Preparation of nHA-coated, porous BCP ceramics
Clinical grade porous BCP ceramics were produced by Gong-Chuang Biofunctional Materials Co., Ltd, Hengyang, China. The specimens used for cell studies were discs of 11 mm in diameter and 2 mm in thickness, those for the flexural strength testing were 5 × 8 × 25-mm cuboids, and those for compressive strength testing were 5 × 5 × 12.5-mm cuboids. All specimens were polished with SiC sand paper (1500 grit), washed with anhydrous ethanol for 30 minutes in an ultrasonic cleaner, and then rinsed with deionized water prior to use.
A hydrothermal deposition method was applied to prepare nHA for coating, using analytical grade reagents purchased from Alfa Aesar. First, 0.3 mol/L ammonium dihydrogen phosphate and 0.5 mol/L nitrate tetrahydrate calcium solution were prepared in deionized water. Then 20 g PVP (polyvinylpyrrolidone) was added to the ammonium dihydrogen phosphate solution, which was heated with stirring until the PVP was completely dissolved, and after the pH value of the solution had been adjusted to 11 with concentrated aqueous ammonia, BCP was added to the solution. Calcium nitrate tetrahydrate solution was added under ultrasonic irradiation at a rate of 2 mL/min into the above reaction mixture, with continued ultrasound for 30 minutes, and the resulting mixture was transferred to the hydrothermal synthesis reactor (Qiang-Qiang Instruments, Shanghai, China) and maintained under hydrothermal conditions at 120°C for 12 hours. After completion of the reaction, the reaction vessel was naturally cooled to room temperature, and then the nHA-coated, porous BCP ceramic samples were removed, filtered, washed with anhydrous ethanol and distilled water alternately, and dried at room temperature before being ready for use.
After completion of the reaction, the precipitate was washed completely with ethanol to remove residual PVP molecules, collected, and dried at 60°C in a vacuum oven. Then a small amount of precipitate was ultrasonically dispersed in anhydrous ethanol, and a drop was taken and dripped onto an ultra-thin carbon film. After drying, the size and shape of the nHA were observed using field emission transmission electron microscopy (FETEM, JEM-2100 F, JEOL, Tokyo, Japan), and the morphology of the coating and pore size distribution on the material surface and section pore wall were observed using field emission scanning electron microscopy (FESEM, Nova NanoSEM 230, FEI Co., Hillsboro, OR, USA). For microstructure characterization, five view fields were examined on each of 12 samples for each specimen (60 fields in total). The phase compositions of the BCP and nHA-coated BCP surfaces were characterized using X-ray diffraction (XRD, D/ruax 2550PC, Rigaku, Japan).
The density and porosity of the specimens were measured according to Archimedes’ method. First, the specimen was dried in a vacuum oven at 60°C for 24 hours to determine the dry weight (W1). Then, the specimen was transferred to a beaker and placed in a vacuum oven at 2 kPa for 15 minutes, followed by slow injection with water until the specimen was completely immersed. Next, the pressure was gradually restored to atmospheric, and then the saturated specimen was placed in a copper wire basket, suspended in a beaker filled with water, and weighed (W2). The specimen was taken out of the water, any water remaining on its surface was removed with wet gauze, and the specimen was weighed again (W3). The porosity was calculated using the following formula: porosity (%) = [(W3 – W1) / (W3 – W2)] × 100%. The density was calculated using the following formula: density (g/cm3) = [W1/(W3 – W2)] × d, where d is the density of water (1 g/cm3).
Characterization of the mechanical properties
The compressive strength of the specimens was tested using a universal testing machine (Dual Column Testing System 3369, Instron Co., Norwood, MA, USA) with a crosshead speed of 0.2 mm/min. Compressive strength (C) was calculated by C = P/A, where P is the critical load and A is the specimen cross-sectional area . The flexural strength of the specimens was measured by the three-point flexural method with a span of 20 mm at a crosshead speed of 0.5 mm/min. Flexural strength (S) was calculated by S = 3 FL/2bd
2, where F is the maximum load, L is flexure span, b is specimen width, and d is specimen thickness . For each material, 12 specimens were tested to determine these mechanical properties.
Isolation and culture of bone marrow MSCs
MSCs were harvested from the bone marrow of 4-week-old New Zealand rabbits, using an extraction method similar to that described in Lim et al.  and Ouyang et al. . The bilateral posterior iliac crests of anesthetized rabbit were disinfected, and 4 ml bone marrow was extracted from the bilateral iliac crests using a puncture needle with saline containing 1000 U/ml sodium heparin and mixed with heparin. The anti-coagulated bone marrow was diluted with 5 times the volume of low glucose Dulbecco’s Modified Eagle Media (DMEM, Gibco, Rockville, MD, USA). The bone marrow mixtures were centrifuged by Percoll density gradient at 1800 rpm for 20 minutes at room temperature, and the nucleated cell layer in the middle was carefully drawn out, added to the DMEM, and cleaned by two 10-minute rounds of 1200 r/min centrifugation. The bone marrow nucleated cells were harvested and then resuspended in control media [DMEM supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA)]. These cells were inoculated in T-75 cm2 flasks at a density of 2 × 105 cells/cm2 in control DMEM and incubated at 37°C in a 5% CO2 saturated humid incubator. After 48 hours, the media was changed for the first time after washing twice with PBS to remove non-adherent cells, and then the media was changed every 3 days. When the cells approached confluence, the cell culture supernatant in the flask was discarded, the cells were washed two or three times with PBS, and 2.5 g/L trypsin/ethylene diaminetetraacetic acid (Gibco) was added for digestion. The cells were subcultured at a ratio of 1:3, and the third generation of cells was prepared for downstream-related experiments. The animal protocol used in the current study compliance with the relevant laws and institutional guidelines, and also was approved by the ethics review committee of the Third Xiangya Hospital, Central South University.
