Characteristics Of Bone Qulity And Soft Tissue Calficiation In OPG-knock-out Mice

Part one Effect of osteoprotegerin gene knockout on mandible mineralization and bone mass in miceObjective:To characterize the changes of bone mineralization and bone mass in mandible in osteoprotegerin gene knockout (OPG-/-) mice.Methods:OPG-/- and wild-type (WT) mice (six-week-old female) were assigned for this study and divided into two groups(n=10 in each goup). They were sacrificed at 4 weeks after fasting for 12h. The right mandible of seven mice was selected for microCT analysis.Results:Mineralization in anterior area (17%) and condyle (10% ) in cortical bone was showed a more significant decrease in OPG-/- group than that in WT group (P<0.05). Trabecular volumetric BMD, tissue BMD, BV/TV, Tb.Th, Tb.N, and Conn.D were all considerably lower than the controls, while BS/BV, Tb.Sp and SMI were markedly higher in the OPG-/- group than that in WT group (P<0.05).Conclusions:OPG-/- induces a dramatic bone loss and micro-architectural deterioration with a decreased cortical mineralization of trabecular bone in an mandible area-specified way. Part two Evalutaion of bone quality in OPG- knockout miceObjective:To investigate the changes of bone mineral density (BMD), microarchitecture, biomechanical property and serum biochemical markers of bone turnover in OPG knockout (OPG-/-) mice?Methods:Bone microarchitecture and loss of bone mass were assessed by hematoxylin-eosin staining, scanning electron microscope and micro-CT. The biomechanical property was determined by a three-point bending test. Serum biochemical markers of bone turnover such as bone alkaline phosphatase (B-ALP), tartrate-resistant acid phosphatase-5b (TRAP-5b) and receptor activator of nuclear factor?B ligand (RANKL) were determined by ELISA. Expression levels of B-ALP in bone tissue were assessed by immunohistochemistry. The number of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining. BMD and BMC were detected by DXA.Results:Compared with wild-type mice, OPG-/- mice showed a loss of BMD and disturbance of microarchitecture. Bone volume fraction (BV/TV), bone surface fraction (BS/BV), trabecular thichness (Tb.Th), trabecular number (Tb.N) and connectivity density (Conn.D) were decreased with the concurrence of increased trabecular separation (Tb.SP) and stucture model index (SMI). Three-point bending test revealed a decrease in ultimate load, ultimate stress, stiff index and elastic modulus. The BMD and BMC were reduced by DXA determination. Serum B-ALP, RANKL and the number of osteoclats were all elevated at the same time?Conclusions:The disturbant bone microarchitecture, lowered BMD and decreased bone biomechanical property were observed in OPG-/- mice, indicating that they happened by a higher bone turnover state in OPG-/ mice. Part three Observation on pathological characteristics of arterial and cardiac muscular calcification in OPG knockout miceObjective:To observe the characteristics of arterial and cardiac muscular calcification in OPG knockout (OPG-/-) mice.Methods:10 week-old (n=10) and 20 week-old (n=10) OPG-/- mice, as well as 10 age-matched wild type (WT) mice were involved in the study. Serum Ca, P, serum bone alkaline phosphatase (B-ALP), and receptor activator of nuclear factor?B ligand (RANKL) were determined. RANKL and BMP protein in arterial and cardiac muscle were observed by immunohistochemistry. The arterial and cardiac muscle were observed by Hematoxylin-eosin staining and tartrate-resistant acid phosphatase (TRAP) staining.Calcium was extracted with 10% formic acid and the colorimetric quantification of calcium was achieved.Results:OPG-/- mice developed arterial and cardiac muscular calcification, which increasd with age. Levels of B-ALP and RANKL in serum were increased, while no significant difference in serum Ca, P.The number of osteoclasts were not found in arterial and cardiac muscle. BMP and RANKL protein was detected in arterial and cardiac muscle of OPG-/- mice but wasn't detected in WT mice.Conclusion:Our findings demonstrated that OPG-/- mice developed arterial and cardiac muscular calcification asscoiated with the elevated ALP activity. It suggested that disturbances of bone remodeling played an important role in arterial and cardiac muscular calcification?...