Preparation of PLGA/PLA mesh scaffolds
Poly (L-lactide-co-glycolide) (PLGA; lactic to glycolic acid molar ratio, 50:50) and poly (L-lactide) (PLA) was purchased from EVONIK. PLGA and PLA fibers, 2–2.5 mm in length, were prepared by using a rotary cutter and their nonwovens were produced via modified wet-laid process. PLGA and PLA fibers were mixed in an aqueous solution with a dispersing agent (1 wt.% Pluronic F127; Sigma-Aldrich) and randomly laid on a wire mesh to filter the liquid. The formed web was subsequently processed through a thermal bonding, in which the web was transferred to a heater and cured at 170 °C for 5 min. The resulting mesh was cut into sheets (4 × 4 × 4 mm, L × W × H) and they were sterilized by soaking in 100 % ethanol under ultraviolet (UV) light.
Cell-derived matrix (CDM) preparation
Collagen type I-overexpression cell line (Col I-293 T-DK) was cultured at the density of 1.3 × 104 cells/cm2 in a 100 mm diameter petri-dish for 4 days in the Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 100 U/mL penicillin and 100 μg/mL streptomycin (P/S). At the time of confluence, the cell culture plates were washed twice with phosphate buffered saline (PBS), incubated briefly in a detergent solution containing 0.25 % Triton X-100 and 10 mM NH4OH (Sigma-Aldrich) at 37 °C, and then subjected to the treatment of 50 U/mL DNase I and 2.5 μL/mL RNase A (Invitrogen) for 1 h at 37 °C. After the decellularization process, the specimens were washed with PBS thoroughly and stored at 4 °C before use.
CDM characterization: protein and DNA content
For CDM analysis, DNA was examined from the organic phase using Trizol® reagent (Invitrogen). 0.3 mL of 100 % EtOH was added to isolate the DNA from each sample and after centrifugation the supernatant was collected. The supernatant was washed twice with 0.1 M sodium citrate in 10 % EtOH, then centrifuged at 2000 g for 5 min at 4 °C. The samples were resuspended in 2 mL of 75 % ethanol and centrifuged again. The pellets were dissolved in 300 μL of 8 mM NaOH and subsequently quantified using a NanoDrop ND 1000 Spectrophotometer (Thermo Fisher Scientific). In addition, BCA protein assay kit (23250, Thermo Scientific) was used according to the manufacturer’s instructions to assess the total protein amount of CDM.
Preparation of CDM-coated mesh scaffolds
After the decellularization, CDM was harvested by gentle pipetting, transferred to 50 mL tubes, and vigorously agitated using a homogenizer (HG-3000, SMT, Japan) until a homogeneous aqueous phase was formed. The polymer mesh scaffolds were then immersed into the CDM suspension solution with a mild agitation and incubated for 24 h. The CDM-coated mesh scaffolds were then freeze-dried overnight. Fibronectin (FN; BD Biosciences)-coated mesh scaffolds were also prepared by soaking the scaffolds in FN solution (50 μg/mL in distilled water) at 37 °C for 1 h. The FN-coated scaffolds were then rinsed with distilled water and freeze-dried. The surface morphology of the FN- and CDM-coated mesh scaffolds was observed via scanning electron microscopy (SEM; Phenom G2 Pro Desktop). In addition, the distribution of the CDM in the mesh scaffolds was visualized via immunofluorescence staining of fibronectin using mouse monoclonal antibody (SC-8422; Santa Cruz Biotechnology) and Alex Fluor 488-conjugated secondary antibody (goat anti-mouse IgG; Invitrogen), respectively.
