Cell Culture and Induction
Bone marrow cells were obtained from male BALB/c mice (6–8 weeks of age). Low-density bone marrow mononuclear cells (MNCs) were isolated by density centrifugation over Histopaque®-1083 (Sigma-Aldrich, US). Cells were then cultured in flask at 37°C and 5% CO2 atmosphere for differentiation of osteoblasts and endothelial cells, respectively. For osteogenic cell induction, cells were cultured in complete media [16, 17] consisting of DMEM supplemented with 10% fetal bovine serum (FBS) (Invitrogen, US), 10 mM β-glycerol phosphate (Sigma-Aldrich, US), 10-4 M L-ascorbic acid (Sigma-Aldrich, US), and 10 nM dexamethasone (Sigma-Aldrich, US), 2 mM glutamine (Invitrogen, US), 100 U/ml penicillin (Invitrogen, US), 100 μg/ml streptomycin (Invitrogen, US). To promote the endothelial phenotype of EPCs, the mononuclear cells were plated onto flasks coated with fibronectin (Sigma-Aldrich, US) and cultured in endothelial cell basal medium-2 (Cambrex, US) supplemented with EGM-2 MV SingleQuot® kit containing 5% FBS, human epidermal growth factor (hEGF), human vascular endothelial growth factor (VEGF), human insulin-like growth factor-1 (IGF-1), hydrocortisone, penicillin (Invitrogen, US), and streptomycin (Invitrogen, US). After 4 days of culture, non-adherent cells were discarded by washing with PBS. When 60% confluence was achieved, cells were subcultured.
Immunocytofluorescence studies were performed to detect the induced endothelial phenotypes. The induced ECs were fixed in 4% paraformaldehyde, permeated with 0.01% Triton X-100 in PBS, and incubated in 1% block serum for 1 h at 37°C. The cells were then incubated for 1 hour with monoclonal antibody against either mouse VEGFR-2 or mouse vWF (Santa Cruz, US). Bound antibodies were detected by incubation with fluorescein-5-isothiocyanate (FITC)-conjugated (Jackson ImmunoResearch, US) (for VEGFR-2) or Alexa Fluor 488-conjugated (Molecular Probes, US) (for vWF) secondary antibody. The cells were examined in fluorescence microscope. 300 μl of Matrigel™ (BD Biosciences, US) mixed with 4 × 104 EPCs-derived ECs at 4°C was dispensed into a 24-well plate and incubated at 37°C until solid. Photographs of capillary-like formation were taken at 7 days of culture in normal condition.
Similar fixation, permeabilization, and blocking processes were performed on bone marrow-derived osteoblasts, followed by the incubation with anti-osteocalcin (Santa Cruz) for 1 hour, and visualization was achieved using avidin-peroxidase complex (ABC kit, Santa Cruz Biotechnology, US). Cells were counterstained with Gill's hematoxylin solution. Calcium deposit produced by osteoblasts was demonstrated using von Kossa staining. After fixation in 4% paraformaldehyde, the cells were incubated with 1% silver nitrate solution (Sigma-Aldrich, US) under ultraviolet light for 20 minutes, and unreacted silver was removed by 5% sodium thiosulfate (Sigma-Aldrich, US). The alkaline phosphatase (ALP) activity of osteoblasts was assayed using an ALP kit (Sigma-Aldrich, US). The induced osteoblasts on slides were fixed in citrate-acetone-formaldehyde solution at room temperature for 1 minute. Following incubation in alkaline-dye mixture for 15 minutes and rinsing in distilled water, the slides were counterstained with hematoxylin solution.
Preparation of HA-PCL Scaffolds
PCL-HA scaffolds were prepared using a particulate leaching technique as described previously . The HA-PCL composite at 2 different component ratios were prepared respectively, with HA (Sigma-Aldrich, US) to PCL (Aldrich, US) at 1:1 (Group A) or 1:4 (Group B) wt/wt. PCL scaffolds without HA were used as a control (Group C). In each group, NaCl particles (particle size 212–355 μm) were used to generate a controlled level of porosity in the matrix with weight ratio to PCL at 16:1 (Group A), 8:1 (Group B) and 4:1 (Group C). PCL (Mn 80000) was dissolved in tetrahydrofuran (Sigma-Aldrich, US) at 10% wt/vol for 12 hours. HA powder (≤ 40 μm particle size) and NaCl particles were mixed to homogeneity in the PCL solution, which was sonicated for 60 seconds until viscous slurry developed. Mixtures were poured into glass dishes to a thickness of 4 mm, and dried at 37°C. After evaporation of the solvent, 1.5 × 1.5 cm squares were cut out and washed in excessive distilled water to leach out the NaCl. All materials were then sterilized in 70% ethanol and dried before biological evaluation. Samples of the PCL-HA scaffolds were gold sputter coated and their morphology was observed using SEM (Hitachi S-2400, Japan) at 15 kV. Energy Dispersive X-ray (EDX) analysis was also conducted to confirm the existence of HA particles on the composite scaffolds. The atomic percentages of calcium and phosphorus were calculated.
