Preparation of silk scaffolds coated with nano-hydroxyapatite
Silk sutures were purchased from Won Corporation (Seoul, South Korea) and silk scaffolds were constructed using a weaving machine. The silk scaffolds were processed by extracting sericin, a glue-like protein that coats the native silk fibroin, using an aqueous solution containing 0.02 M Na2CO3 and 0.3% Ivory detergent. 0.15 g of nano-hydroxyapatite (Sigma) was dissolved in 10 ml of PBS and 1ml of the solution was dried on the silk scaffold (0.8×1.2 cm) in air. The scaffolds were then soaked in a 1% type atelocollagen solution (Bioland, Korea) and lyophilized in a freeze dryer (Samwon Freezing Engineering Co., Korea) at −80°C for 48 h. The silk scaffolds were incubated in 20 ml of 40% (v/v) ethanol containing 50 mM 2-morpholineoehtane sulfonic acid (MES, Fluka Chemic AG) (pH 5.5) for 30 min at room temperature. The silk scaffolds were then immersed in 20 ml of 40% (v/v) ethanol containing 50 mM MES (pH 5.5), 24 mM 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (Fluka Chemic AG) and 5 mM N-hydroxysuccinimide (Fluka Chemic AG) for 12 h at room temperature. Once the reaction was complete, the composite silk scaffold was washed twice in 0.1 M Na2HPO4 (pH9.0) for 12 h. The scaffolds were washed twice in 1 M NaCl for 6 h, then in 2 M NaCl for 2 days and finally rinsed with distilled water. The washed scaffolds were lyophilized again and sterilized with γ-irradiation at 15 KGy.
Primary cell culture
DPCs
Extracted third molars were used in experiments with the approval of patients. The patients provided informed consent for their tissue being used in accordance with guidelines approved by the Institutional Review Board, College of Dentistry, Yonsei University (IRB No. 2-2010-0009). The extracted third molars were washed with PBS containing antibiotic antimycotic (AA) (Welgene, Korea) for 3 mins after washing with 70% ethanol. The third molars were wavered with pliers and the dental pulp was removed. The pulp tissues were digested in a solution of 3 mg/mL collagenase type I (Sigma St. Louis, Mo., USA) and 4 mg/mL dispase (Siga, St. Louis, MO., USA) for 12 h at 4°C. The pulp tissue was then incubated in 3 mL of 0.25% trypsin for 15 min at 37°C. After trypsin digestion, the trypsin solution was diluted in alpha Modified Eagle’s Medium (α-MEM, Sigma) containing 10% fetal bovine serum (FBS, BioWhittakerTM, Cambrex, MD, USA). The cells were isolated from the dental pulp by pipetting. After centrifugation at 800 rpm for 5 min, the supernatant was removed and added to 10 mL of α-MEM. The cell suspensions were seeded in 100 mm culture dishes.
PDLCs
Human periodontal tissue was obtained from several extracted third molars of patients who had given their informed consent (IRB No. 2-2010-0009). The extracted third molars were washed with PBS containing antibiotic antimycotic solution (AA) (Welgene, Korea) for 3 min after washing with 70% ethanol.
The periodontal tissue was removed from the roots of the teeth and then divided into small pieces with scissors and tissues were digested in a solution of 3 mg/mL collagenase type I (Sigma, St. Louis, MO, USA) and 4 mg/mL Protease (Sigma, P 3417, USA) for 12 h at 4°C. Then, PDL tissues were incubated in 3 mL of 0.25% trypsin for 15 min at 37°C. After trypsin digestion, the trypsin solution was diluted with alpha Modified Eagle’s Medium (α-MEM, Sigma) containing 10% fetal bovine serum (FBS, BioWhittakerTM, Cambrex, MD, USA).
After centrifuging at 800 rpm for 5 min, the supernatant was removed and pipetted with 10 mL of α-MEM. The cell suspensions were seeded in 100 mm culture dish. The PDL fibroblasts from third to five passages were used.