In vitro culture of UCB-MSCs and proliferation assay
Human umbilical cord blood mesenchymal stem cells (UCB-MSCs) were kindly provided by MEDIPOST Co (Seoul, Korea). UCB-MSCs were cultured in Minimum Essential Medium alpha medium (α-MEM) supplemented with 10 FBS and 1 % P/S. Cells were seeded at 5 × 105 cells in culture flasks and maintained at 37 °C in a humidified 5 % CO2 atmosphere with a medium change twice a week. Passage 9 UCB-MSCs were used throughout the experiments. Mesh scaffolds were transferred into non-adherent 24-well tissue culture plates, onto which UCB-MSCs were slowly inoculated at a density of 5 × 104 cells per scaffold. Cells were allowed to adhere for 3 h and cultured in a growth medium for up to 5 days. Three different test groups (n = 3, per group) were prepared: 1) plain mesh (control), 2) fibronectin-coated mesh (FN-mesh), and 3) CDM-coated mesh (CDM-mesh). Cell proliferation was evaluated on 2nd and 5th day of culture using CCK-8 assay (Dojindo, Japan). Aliquots from each sample (100 μL) were transferred into a 96-well plate and measured for the absorbance at a wavelength of 450 nm using a Multiskan microplate reader (Thermo Scientific).
Osteogenic differentiation of UCB-MSCs
Osteogenic differentiation of UCB-MSCs was induced in the presence of osteogenic supplements such as 10 % FBS, 1 % P/S, 50 μg/mL ascorbic acid, 0.01 M glycerol-2-phosphate, 50 ng/mL bone morphogenetic protein (BMP)-2 and 100 nM dexamethasone for 1 and 3 weeks, respectively. Medium was changed every 2 or 3 day.
Alkaline phosphatase (ALP) activity
ALP activity of each group (n = 4 per group) after osteogenic induction for 3 weeks was analyzed using a Lab Assay ALP kit (Wako Pure Chemicals, Japan). Samples were incubated in the lysis buffer (0.1 % Triton X-100 in PBS) for 30 min at 37 °C. 50 mL of the lysis solution was added to 2 mg/mL of p-nitrophenyl phosphate (Sigma 104 tablet) in 0.1 M Tris–HCl buffer (pH 8.5). The absorbance was measured at 405 nm and normalized to the total amount of proteins in each sample lysate, which was assessed via BCA assay (Thermo Scientific).
Quantitative real-time polymerase chain reaction (q-PCR)
Gene expression of osteogenic markers, such as bone sialoprotein (BSP), collagen type I (Col I), ALP, and osteocalcin (OC) was analyzed via quantitative real-time PCR. Total RNA was isolated using a Trizol® reagent (Invitrogen) extraction method. The extracted samples were subsequently quantified using a NanoDrop ND1000 Spectrophotometer (Thermo Fisher Scientific). cDNA synthesis was performed using a Maxime RT premix kit (Intron). All polymerase chain reactions were carried out using ABI Prism 7500 (Applied Biosystems) and gene expression level was quantified using SYBR Green (RR420A, TaKaRa). Relative gene expression level was calculated by the delta delta Ct method. The primer sequences of the target genes are as follows BSP: CAACCACCCTCTTCACCACT (forward) and GATCTTCTGGGGTGGTCTCA (reverse); ALP: ATGGGATGGGTGTT CCTACA (forward) and GTCTTAGAGAGGGCGACGTG (reverse); Col I: CAAGAACCC CAAGGACAAGA (forward) and GAATCCATCGGTCATGCTCT (reverse); OC: CCAGTT CTGCTCCTCTCCAG (forward) and GCCCACAGATTCCTCTTCTG (reverse) Housekeeping gene is glyceraldehyde-3-phosphate dehydrogenase (GAPDH): GGGCTCTCCAGAACATCATC (forward) and TTCTAGACGGCAGGTCAGGT (reverse).
Harvested samples at 1 and 3 weeks were fixed in 4 % paraformaldehyde for 24 h, dehydrated, embedded in paraffin wax, and cut into 10 μm thickness. Those thin sections (n = 4 per group) were then subjected to Alizarin Red, ALP and von kossa staining, respectively.
Data are expressed as mean ± standard deviation. Statistical significance is determined via one-way analysis of variance (ANOVA) with a posthoc, Bonferroni’s multiple comparison test (GraphPad Prism 5, La Jolla, CA). Statistical significance is marked as * (p < 0.05), ** (p < 0.01), or *** (p < 0.001).