Seeding of MSCs onto BCP and nHA-coated BCP scaffolds
Prior to cell culture, the BCP and nHA-coated BCP discs were sterilized in a low temperature plasma sterilizer (STERRAD 100S, Irvine, CA, USA). Immediately before cell seeding, the nHA-coated BCP and BCP scaffolds were soaked in osteogenic media [control media further supplemented with 10 nmol/L dexamethasone (Sigma), 200 μmol/L L-ascorbic acid-2-phosphate (Sigma), and 10 mmol/L β-glycerol sodium phosphate (Sigma)]  for 24 hours. MSCs were suspended in osteogenic media. Twenty thousand cells diluted into 2 ml osteogenic media were added to each well of a 24-well culture plate containing a BCP or nHA-coated BCP scaffold and maintained in a 5% CO2 humidified incubator at 37°C. The media was changed every 2 days.
FESEM of MSCs attached to scaffolds
After culture of MSCs on BCP or nHA-coated BCP scaffolds for 1 day, samples were washed with PBS, fixed with 2.5% glutaraldehyde, dehydrated by an alcohol gradient, soaked in osmium tetroxide, dried, and coated with gold for observation by FESEM.
After 1 day or 14 days, the media was removed and the cells cultured on BCP and nHA-coated BCP discs were washed with PBS. The samples were treated with the dye solutions of the LIVE/DEAD Cell Imaging kit (Molecular Probes, Eugene, OR, USA) for 15 minutes at room temperature for staining of live and dead cells. Green fluorescent live cells and red fluorescent dead cells were viewed by epifluorescence microscopy (IX71, Olympus).
Cell proliferation was calculated by measuring two parameters, the percentage of live cells (P
Live) and cell attachment (C
Attach), according to previously established methods [24, 33]. Images were taken of three randomly selected fields of view for each sample (3 fields of view × 10 samples = 30 photographs per scaffold type), and live and dead cells were counted in each image. P
Live was calculated by dividing the number of live cells by the sum of the numbers of live and dead cells. C
Attach represents the number of live cells attached to the specimen and was calculated by dividing the number of live cells by the sample area. It is important to consider both P
Live and C
Attach, because P
Live will be high if few dead cells are present even if only a small number of live cells are present. By contrast, C
Attach provides an absolute measure of cell survival on the scaffolds.
Metabolic activity of the cells was analyzed using the MTT mitochondrial reaction, which is based on the ability of live cells to reduce a tetrazulium-based compound, MTT, to a purplish formazan product. Cell viability is proportional to the amount of dehydrogenase activity within the cells. After 14 days, BCP and nHA-coated BCP cell culture samples were transferred to new 24-well plates, washed twice with PBS, and incubated in 1 ml PBS and 100 μl MTT (5 mg/ml) solution at 37°C for 4 hours. The solution was then removed by aspiration, and 1 ml DMSO was added to completely dissolve the formazan. Two hundred-microliter samples of the final sample solutions were transferred to 98-well plates, and absorbance at 490 nm was measured with a microplate reader (Wallac Victor 31420 Multilabel Counter, Turku, Finland).
Osteogenic differentiation of MSCs on BCP and nHA-coated BCP scaffolds
After 14 days of MCS culture on each scaffold type, the culture supernatant in each well was aspirated, and the samples were washed twice with PBS before transfer to a new 24-well plate. In the new plate, 0.2 ml de-ionized distilled water was added to each well before storage at –20°C overnight, followed by three freeze–thaw cycles (–80°C and room temperature for 30 minutes each) to disrupt the cell membrane. Samples were then transferred to 1.5-ml centrifuge tubes and subjected to 5000 g centrifugation at 4°C for 5 minutes before careful collection of the supernatant.
Three osteogenic markers were detected: alkaline phosphatase (ALP), collagen type I (Coll I), osteocalcin (OC). ALP activity, Coll I production, and OC production were quantified using the Rabbit Total Alkaline Phosphatase (TALP) enzyme-linked immunosorbent assay (ELISA) Kit, the Rabbit Collagen Type I, Col I ELISA Kit, and the Rabbit Osteocalcin/bone gla protein ELISA Kit (all from Sino-American Biotechnology, Wuhan, China), respectively, according to the manufacturer’s instructions. The absorbance of the solution in each well was measured using a microplate reader (Wallac Victor 31420 Multilabel Counter) at 450 nm, and the ALP, Coll I, and OC concentrations were calculated according to standard curves established based on the standard density as the abscissa and the optical density value as the ordinate, with MSCs cultured on tissue culture plates in a control media serving as a control.
All data obtained are presented as mean ± SD values. One-way and two-way analyses of variance (ANOVAs) were carried out to detect significant effects of the experimental variables. Tukey’s multiple comparison tests were used with a P value of 0.05.