Increased metabolism of bone collagen in post-menopausal female osteoporotic femoral heads

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Abstract

Our previous studies demonstrated that the residual collagen in osteoporotic bone was not normal but possessed higher levels of lysine hydroxylation and modified cross-linking. However, the mechanism for these changes was not clear. In the current investigation, an assessment of bone collagen metabolism in osteoporosis (OP) revealed an increase in the overall metabolism of collagen relative to age-matched controls. The increased metabolism accounts for the observed post-translational modifications of collagen which lead to a more fragile bone matrix. The rate of bone metabolism is therefore an important aspect of the pathogenesis of osteoporosis, the greater the turnover the greater the propensity of a more fragile tissue. Clearly, the quality of bone tissue does not depend solely on adequate bone density but also on the state of the collagenous matrix.

Introduction

An estimated one-third of post-menopausal Caucasian women will experience a bone fracture, and with health costs running into thousands of millions of pounds osteoporosis (OP) warrants an extensive research programme into its aetiology. Dual energy X-ray absorptiometry (DXA) of bone density currently provides the most realistic correlation with fracture risk, but it is generally accepted that bone strength cannot be predicted accurately by a simple assessment of bone density (Wilkin & Devendra, 2001). This suggests that a second parameter may be just as important and since bone is basically a two phase system of collagen and mineral there could be a change in the quality of the collagenous framework. Indeed, our previous studies on bone collagen in OP human femoral heads demonstrated that the quality of the collagen is compromised, significant post-translational modifications occurred which could seriously affect the properties of the fibre (Bailey et al., 1992, Bailey et al., 1992). In particular, the increase in lysine hydroxylation, which leads to thinner fibrils (Torre-Blanco et al., 1992), and the reduction in cross-links would lead to an increase in bone collagen fragility. These findings were recently supported by Kowitz, Knippel, Schuhr, and Mach (1997). Similarly, increased lysine hydroxylation has also been reported in vertebral bodies from OP subjects (Oxlund, Mosekilde, & Ortoft, 1996). These findings correlated with a decrease in fibril diameter and a decrease in bone strength. However, the mechanisms responsible for these changes were not clear. We have also previously shown that in non-OP subjects there are no changes in the biochemical properties of collagen over a wide age range, despite a decrease in bone density and mechanical strength, both of which correlated with a decrease in collagen content (Bailey et al., 1992, Bailey et al., 1992). Consequently, making the assumption that if all cancellous bones are effectively the same any changes observed in OP are likely to be due to the disease process rather than a function of ageing. The normal growth and remodelling of bone requires the prior synthesis of collagen in the form of almost crystalline fibres within which fine crystals of calcium Apatite can form. It is therefore crucial that the structure of the new replacement collagen prior to calcification is identical to the pre-existing matrix since a modified collagen molecule could result in a weakened scaffold and poor mineralisation.

It is readily accepted that there is a change in the environment of bone cells in post-menopausal OP due to the dramatically reduced levels of oestrogen, and consequently changes in the levels of many other local and systemic factors. Many growth factors and cytokines have been implicated in the pathogenesis of OP, several of which have been demonstrated to have specific effects on collagen metabolism and synthesis. TGF-β for example, an inhibitor of osteoclast formation and activity is decreased in OP (Pfeilschifter et al., 1998). Osteoblasts have also been reported to change their phenotypic expression with both age and OP, and have a reduced ability to proliferate in OP (Byers, Denton, Hoyland, & Freemont, 1997). Although no amino acid mutation has been identified, a polymorphic Sp1 binding site in the collagen type I α1 gene has been associated with reduced bone density in man (Grant et al., 1996). It is thought that an Sp1 polymorphism may affect Coll1A1 gene transcription or be in linked disequilibrium with another mutation that also affects gene transcription. Such allelic differences may result in changes in the turnover of type I collagen, and it is not inconceivable that they may also effect the nature of the collagen produced. It is therefore possible that in OP, in addition to altered collagen turnover, the modified osteoblast environment post-menopause may precipitate qualitative changes in the newly synthesised matrix. There are many instances where changes in the cellular environment induces alterations in both the genetic types of collagen normally present, and the post-translational modifications of the collagen produced, as occurs for example in bone fracture (Liu, Yang, al-Shaikh, & Lane, 1995). Osteoblast environmental changes may account for the increased lysine hydroxylation and reduced cross-linking. Alternatively, since increased hydroxylation is generally associated with increased collagen turnover the observed effect may also be due to an increased rate of turnover despite the overall increased resorption in OP. The present investigation was carried out to determine whether there is indeed an increased turnover of bone collagen in OP subjects that could affect the quality of bone tissue.

Section snippets

Materials

Femoral heads (12) were collected from elderly females (65–90 years) immediately after hip arthroplasty following OP fracture at the Bristol Royal Infirmary. Seven normal femoral heads (age range 60–85 years) were obtained from Sheffield University Hospital during routine post-mortem. All samples were obtained from individuals with no history of cancer, renal or hepatic disease or drug abuse. All samples were initially stored refrigerated for 24 h prior to long-term storage (2 months) at −20 °C.

Preparation of samples

PICP

The processing of type I pro-collagen molecules requires the removal of the extension peptides by specific proteinases. The concentration of PICP therefore accurately reflects the stoichiometric synthesis of type I collagen. Fig. 1 illustrates the extent of type I collagen synthesis between normal and osteoporotic bone tissue. The concentration of PICP expressed in relation to extracted protein, was found to be significantly greater (P<0.001) by approximately three-fold (mean concentration of

Discussion

Analysis of the rate of matrix turnover between normal and OP bone from human femoral heads revealed some surprising results. The extent of collagen synthesis, as determined by analysis of the propeptide (PICP), increased hydroxylysine and immature cross-links was significantly increased in OP femoral heads. It is possible that some of the PICP may have been retained within a fraction of the insoluble matrix and the PICP levels underestimated. Alternatively the metabolic clearance of this

Acknowledgements

The authors wish to express their gratitude to the following: Mr. Trevor Sims and Mr. Nick Avery of the Collagen Research Group, University of Bristol for the collagen cross-link and hydroxylysine analyses. We would also like to thank the Biochemistry Unit, Langford for analysis of tissue samples for calcium, total protein and alkaline phosphatase. Dr. Jason Mansell is indebted to the Arthritis Research Campaign for their financial support.

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