Seeding of cells in silk scaffolds
DPCs and PDLCs, suspended in alpha Modified eagle’s Medium (α-MEM, Sigma) supplemented with 10% fetal bovine serum (FBS, BioWhitekerTM, Cambrex Bioscience Walkersville, Inc., MD), were seeded onto the silk scaffolds. To obtain a very high cell seeding density, the cells were seeded onto the silk scaffolds under a dried condition. 100 μl of a harvested suspension – containing 2.8 × 104 human dental pulp cells or 1 × 106 human PDL cells – and silk scaffolds were placed in the petri dish in a humidified 5% CO2 incubator, and 3h later, 10 ml of α-MEM was added to the petri dish. The medium was replaced every 3 days for 7 days. After incubation for additional 7 days, the scaffolds were cultured in differentiation medium, which was replaced every 2 days for 3 weeks. The differentiation medium for DPCs was alpha Modified Eagle’s Medium (α-MEM, Sigma) supplemented with 10% fetal bovine serum (FBS, BioWhittakerTM, Cambrex, MD, USA), 1% AA, 100 nM dexamethasone, 0.05 mM Ascorbic acid and 10 mM β-glycerophospate. The differentiation medium for PDLCs was α-MEM containing 10% FBS, 10 mM β-glycerophosphate (Sigma), 50 μM L-ascorbate2-phosphate (Sigma) and 10−7 M dexamethasone (Sigma). The result of cell-seeding procedure was checked with scanning electron microscopy.
Animals
Eight male New Zealand White rabbits weighing 2.8-3.2 kg and age 16–20 weeks were used in this study. Animal selection and management, surgical protocol and preparation followed the routines approved by the Institutional Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea.
Study design
Four circular defects (8mm diameter) were created in each animal, which were randomly allocated to the four study groups including PDLC-silk scaffold group (PDLSS group), DPC-silk scaffold group (DPSS group), normal saline-soaked silk scaffold group (SS group) and an empty control group. The animals were euthanized at either 2 or 4 weeks postsurgery.
Surgical protocol
General anaesthesia was induced in all animals using intramuscular injections of zoletil (15 mg/kg) and rompun (5 mg/kg). The head of the rabbit was shaved and disinfected using povidone iodine prior to local anaesthetic injections at the surgical site using 2.2 ml Lidocaine Hydrochloride 2% with adrenaline 1:80,000. An incision was made along the midline from the frontal bone to the occipital bone and a full-thickness flap was elevated. Under copious saline irrigation, four standardized round defects 8 mm in diameter were created using a trephine bur (Figure 1). The resected bones were removed carefully to avoid injury to the underlying brain tissue. The defects were filled with the experimental materials according to the assigned group. The flaps were repositioned and sutured with resorbable suture material.
The animals were sacrificed at either 2 or 4 weeks postsurgery. The skin flaps were reflected to expose the periosteum and the entire calvarium was resected and harvested from each animal.
Histologic processing
Block sections of the surgical sites were fixed in 10% formalin for 10 days. The fixed specimens were decalcified in 5% formic acid for 14 days and then embedded in paraffin. Serial 5 μm thick sections were cut along the midline of the calvarial defects. Only sections located at the middle of the defects were selected, and stained with hematoxylin-eosin for histologic and histometric analysis.
Analysis methods
Histomorphometric analysis
After the conventional microscopic examination, computer-assisted histometric measurements in the calvarial defect were performed using an automated image analysis system (Image-Pro Plus; Media Cybernetics, Silver Spring, MD). The following parameters were measured from each histologic section of the defect areas.
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Total augmented area (mm2) – Total sum of the area of new bone, residual particles, connective tissue, adipose tissue, blood vessels within the defect area.
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Residual material (mm2) – Area of the remaining silk scaffold within the defect.
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New bone (mm2) – Area of newly formed bone within the defect.
Statistical analysis
The statistical analysis was performed using a commercially available software program (SPSS 18.0, SPSS, Chicago, IL). Histomorphometric records from the calvarial defect samples were used to calculate the mean and standard deviation (SD) values of groups. One-way analysis of variance (ANOVA) was used to analyze the difference between the groups at 2 and 4 weeks. Statistical significance was considered when P <0.05.
SEM observations
The morphology of nHA-coated scaffolds before and after the cell-seeding procedure were observed by scanning electron microscopy (SEM; S-3000N, Hitachi, Tokyo, Japan) at an accelerating voltage of 30 kV.
Clinical and histological findings
Throughout the healing periods, the animals were carefully monitored for adverse reactions around the surgical site. The specimens were examined by a single, blinded examiner with the aid of a binocular microscope (DM LB, Leica Microsystems, Wetzlar, Germany) equipped with a camera (DC300F, Leica Microsystems). Images of the slides were acquired and saved as digital files.