Materials
PCL (Mw 80,000 Da) and tetraglycol (glycofurol) as a nontoxic solvent for PCL were purchased from Sigma-Aldrich (USA). All other chemicals were analytical grade and were used as received. Ultrapure grade water (>18 mΩ) was purified using a Milli-Q purification system (Millipore Co., USA). For animal study, the porous PCL beads were sterilized by ethylene oxide (EO).
Preparation of porous PCL beads
Porous PCL beads were simply prepared by spray/precipitation method using double nozzle spray. PCL pellets were dissolved in tetraglycol at 90°C (15 wt%), and the PCL solution was immediately transferred in a 10 mL syringe. The warm solution was sprayed through a double nozzle spray with N2 purging of 2.5 L/min (outer nozzle) into 50% ethanol solution (coagulation solution) to induce the solidification (precipitation) of PCL solution (Figure 1). Feeding rate of the solution was fixed to 60 mL/h (inner nozzle, using syringe pump). The syringe and double nozzle spray were heated (90°C) using a heating system equipped with heating tape (PID temperature controller, Model, TC130P; heating tape, Model, HT2510; Misung Scientific, Korea) to prevent precipitation of PCL during the process. The distance of tip-to-coagulation solution was 20 cm. The precipitated PCL beads were maintained at coagulation solution for 6 hrs, then the PCL beads were washed out in excess water for 24 hrs to remove residual tetraglycol and ethanol. The PCL beads were obtained by centrifugation and dried in a vacuum oven overnight, and the beads were separated in different size ranges (53 ~ 100, 100 ~ 200, 200 ~ 300, 300 ~ 425, 425 ~ 500, 500 ~ 600 μm) using standard testing sieves (Chunggye Industrial Co., Korea).
Characterization of porous PCL beads
Morphology observation and porosity measurement
The morphology of prepared porous PCL beads was observed by a field emission scanning electron microscope (FE-SEM; Model S-4300, Hitachi, Japan). The cross-sectional specimen was prepared by cutting them using a blade after being frozen in liquid nitrogen. The porosity of the PCL beads was estimated using mercury porosimetry (Poresizer 9320; Micromeritics, USA). To determine the porosity, it was assumed that the surface tension of mercury is 480 dyne/cm [17].
Preliminary animal study
Sprague–Dawley (SD) rats (~250 g) were selected as an animal model to estimate the bone reconstruction potential by the use of porous PCL beads. The animal study was permitted from the Animal Care Committee of the Hannam University in Korea, and all surgical procedures were performed according to the guidelines. The rats were completely anesthesized with intramuscular injection of tiletamine/zolazepam (10 mg/kg; Zoletil 50®, Virbac Laboratories, France) and 2% xylazine hydrochloride (2 mg/kg; Rumpun®, Byely, Korea), and placed in the prone position. The left leg was shaved and the skin surface discontaminated with 7% tincture of iodine. The front skin of the mid-femur in rats was incised straight and longitudinally at 30 mm in length. After splitting the muscle, the periosteum was stripped to expose the femoral bone surface. Two drill-holes (each hole, ~4 mm in diameter) were created through the femoral cortex using a small tungsten carbide dental bur with a diameter of 0.8 mm in the anterior portion of the diaphysis of one femur (10 mm position from the knee joint) (Figure 2). The bone defect was prepared with a very gentle surgical technique and continuous internal cooling with physiological saline solution. Then the porous PCL beads were implanted into the defect, and the wound was closed in two layers using 4–0 vicryl and 4–0 nylon sutures. The blank (w/o any treatment) was also studied as a control group. During surgery and 24 hrs later, all animals received subcutaneous injections of antibiotics (sulfadoxin and trimethoprin, 5:1, 15 mg/kg) to minimize the risk of infections. At 4 weeks after implantation, the rats were sacrificed by an overdose of CO2 gas. For histological observation, the bone defects with surrounding femoral bone were harvested, and the specimens were fixed with 4% formaldehyde and decalcified in 10% formic acid. After dehydration procedure of the fixed specimens in a graded series of alcohol, the specimens were embedded in paraffin and cut into 5 μm transverse sections in the defects. The sections were stained with Hematoxyline & eosin (H&E) and Masson’s trichrome (MT) for observation by light microscopy (Model BX50F4, Olympus). For radiographic evaluation, the tissue specimens harvested from the femur of rat were frozen, and placed on an automatic axially-moving turntable to scan using a micro-computed tomography (μ-CT) system (Skyscan 1176, Skyscan N.V., Belgium)).