Production of BC membrane
Acetobacter xylinum (ATCC 52582) obtained from the American Type Culture Collection (Rockville, MD) was cultivated in 20 mL of medium in 100 mL flasks for 120 h at 28°C in static culture. The nutrient medium contained 2 wt% glucose, 0.5 wt% peptone, 0.5 wt% yeast extract, 0.27 wt% disodium hydrogen phosphate and 0.115 wt% citric acid. Bacterial cellulose pellicles were harvested and cleaned by immersion in 2 wt% NaOH solution at 80°C for 1 h. The pellicles were then immersed in 1 wt% NaClO solution for 30 min, washed with deionized water and sterilized by autoclaving (121°C for 15 min).
Production of BC supplemented with HA (BC/HA)
Two methods were used to introduce HA into BC.
Surface deposition of HA onto BC. HA was formed in BC hydrogel by performing alternating incubation cycles with calcium and phosphate solutions (Hutchens et al.,
2006). Briefly, BC membranes were incubated in a solution of CaCl2 (11 g/L) at pH 4.83 under agitation in an orbital shaker for 12 h at 23°C. The membranes were rinsed with deionized water and then incubated in a Na2HPO4 solution (8.52 g/L) for 12 h. The samples were then rinsed in deionized water and dried at 60°C to constant weight. The mass of HA in the cellulose was determined by subtracting the total composite weight from the average weight of control cellulose membranes. HA weight percentages were calculated by dividing the mass of the HA by the total composite weight. This was estimated at 50% of HA in the BC/HA membrane.
Hydroxyapatite mixture with the bacterial cellulose pulp. BC hydrogel was mechanically cut into slices to generate a BC pulp of 10% cellulose. After homogenization, BC solution was supplemented with hydroxyapatite at a 0.33% as previously reported (Varma and Babu
2005). The mixture was then vigorously stirred, poured into rectangular molds and dried at room temperature before use.
X-ray photoelectron spectroscopy (XPS) analysis
The surface chemical elements of BC, BC/HA(50%) and BC/HA(0.3%) membranes were analyzed with a PerkinElmer PHI 5600 XPS (Eden Prairie, MN) with a standard magnesium X-ray source (1253.6 eV). The emitted photoelectrons were detected at a 45° take-off angle and analyzed with a hemispheric electron energy analyzer operated at a pass energy of 187.9 eV for the survey scans. For each membrane three locations of 0.8 × 0.8 mm2 each were analyzed and averaged. The vacuum in each sample chamber was maintained at 10–10 torr during measurement. All measurements were done at the air-facing side of the membranes, which is the side that faced air during membrane preparation.
Saos2 osteoblast-like cells, a human osteosarcoma cell line with osteoblastic properties, were used in this study. The osteoblasts were cultured in a 3:1 mixture of Dulbecco-Vogt’s modified Eagle’s (DME) medium and Ham’s F-12 (H) (Invitrogen Life Technologies, Burlington, ON, Canada) supplemented with 24.3 μg/ml adenine, 10 μg/ml human epidermal growth factor (Chiron Corp., Emeryville, CA, USA), 0.4 μg/ml hydrocortisone (Calbiochem, La Jolla, CA, USA), 5 μg/ml bovine insulin, 5 μg/ml human transferrin, 2 × 10-9 M 3,3’,5’-triiodo-L-thyronine, 100 U/ml penicillin, 25 μg/ml gentamicin (Schering, Pointe-Claire, QC, Canada), and 10% foetal calf serum (NCS, fetal clone II; Hyclone, Logan, UT, USA). Sub-confluent cell cultures were trypsinized; cells were split 1:10 to maintain cell growth, and were subsequently incubated at 37°C in a humid 8% CO2 atmosphere.
Osteoblasts were seeded onto the different BC membranes at 5 × 105 for two days. Adherent cells were subjected to Hoechst staining. The samples were first fixed with methanol/glacial acetic acid (75/25) for 3 × 15 min, and washed 3 times with PBS. They were then incubated with Hoechst 33342 (H42) (Riedel de Haen, Seele, Germany) (1 μg/ml) in PBS for 15 min at room temperature in a dark atmosphere. After three washes with deionized water, the samples were observed and photographed using an epifluorescence light microscope (Axiophot, Zeiss, Oberkochen, Germany).