Cells Culture on Scaffolds
Induced osteoblasts or endothelial cells in 50 μl suspensions (3.5 × 106 cells/ml) were respectively loaded onto each scaffold in 6-well plates. The scaffolds were left undisturbed in a 37°C incubator for 3 hours to allow cells to attach to the scaffold, after which the cells-materials complex were kept in culture using the original osteogenic or endothelial media. At day 7 the samples were harvested for and biochemical evaluation.
For morphological examination, cells-materials complex were fixed with 1.5% glutaraldehyde (Fisher Scientific, US) for 30 min at 4°C. The samples were exposed to 2% osmium tetroxide (Sigma-Aldrich, US) for 30 min. Following rinse in distilled water, they were dehydrated through a graded series of ethanol (50, 70, 90, and 100%) for 2–5 min. The dehydration was completed in hexamethyl disilazane (Fluka, Germany) for 10 minutes. After air-drying and sputter coating with gold, the cells morphology on the PCL-HA scaffolds was evaluated using SEM at 10 kV.
Assessment of Cell Viabilities and Functions on Scaffolds
For biochemistry assay, each type of cells was seeded on 30 scaffolds per group. Cell viability was evaluated by analyzing the mitochondrial activities of the cells. The Alamar Blue assay (BioSource, US) was used to determine the mitochondrial activity after 7 days of cell culture. The cells-materials complexes were washed in Phosphate-Buffered Saline (PBS) in 6-well plate. 3 ml of new conditioned media supplemented with 200 μl of Alamar blue was added to each well. Incubation was continued at 37°C, 5% CO2, for 3 hours. The culture medium was then transferred to a 96-well plate and read on a spectrofluorometer (excitation wavelength 530 nm, emission wavelength 590 nm). The Alamar blue absorbance/mg of DNA values was calculated for each sample.
Cell amount were determined by a fluorometric quantification of DNA in the cells-materials complexes. After the Alamar Blue assay, the cell-scaffolds were rinsed with PBS, followed by 4 times of freezing (-80°C) and thawing (37°C) cycles for 15 minutes each. The scaffolds were then homogenized in 1.4 ml of cold 10 mM EDTA solution (Sigma-Aldrich, US). The pH of the samples was adjusted to 7.0 by adding 1 M KH2PO4 prior to the addition of 1.5 ml of the 200 ng/ml Hoechst 33258 fluorescent dye (Sigma-Aldrich, US). 100 μl of supernatant sample were read with an excitation set at 350 nm and an emission at 455 nm on a spectrofluorometer. The DNA concentration in the samples was determined against a DNA standard curve that was plotted according to a series of 100 μl of calf thymus DNA (Sigma, US) in a range of concentrations from 0.15265 to 20 μg/ml. The DNA values were used to normalize the cell viability and other cell function parameters.
Production of ALP by osteogenic cells was measured using a spectrophotometer. After the previous freeze-thaw cycle and DNA assay, 50 μl of the sample was transferred to a fresh 96-well plate and 50 μl of p-nitrophenyl phosphate solution (Sigma, US) was added to each sample. Following incubation for 5 min at 37°C, the production of p-nitrophenol in the presence of ALP was measured by monitoring light absorbance at 405 nm. The measurement of the ALP assay was normalized against the amount of total DNA in each sample.
The nitric oxide generated by endothelial cells on scaffolds was assessed using the Nitric Oxide Colorimetric Assay kit (Calbiochem, Germany) in accordance with the manufacturer's instructions. The presence of nitric oxide in the culture media of endothelial cells-materials complex was determined by detecting the colored product spectrophotometrically. The absorbance was read at 540 nm. The nitric oxide concentrations for samples were calculated according to the standard curve. Cellular nitric oxide amount was normalized by total DNA each sample.
All experiments were replicated three times to ensure the reproducibility, and all data was presented as the mean ± standard deviation. Single factor analysis of variance (ANOVA) with a post hoc LSD from SPSS™ (Student Version 10.0.5, Chicago, IL) was used to assess the statistical significance among groups, which was defined as p < 0.05.