In vitro studies with bone in metabolic bone disease

McLoughlin, Patricia (1982) In vitro studies with bone in metabolic bone disease. PhD thesis, University of Glasgow.

Abstract

Improvements in living standards and health care have caused a great increase in the number of elderly individuals in our community. The problems associated with age are many, but one in particular is of interest in this thesis; bone loss and the problems of metabolic bone disease, leading to various studies concerned with their effects on bone and bone metabolism. Organ culture preparations of neonatal mouse calvaria were used to look at several aspects of bone metabolism. By preparing standard cultures of neonatal mouse calvaria, but with the bone prelabelled with 45Ca and the culture medium labelled with 47Ca, it was possible to look at the effects of human serum from patients suffering from metabolic bone disease on bone turnover in vitro. The results of this study proved interesting, suggesting that such sera act on bone to produce particular patterns of formation and resorption, reminiscent, in general, of the symptoms of the bone disease in vivo. Although there are other and better ways of diagnosing metabolic bone disease, such as serum biochemistry and bone histology, this method could be useful in assessing the development of any disorder. Human serum is abundant and easy to obtain, unlike human bone, and the cultures are easy to prepare; the effects of any drug therapy on bone turnover can be seen quickly, by monitoring 45Ca and 47Ca in the culture medium, and by constant monitoring of serum biochemistry, it may be possible to understand how particular drug therapies affect bone. The enzyme alkaline phosphatase stimulated a lot of interest. First of all the relationship between serum alkaline phosphatase and the level of the enzyme within bone itself was studied, showing a strong correlation between the two parameters and supporting the study of serum alkaline phosphatase levels as a tool in the diagnosis of metabolic bone disease. Dialysis osteomalacia is thought to be caused by the inhibition of alkaline phosphatase, through the build-up of aluminium. By studying the effect of aluminium in vitro, it was found that it does inhibit alkaline phosphatase, the inhibition of which is accompanied by high.levels of free calcium in the culture medium, showing a strong inverse relationship between alkaline phosphatase and calcium and supporting the role of the enzyme in mineralisation. This relationship was also supported when alkaline phosphatase and free calcium levels v/ere compared-in cultures containing foetal calf serum and with human serum. Other studies concerned with metabolic bone disease involved the assay of two different and unrelated enzymes: collagen prolyl hydroxylase and creatine kinase. Collagen is the main structural component of bone and collagen prolyl hydroxylase is the enzyme responsible for the hydroxylation of proline, without which the classic triple helix structure cannot be formed. By assaying homogenates of bone obtained by biopsy of the iliac crest, it was hoped to discern any differences in collagen metabolism between the different metabolic bone diseases. Unfortunately, for a variety of reasons, it was not possible to pursue this line of study as much as was desired. The assay of creatine kinase was more successful. Creatine kinase is the enzyme involved in the breakdown of the high energy molecule creatine phosphate to form ATP and is particularly abundant in skeletal muscle. Normal serum has little or no creatine kinase content, but the level has been found to change with different disorders, particularly those related to muscle weakness. As many metabolic bone diseases are severely debilitating, it was decided, looking at homogenates of striated muscle, to see if there is any pattern of creatine kinase activity in different metabolic bone diseases. Results showed no difference in creatine kinase levels in striated muscle in different metabolic bone diseases, but showed higher levels in the muscle of males than in females and increasingly high levels of the enzyme, for both sexes, with increasing age. Previous work, looking at serum creatine kinase levels, related the sex-difference to the inhibition of the enzyme by oestrogen in pre-menopausal women. The culture of human bone fragments is possible, but not desirable as long as uniform samples, containing equivalent amounts of cells and bone, of a suitable size are difficult to obtain. Human bone cell cultures are more desirable as they would be easier to reproduce and control. Although unsuccessful here, new technology will aid the culture of human bone cells and these cultures will play an important role in the study of metabolic bone diseases.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: I T Boyle
• Keywords: Medicine
• Date of Award: 1982
• Depositing User: Enlighten Team
• Unique ID: glathesis:1982-73358
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73358