Materials
CS (200–800 cP, 1 % in 1 % acetic acid, Brookfield (lit.), degree of deacetylation is 75–85 %) was purchased from the Sigma Chemical Company and used without further purification. Paraffin with a melting point between 53 and 57 °C, gelatin, and N-hexane were obtained from Tianjin Kemiou Chemical Reagent Company.
Preparation and characterization
HA fabrication and characterization
HA can be produced from natural resources like corals, bovine bone, porcine bone [22] or cuttlefish bone. High temperature heat-treatment of animal bones was found to be most often used technique to produce natural HA. The porcine cancellous bone was collected from nearby butcher shops and all of the attached meat and fat were removed and cleaned from the bones. The ends of the cancellous bone were cut into slices about 2 mm, then cyclic soaked in hydrogen peroxide and ethyl ether to be degreased, deproteined, then being washed and dried. The dried degreased and deproteined bone was calcined (1000 °C, 3 h) to prepare the true bone ceramic (TBC), then the TBC was being ball milled at 230 r/min for 2.5 h.
Fourier transform infrared (FTIR) spectroscopy (NICOLET380 FT-IR) was performed in the wave number range 4000–400 cm−1. The sample was mixed with KBr and pressed into a pellet. The solid pellet was used for FTIR spectroscopy.
X-ray diffraction (XRD) (D8 DISCOVER, BRUKER company, German) was carried out to determine the crystal phases of the HA using monochromatic Cu Ka radiation at 40 kV. The 2θ scan range was 10–45°.
The particle size of HA was detected by laser particle size analysator. (LSPOP(6), OMEC company, Zhuhai, China).
Preparation of paraffin spheres
Paraffin spheres were prepared by emulsifying the paraffin in a heated 0.5 % (g/ml) gelatin solution and quenching the associated emulsion in ice water. 4 g gelatin was dissolved in 800 mL heated deionized water approximately 80 °C, and then 40 g paraffin was added into the solution. The mixture was mechanically stirred at 350 rpm to form a well dispersed suspension. Two hours later, 600 mL ice water was poured into the stirred suspension to solidify paraffin spheres. The suspension containing the paraffin spheres was then separated and subsequently rinsed with deionized water for several times. The paraffin spheres were sieved by standard sieves (50–90 mesh), after being dried in air, the paraffin spheres were collected and stored in a vacuum desiccator for further use.
The paraffin spheres were observed under the optical microscope (Olympus BX51, Japan) and the images were captured by a digital camera (Olympus DP71, Japan).
Preparation of micro-HA/CS composite porous scaffolds
Paraffin microspheres were used as the porogen in the fabrication of the scaffolds. Polymer scaffolds were fabricated by the technique of compression molding and particulate leaching method [21, 23]. The fabrication scheme of scaffolds was shown in Fig. 1.
In brief, micro-HA was dispersed in deionized water by ultrasonication, then CS and acetic acid was added (The final acetic acid concentration of the solution was 1 %.) under stirring for 1–2 h at room temperature until a homogeneous solution was obtained. Sieved porogen particles were added into the polymer solution to form a paste-like mixture which was then pressed into a PTFE mould and kept in a vacuum oven for 72 h to remove residual solvents. The composite sample was then taken from the mould and the porogen particles were removed using Soxhlet extractor with N-hexane as the refluxing solvent for 24 h. The resulting porous scaffolds were air-dried for 24 h, vacuum-dried for another 24 h and stored in a desiccator until further characterization.
Characterization
Scanning electron microscopy (SEM) observation
SEM was carried out to determine the effects of the paraffin spheres content on the pore structure of the composite scaffolds. Slices were cut from the porous scaffolds using a sharp blade for cross-section observation by SEM (LEO 1530VP, German) at an accelerating voltage of 10 kV. The samples were coated with gold prior to SEM observation.
Porosity measurement
The porosities of the scaffolds were determined by X-ray microcomputed tomography (Micro-CT) using a SkyScan 1172 system (Skyscan, Belgium) at 7 μm spatial resolution with an integration time of 2 s.
Water absorption rate
A known weight of dry scaffold (Wdry) was immersed in a 6 well cell culture plate filled with 0.1 M PBS solution at room temperature. The samples (n = 6) were removed from the 6 well cell culture plate after 24 h and weighed (Wwet). Water absorption rate of the scaffolds was calculated by the following equation:
$$ {\text{Water absorption rate}} = \left( {{\text{W}}_{\text{wet}} - {\text{W}}_{\text{dry}} } \right)/{\text{W}}_{\text{dry}} $$
Mechanical compress testing: stress–strain cyclic loading
The samples were tested on an Instron Model 5865 Materials Testing Machine (Instron Co., USA) at the ambient temperature. A thickness gauge was used to measure the thickness of each sample in four locations and the average thicknesses were used as inputs in the resulting stress–strain analysis. Cuboid scaffolds of 10 mm in width and 2–5 mm in height were compressed.
For all of the cyclic loading tests conducted, deformations of the samples were defined by the strain ε = ∆L/L0, where ∆L is the decrease in sample thickness relative to the initial thickness, L0. The resulting stress on each sample is defined by σ = F/A, where F is the compressive force and A is the cross-sectional area of the sample. In order to compare the time-dependent behavior, each cyclic loading test consisted of 15 compression cycles. Applied a preload of 0.1 N and compressive strain of 10 % was chosen for all samples.
Additionally, each sample was tested under four levels of loading frequency, 0.1, 0.5, 1 and 1.5 Hz. For each material group three samples were tested and the average and standard deviation were calculated. Samples were compressed under a sinusoidal strain defined by ε = ∆ε sin(ωt), where ∆ε and ω are the respective strain amplitude and frequency. The response (a strain) though sinusoidal is not in phase with the developed stress, and lags behind the stress by phase angle 90. Reproducible stress–strain curves were plotted and showed in the display. Results of the cyclic loading tests were analyzed by calculating both the instantaneous and steady-state tangent moduli, Eint and Ess, which correspond to the peak stresses at the first and last cycle relative to the respective strain amplitudes.
Hysteresis analysis
Hysteresis of the energy dissipating capabilities is a fundamental property of all viscoelastic tissues. In order to determine how the paraffin spheres content affected the mechanical property of composite scaffolds, integrals were carried out over each hysteresis loop and were recorded as the amount of energy dissipation per unit volume of material.
Statistical analysis
SPSS11.5 was used to evaluate the significant differences among the six groups. Data were presented as mean ± standard error. In all cases, the results were considered statistically different at p < 0.05.
