Tissue area (TA) was defined as the area of an extraction socket and surrounding bone Tibial bone defects and tooth extractions The osseous defects were created in the tibia and jaw (Fig. 1b, c) under ketamine/xylazine general anesthesia. selleck chemicals An ∼10-mm incision was made below the knee joint. The periosteum was reflected to expose the bone surface distal to the proximal metaphysis. A hole (1 mm in diameter) was drilled at 4.5 mm distal to the growth plate using a round bur <2,000 rpm under copious irrigation. Primary closures

were achieved using surgical staples. Next, the maxillary right second molar was extracted using dental instruments. The extraction sites were left as open wounds as clinically performed in human tooth extractions. Microcomputed tomography At killing, the maxillae and tibiae were dissected, fixed in 10 % formalin, and analyzed using microCT (μCT-100; Scanco Medical AG, Bruttisellen, Switzerland) CRT0066101 research buy to assess bone parameters in extraction sockets and tibial defects as well as in intact bone. The maxillae and tibiae were scanned at 10-μm voxel resolution

with an energy level of 70 kV. The extraction sockets were segmented by the semi-manual contouring method [21]. The tibial defects were scanned from 3.7 to 5.9 mm distal to the growth plate (Fig. 1b). To establish baseline bone responses to the treatment, the metaphyseal bone of the proximal tibiae from 1.2 to 3.5 mm distal to the growth plate and the interradicular bone between the mesial and distal roots of the maxillary first molars (Fig. 1c) were Momelotinib manufacturer assessed as well [22]. Data were analyzed using the

built-in Scanco software. Histomorphometry Amylase The maxillae and tibiae were demineralized in 10 % ethylenediaminetetraacetic acid at 4 °C, paraffin embedded, and sectioned at 5 μm. Hematoxylin and eosin, tartrate resistant acid phosphatase, and Masson’s trichrome staining was performed following standard protocols and/or manufacturer’s instructions (386A, HT15, Sigma-Aldrich, St. Louis, MO) [23]. Blood vessels in the extraction wounds were immunohistochemically stained. Briefly, sections were deparaffinized, nonspecific protein blocked, and incubated overnight with a rabbit von Willebrand factor (vWF) antibody (ab6999, Abcam, Cambridge, MA). Goat anti-rabbit IgG (AP307, Millipore) was used as a secondary antibody and proteins developed with 3,3-diaminobenzidine (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to assess apoptotic cells in the tibial defects (FragEL™ DNA Fragmentation Detection Kit, EMD Chemicals, Gibbstown, NJ). Empty osteocyte lacunae were quantified within 100 μm depth of bone surface. Necrotic bone area, defined as the portion of bone with greater than or equal to 10 adjacent empty osteocyte lacunae, was measured [24].