2.3. In vivo study of critical-sized rat calvarial defect model

Procedures involving animals were conducted in accordance with the guidelines of the Institutional Animal Experiment Department of Shanghai Jiao Tong University (Shanghai, China; Animal Ethics Approval #201040) and principles of laboratory animal care (NIH publication number 85–23, revised 1985) and ethics committee specifically approved this study.

All surgical procedures were performed on eight-week old male Sprague–Dawley rats, weighing approximately 300–350 g and all rats acclimated for 14 days prior to surgery. The animals were anesthetized with 5% ketamine. Following anesthesia, the surgical areas were shaved and disinfected with povidone-iodine. A 1.0–1.5 cm vertical incision was made on the proximal-medial area of the calvaria and the soft tissue and the periosteum were elevated to expose calvaria. Unilateral critical-sized defect was created in each rat with micro drills (Stryker, Mahwah, NJ, USA) of 5 mm in diameter at low rotation speed with constant irrigation. The 32 rats were randomly allocated to the following graft study groups: (1) 3DP PLA/HA (n = 8), (2) β-TCP (n = 8), (3) DBM (n = 8) and (4) blank control (n = 8). The periosteum was repositioned and sutured with a 5–0 PDS suture. Then the skin was sutured by 4–0 silk suture. Following the operation, the animals were allowed rat food and water ad libitum. After four or eight weeks, the animals were sacrificed with carbon dioxide gas suffocation.

In order to assess local inflammatory reaction after implanting different kinds of materials, blood samples were collected and analyzed with a routine blood test. At 12 days, four weeks, six weeks and eight weeks after surgery, blood was sampled from heart puncture in tubes containing ethylenediaminetetraacetic acid (EDTA). With the help of a counting chamber, total leukocyte and red cells numbers as well as hemoglobin content were assessed. Differential counts of white blood cells including lymphocytes, monocytes, eosinophils, and basophils were determined

After sacrificing the animals at four and eight weeks post-operatively, the skulls were dissected out and fixed in neutral 10% formalin then examined with micro-CT (Scanco Medical AG, Basserdorf, Switzerland). Besides, micro-CT scanned at 10 μm resolution for undecalcified tissue samples and original materials that were used to evaluate new bone formation and materials degradation rates. Three-dimensional images were reconstructed using 3D Creator software. Three-dimensional measurements of the amount of bone volume per total volume (BV/TV) and residual materials volume fraction (RMvF) [38] in the bone defect were also calculated using the analysis software.

Subsequent to μCT analysis, the specimens were decalcified by immersing in 10% EDTA for four weeks at 37°C and during this period the solution was changed three times.

Decalcified specimens were dehydrated in ascending grades of ethanol and embedded in paraffin. To compare and visualize new bone formation, 5 μm thick serial slices with a microtome were sectioned starting from the middle region of the defect area. Sections in all groups were stained with hematoxylin and eosin (HE). Immunohistochemical staining was further carried out to assess and analyze the formation of new bone. Sections were then incubated with the primary antibody: against osteocalcin (OC) and type I collagen (COL-1) (Abcam, Cambridge, MA, USA) at 4°C overnight. After rinsing with the PBS buffer, the HRP-conjugated secondary antibody and 3,3′-diaminobenzidine (DAB) solution was used to stain the sections, and then cell nuclei were to identified with hematoxylin. Immunohistochemical experiment was performed on four samples per group (n = 4). The images were acquired using a microscope (Leica DM 4000B, Wetzlar, Germany) with BioQuant OSTEO II software (BioQuant Image Analysis Corporation, Nashville, TN, USA). The OC and COL-1 positive areas were calculated using Image-Pro Plus 6.0 software (SPSS 17.0, Chicago, IL, USA) to evaluate the formation of new bone.