Lactate dehydrogenase assay
Since LDH is a soluble cytosolic enzyme that is released into the culture medium following loss of membrane integrity resulting from either apoptosis or necrosis (Melo et al.,
), it was used in our experiments to assess possible cytotoxicity resulting from BC membranes. Osteoblasts were seeded onto BC and BC/HA membranes and then cultured for 2 days. At this time medium was refreshed and cells were cultured for an additional two and four days. Culture supernatant was collected at days two and four for LDH assessment; 4 and 6 day cultures respectively after seeding. LDH activity was measured using an LDH cytotoxicity assay (Promega, Madison, WI), per the manufacturer's protocol. Briefly, 50 μl of each supernatant were transferred to a 96-well flat-bottom plate and supplemented with 50 μl reconstituted substrate mix. The plate was incubated in the dark at room temperature for 30 min. This assay is based on the conversion of L-lactate and NAD to pyruvate and NADH by released LDH (Gleitz et al.,
). To stop the reaction a volume of 50 μl of an acid solution was added to each well, after which approximately 100 μl of each reacted solution was transferred to a 96-well flat-bottom plate, and absorbance read at 490 nm with a X-Mark Microplate Spectrophotometer (Bio-Rad, Mississauga, ON, Canada). A positive control for total LDH activity release was added to the experiment. Both positive (PC) and negative controls (NC) were included. LDH release was calculated using the following formula:
MTT assay of osteoblasts cultured on BC membranes
Osteoblasts (5 × 105) were cultured on BC/HA membranes for 2, 4 and 6 days before being subjected to MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide)] assay as we previously reported (Denizot and Lang
1986). BC membranes that do not contain hydroxyapatite were used as control. A second control, i.e., BC/HA and BC membranes that were not seeded with osteoblasts, was also included. This allows for the measurement of non-specific adsorption of MTT to the BC membranes. Osteoblasts on each membrane were cultured in the presence of 1% (v/v) MTT solution (5 mg/mL) for 4 h, after which the supernatant was removed and the cultures were washed with PBS. Then 1 mL of HCl in isopropanol (0.04N) was added and incubated for 15 min. At the end of the incubation, 200 μL (in triplicate) of solution was transferred from each membrane to a 96-well flat-bottom plate, and the absorbance of the MTT (formazan) was determined at 550 nm using an ELISA reader (Model 680, BioRad Laboratories, Mississauga, ON, Canada).
Alkaline phosphatase assay
Alkaline phosphatase (ALP) activity in the supernatant of osteoblast cultured membranes was measured. In preparation for this assay, Saos2 cells (5 × 105) were seeded on BC, BC/HA(50%) and BC/HA(0.3%) membranes and allowed to adhere overnight. Medium was refreshed and cells were cultured for 2 and 4 days at 37°C in a 5% Co2 humid atmosphere. At the end of each culture period, medium was collected and centrifuged twice at 3000 rpm for 10 min to eliminate cell debris and ALP enzyme activity was assayed. One hundred μL of collected culture medium was supplemented with 100 μL of substrate solution (100 mg of 4-nitrophenyl phosphate disodium salt hexahydrate in 25 mL H2O) and 20 μL of alkaline buffer solution (1.5 M 2-amino-2-2 methyl-1-propanolol at pH of 10.3) in a 96 well plate, and the plate was incubated for 1 hour at 37°C. The reaction was stopped by adding 100 μL 0.3 M NaOH to each well. Optical density was read at 405 nm using an X-Mark microplate spectrophotometer (Bio-Rad, Mississauga, ON, Canada) and translated into ALP enzyme activity using a standard curve generated with p-nitrophenol (Sigma-Aldrich) ranging in concentration from 0 to 20 mM.
Qualitative and quantitative analyses of nodule formation
Osteoblasts (5 × 104) were cultured on BC and BC/HA membranes for 3 and 4 weeks. Membranes were washed twice with PBS and then stained with Alizarin Red S (ARS) solution for 2 min before being washed three times with sodium acetate buffer solution (pH 6.3) as we have previously reported (Meng et al.,
2011). Mineral nodules were documented by photomicrography at random locations on each membrane. To quantitatively assay nodule mineralization, the ARS stain was dissolved with cetyl-pyridinium chloride (CPC) (Fisher Scientific, Ottawa, ON, Canada) for 1 h under gentle agitation. One hundred microliters of the CPC from each membrane were transferred to a 96-well plate and diluted with 100 μL of water. Absorbance of ARS was determined at 570 nm by means of the X-Mark Microplate Spectrophotometer (Bio-Rad, n = 4).
Scanning electron microscopy (SEM) analysis
Osteoblasts (5 × 105/cm2) were cultured on BC and BC/HA membranes for 2, 4 and 6 days. The membranes were rinsed three times with PBS, fixed in 4% paraformaldehyde for 15 min, and rinsed again four times in distilled water. Dehydration was performed in a series of ethanol solutions of increasing concentrations (50, 70, 90, and twice at 100%), with a 5-min dehydration treatment in each solution. The dehydrated specimens were kept overnight in a vacuum oven at 25°C, after which time they were sputter-coated with gold and examined with a JEOL 6360 LV SEM (Soquelec) operating at a 30 kV accelerating voltage. The experiment was repeated four times and representative photographs were taken (n = 4).
Energy dispersive X-ray spectroscope (EDX) analysis
Osteoblasts (5 × 105/cm2) were cultured on BC and BC/HA membranes for 2, 4 and 6 days. The specimens were then subjected to EDX analyses. The specimens were fixed with ethylene glycol for 30 min and dried at 50°C. Following sputter coating with Au-Pd, the specimens were analyzed by means of EDX using a JEOL 840-A SEM (JEOL, Tokyo, Japan).
Experimental values are presented as means ± SD. The statistical significance of differences between the values was evaluated using a one-way ANOVA. Posteriori comparisons were done using Tukey’s method. Normality and variance assumptions were verified using the Shapiro-Wilk test and the Brown and Forsythe test, respectively. All of the assumptions were fulfilled. Data were analyzed using the SAS version 8.2 statistical package (SAS Institute Inc., Cary, NC, USA). Results were considered significant at < 0.05.