It is well known that bone mass density decreases with age. Age-related bone mass loss is ascribed to several factors. Nonenzymatic glycation has been proposed as a new potential factor in the loss of bone during aging. In this study we evaluated the concentration of pentosidine, an advanced glycation end product, in cortical and trabecular bone and in the plasma of subjects undergoing orthopedic surgery. The relationship between these parameters and a clinical index of osteoporosis was also studied. Samples of bone and plasma of 104 nondiabetic subjects (74 women and 30 men), 72 +/- 1 years old, were studied. Pentosidine was determined by HPLC after decalcification and hydrolysis. The radiologic Singh index was evaluated blindly by orthopedic surgeons to provide the degree of osteoporosis. Pentosidine concentration of cortical bone shows a significant exponential increase with age (r = 0.610, P < 0.001). This increase, however, is not seen in the trabecular bone, which is characterized by a large spread in the data. Interestingly the concentration of cortical pentosidine is also related to the Singh score (r(s) = -0.274, P < 0.01). Plasma pentosidine has a significant exponential correlation with age (r = +0.339, P < 0.001) and a linear correlation with the cortical bone pentosidine (r = +0.248, P < 0.05). This study demonstrates that pentosidine increases exponentially in cortical bone during aging, and is thus a good biomarker for the degree of bone mass density loss. The trabecular bone concentration of pentosidine is more variable, probably because of the turnover rate and the local environment; plasma pentosidine might provide information on the bone turnover rate.
Osteoporosis, a multifactorial and progressive skeletal metabolic disease, is characterized by low-mass density and structural deterioration of bone micro-architecture that leads to enhanced bone fragility and increased susceptibility to fractures. Recently, it has been proposed that age-related bone loss could be correlated with the glycoxidative process. The aim of the present study was to investigate the in vitro effects of pentosidine, a glycoxidative end product, on human osteoblasts (HOb). The mineralization rate, the specific bone markers (alkaline phosphatase [ALP], collagen Ialpha1 [COL Ialpha1], osteocalcin [BGP]), and the human receptor for advanced glycation end products (RAGE) gene expression have been evaluated. Pentosidine incubation of HOb caused a significant decrease in ALP, Col Ialpha1, and RAGE mRNA levels, but only the RAGE gene expression decreased with no dose dependency. Moreover, pentosidine incubation of osteoblasts hampered the formation of bone nodules. No effect was observed on BGP gene expression under all experimental conditions. Our data gives further support to a detrimental effect of AGEs on bone that leads to functional alterations of osteoblasts. This study addresses a crucial role of protein glycoxidation in the bone mineralization process. AGEs formation and accumulation in bone may be one of the first pathogenetic steps of bone remodeling in aging and in age-related diseases, leading to enhanced bone mass loss.
This study describes the adhesion of human osteoblasts, cultured in vitro, to proteins of the extracellular matrix, the biosynthesis of integrins, their topography and organization in focal contacts. The adhesion of osteoblasts to laminin, type I collagen, vitronectin and fibronectin was 77-100%, in 2 h and at 55 nM substrata concentration, and it was accompanied by spreading of the cells. Adhesion to fibronectin (FN), laminin (LN) and type I collagen (COL) was inhibited by antibodies to the beta 1 integrin and antibodies to the alpha 5 chain affected adhesion only to fibronectin. Using a panel of polyclonal antibodies against alpha 2, alpha 3, alpha 5, alpha v, beta 1 and beta 3 integrins we detected synthesis of alpha 3 beta 1, alpha 5 beta 1, alpha v beta 3, and an alpha v beta 1-like dimer by immunoprecipitation of metabolically labelled cell lysates. Studies of immunolocalization demonstrated the presence of the same integrins identified in lysates, plus alpha 4, alpha 1 and beta 5 subunits. In cells adhering in the presence of serum we showed organization of beta 3 and alpha v integrins in focal contacts. In cells adhering to fibronectin alpha 5 and beta 1 integrins were localized in focal contacts. In cells spread on laminin or type I collagen none of the integrins investigated was localized in focal contacts.
We established cultures of cells growing out from adult bone chips and maintained them through 12 passages in culture. The cultures showed osteoblastic phenotype accompanied by synthesis of collagen type I, osteonectin, alkaline phosphatase, and osteocalcin. We report the characterization of 21 clones obtained from three different individual primary cultures. We studied the expression of osteonectin, alkaline phosphatase, collagen, and osteocalcin in the clones. Metabolic labeling showed production of type I collagen and of osteonectin in all clones studied. In two-thirds of the clones and in mass cultures alkaline phosphatase was not detected at passage 2, but it was detected in increasing amounts at later passages in culture. The clones attained different but detectable levels of expression of this marker by passage 8. The different levels in the expression of alkaline phosphatase in positive clones may be because they were derived from cells at different stages of osteoblastic maturation or due to small changes in microenvironment. The alkaline phosphatase-positive clones were tested for osteocalcin, and they showed measurable expression only at passage 10. A third of the clones obtained were negative for alkaline phosphatase during 12 passages in culture. The obtainment of clones unable to produce alkaline phosphatase may be due to loss of differentiating potential under the in vitro culture conditions. The growth rate and potential of all clones studied were similar through 12 passages in culture, regardless of their potential for expression of alkaline phosphatase.
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