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Bone matrix turnover and balance in vitro. II. The effects of aging.

The rates of both formation and resorption of bone collagen may be accurately quantitated by kinetic analysis of hydroxyproline metabolism in vitro. Using this approach we have studied the changes in bone collagen turnover with age in the rat. The rates of synthesis and resorption of collagen decline with age although the resorptive activity per cell increases up to 6 months of age. The solubility of collagen declines with age. The fraction of the newly synthesized collagen which is deposited as matrix declines dramatically with age revealing a new and hitherto unsuspected aspect of the osteoporotic process. The collagen balance becomes progressively more negative over the 1st 6 months of life. These results indicate that even in an animal who is not subject to clinical osteoporosis, biochemical measurement reveals that such a trend exists. The application of this approach to human subjects is feasible and has important implications.

Aging↗

The development and identification of constructing tissue engineered bone by seeding osteoblasts from differentiated rat marrow stromal stem cells onto three-dimensional porous nano-hydroxylapatite bone matrix in vitro.

The purposes of this study were to develop a new cultural method for the rat bone marrow stromal cells (MSCs) to differentiate into osteoblasts well in vitro, and to investigate the feasibility of using MSCs as seed cells and three-dimensional porous nano-hydroxylapatite as scaffolds for constructing tissue-engineered bone. MSCs of rats were isolated, cultured, induced to differentiate into osteoblasts, and then observed with inverted microscopy. Histochemical staining and radio-immunological analysis were applied for identifying MSCs. Whereafter MSCs were seeded onto three-dimensional porous nano-hydroxylapatite scaffolds, and scanning electron microscopy was applied to evaluate their growth on scaffolds. Results showed that MSCs were typical fibroblast-like and possessed a better proliferating capability; the activity of alkaline phosphatase (ALP) and the secretion of osteocalcin of MSCs were produced gradually and increased continuously; the cells seeded on three-dimensional porous nano-hydroxylapatite scaffolds adhered, proliferated and differentiated well. These results demonstrated that the new improved culture method had the advantages of short isolating time, less risk of contamination and higher efficiency and accordingly was conducive to MSCs proliferating and differentiating into osteoblasts, and that it was advantageous to constructing tissue-engineered bone using MSCs as seed cells and three-dimensional porous nano-hydroxylapatite as scaffolds.

Animals↗

Differentiation of osteoblasts in three-dimensional culture in processed cancellous bone matrix: quantitative analysis of gene expression based on real-time reverse transcription-polymerase chain reaction.

Processed bovine cancellous bone (PBCB) is an attractive material for tissue engineering of bone. It is biocompatible, osteoconductive, nonimmunogenic, and porous and its biomechanical properties are close to those of native bone. In this study, differentiation of primary rat osteoblasts (rOBs) incubated on PBCB was investigated in vitro. rOBs were isolated and expanded in two-dimensional culture. Expanded rOBs were seeded into PBCB disks and cultured either in basal medium (BM) or differentiation medium (DM) containing ascorbic acid, beta-glycerol phosphate, and dexamethasone. Alkaline phosphatase (ALP) activity and RNA expression of ALP, bone sialoprotein (BSP), collagen type I (COL1), osteocalcin (OC), and osteopontin (OPN) were assessed by chemiluminescence assay and quantitative real-time RT-PCR over 14 days. Histologic analysis was performed on day 14. ALP increased over the observation period independent of stimulation. OPN and BSP expression was significantly higher in the DM group whereas COL1 and OC expression was significantly higher in the BM group. Matrix calcification was detectable only in the DM group by von Kossa stain. The observed expression patterns suggest a physiological response of rOBs to the differentiation stimulus. PBCB is a suitable matrix for in vitro differentiation of osteoblasts. Cell-seeded PBCB is a potential osteogenic construct for in vivo application.

Alkaline Phosphatase↗

Changes in phosphoproteins of chicken bone matrix in vitamin D-deficient rickets.

Vitamin D-deficiency and rickets was produced in growing chicks. The resulting decrease in mineralization of whole bone and of fractions separated by density centrifugation was accompanied by a very significant decrease in the contents of O-phosphoserine and O-phosphothreonine. Likewise, the total amount of O-phosphoserine and O-phosphothreonine and the concentrations of these phosphoamino acids in EDTA extracts and in fractions obtained by molecular sieving was also reduced. These data provide the first in vivo evidence that phosphoproteins may be critically involved in the calcification of bone.

Animals↗