed using the SPSS statistic software(realese 24.0.

0.0 for Mac). First, a Shapiro-Wilk test was used to ascertainthe data normality and Levene test for test the homogeneity of variances. The data following a normal distribution, parametric tests were used,namely one-wayANOVA test followed by Tukey’s HSD test.  When the normality and variance homogeneity wererejected, non-parametric tests were used, namely a Kruskal-Wallis test followedby Tukey’s HSD test. A p < 0.01 was consideredstatistically significant in the analysis of the results.