001), and 3-fold wider
Rea at the calcified cartilage/bone plate (p<0.001), and 3-fold wider hole area at deeper levels (p<0.001 vs controls, Figure 9B). By micro-CT, high molecular weight chitosan induced a particularly strong bone resorption at 0.5mm and 1.5mm depths (p<0.05, 150K vs 40K hole area, Figure 3-(2,4-Dichlorophenoxy)azetidine 9B), which was consistent with histomorphometry measures in collagen type I-stained sections showing a larger area of granulation tissue in 150K-treated defects than in both 40K and 10K-treated defects after 21 days (p<0.05, data not shown). Micro-CT analysis of hole depth also showed that middle control bone defects were significantly shallower than the 40K-treated middle hole (p<0.05, Figure 9C). The strong resorption in treated defects was accompanied by the synthesis of new woven bone and angiogenesis (Figure 10). Although the treated holes were bigger than the initial 2 mm deep, 1.4 mm diameter drill holes (Figure 9B, Figure 10A), subchondral new woven bone was identified by characteristic high-density osteocytes with a largercell diameter compared to small osteocytes in peripheral nonremodeled trabecular bone (Figure 10B). Blood vessels were detected in the granulation tissue of treated defects (Figure 10B). The synthesis of new woven bone demonstrates that the implant has induced remodeling and not exclusively osteoclastic resorption. In control defects, no signs of bone resorption were present (Figure 9B, Figure 10D) and the subchondral bone contained large blood vessels, low levels of new woven bone, as chondrocytes fused to bone (Figure 10E) which indicates that endochondral ossification is underway. Osteoclast-induced bone remodeling in the bone plate area of treated defects was associated with a 10-fold greater lateral integration of treated repair tissues with the bone plate compared to control defects with detached repair (p<0.05, Figures 11 and 12). The mean lateral detachment was <35 m deep in treated holes versus 350 m deep in control drill holes in sections immunostained for collagen type II (Figure 11A-C, Figure 12), collagen type PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/4155310 ILafantaisie-Favreau et al. BMC Musculoskeletal Disorders 2013, 14:27 http://www.biomedcentral.com/1471-2474/14/Page 10 ofFigure 7 Distribution of repair tissues in treated defects and volume density of neutrophils and stromal cells. Relative crosssectional area of apoptotic, neutrophil-rich, neutrophil+stromal and stromal tissues in sections collected (A) at the edge and (B) through the middle of treated defects in collagen type I-stained cryosections (N=4). Apoptotic tissues cover a larger area in the middle of 150Ktreated defects after 21 days than 40K and 10K-treated defects (p=0.0417, p=0.0515, respectively). (C) Volume density (Vv) of neutrophils and stromal cells in 40x magnification pictures of neutrophil-rich, neutrophil+stromal and stromal tissues in the middle of day 21 1-(4-Bromo-2-pyridyl)piperazine treated defects of collagen type I-stained cryosections (N=4).(Figure 11D-F), and stained PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 for Safranin O (Figure 4L). Given that the bone plate (including the calcified cartilage) is 400 m thick in skeletally mature rabbits [23,24], these data showed that the marrow-derived fibrocartilage repair in control defects was generally not integrated with the bone plate. All three chitosan formulations reproducibly induced the same improved level of bone repair tissue integration.Discussion This study demonstrated that subchondral chitosan implants sequentially draw neutrophils and undifferentiated bone marrow-derived stromal cells into dr.