Calcification in Biological Systems Calcification in Biological Systems Editor Ermanno Bonucci Professor of Pathological Anatomy Department of Human Biopathology University “La Sapienza” Rome, Italy CRC Press Boca Raton Ann Arbor London Tokyo Library of Congress Cataloging-in-Publication Data Calcification in biological systems / Ermanno Bonucci, editor, p. cm. Includes bibliographical references and index. ISBN 0-8493-5735-7 1. Calcification. I. Bonucci, E. QP535.C2C25 1992 591.4'7—dc20 91-36813 CIP This book represents information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Every reasonable effort has been made to give reliable data and information, but the authors and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. All rights reserved. This book, or any parts thereof, may not be reproduced in any form without written consent from the publisher. Direct all inquiries to CRC Press, Inc., 2000 Corporate Blvd., N.W., Boca Raton, Florida 33431. ©1992 by CRC Press, Inc. International Standard Book Number 0-8493-5735-7 Library of Congress Card Number 91-36813 Printed in the United States of America 1234567890 Printed on acid-free paper PREFACE Calcification is one of the most important biological processes for the life of the organisms, being involved in at least two of the most prominent physiological activities: control of calcium homeostasis, indispensable for all cellular functions; and assembly of hard structures, necessary for soft tissue support, protection, and movement. On the other hand, calcification is diffuse both in the animal and vegetal kingdoms and its different aspects are almost as numerous as the different calcified structures. This had led to a large number of investigations carried out with different methods and techniques in various biological systems, from unicellular organisms to superior verte­ brates. Although an impressive amount of information has been produced by these studies, the calcification process largely remains a biological enigma. This can be due, at least in part, to the fact that the multidisciplinary character of the investigations, the frequent overspecialization of the methods applied to them, and the consequent fragmentation of pertinent information, do not easily achieve a synthesis of available results and a selection of those which could be useful to solve the problem. Consequently, it becomes extremely difficult to compare results from different sources and to attempt their as­ sembly into a Unitarian theory which might explain the mechanism of inorganic substance deposition in calcifiable matrices. Calcification in Biological Systems gathers in a single volume results, experiences, opinions, and conclusions drawn from studies of different cal­ cified systems. Its principal aim is to collect the essential data concerning the various and dissimilar aspects of the calcification process and to make them available to readers who perform investigations in different biological fields and consequently have different scientific knowledge, mentality, and back­ ground. On this basis, the presence in the same volume of papers ranging from basic molecular problems to general structural questions, and concerning systems as different as unicellular organisms and human skeletal tissues, or in vitro cultures and urinary stones, should not appear dispersive and useless. Such a collection of dissimilar papers can furnish elements to reach a com­ prehensive view of current information on calcified matrices and the calci­ fication process, and above all can help in solving what is perhaps the major problem connected with biological calcifications, that is, if there are as many calcification mechanisms as calcified tissues, or if there is a common, although not identical, basic mechanism which can be operative in all of them. Ob­ viously, Calcification in Biological Systems does not pretend to give an answer to this fundamental question, or to treat in detail all the aspects connected with different types of biological calcification. However, it is hoped that it will not fail its major objective, which is that of furnishing sufficient inter­ acting information to investigators working in fields too different to allow easy exchange of ideas, results, discussion, and collaboration. The first chapter concerns the dynamic of calcium-phosphate precipitation in calcifying tissues. Of course, biological calcifications cannot be thought of as simple physical processes of calcium and phosphate precipitation like those which can occur in oversaturated solutions in vitro, because the organic components of the calcifying matrix can interact with the inorganic ions and can modify their solubility properties. However, knowledge of the basic mechanisms which regulate inorganic ion dynamics is fundamental for un­ derstanding the basic mechanism of the calcification process, especially if the physicochemical factors which lead to mineralization are considered to­ gether with the possible constraints specific biological factors can have on crystal formation. The second chapter deals with the role of collagen fibrils in calcification. The close relationship of mineral substance with collagen periodic banding, recognizable in bone, dentin, tendons, and other calcifying tissues, suggests that collagen fibrils have a primary role in calcification. This possibility, still under discussion, has been the subject of innumerable studies which, besides direct information on collagen fibrils, have allowed significant insight into the role of other matrix components in calcification, especially noncollagenous proteins. Although the structure and composition of the main calcifiable matrices are long known, this is a relatively new subject. Its development can lead to important discoveries on the organic-inorganic relationships during calcification, and can connect the process, as it occurs in collagenous matrices, to that which takes place in matrices which lack collagen fibrils. The following chapter deals with a related subject, i.e., proteoglycans and calcification. In spite of the observation that proteoglycan molecules are always present, although at different concentrations, in tissues which calcify, their role in calcification is still debated. In particular, it is still dubious if they inhibit or induce calcification, or if they inhibit the process in their native state and permit or facilitate inorganic deposition after their depolymerization. The use of new dye techniques of proteoglycan stabilization and of electron microscopy offers further possibilities of studying the organic-inorganic re­ lationships in calcified matrices and the role of proteoglycans in calcification. The role of lipids in osteogenesis, and of cell signaling in calcification, are the subjects of the fourth chapter. Both biochemical and histochemical investigations provide clear, although indirect evidence for a role of lipids and proteolipid complexes in calcification. However, knowledge on this sub­ ject is still incomplete, probably because the study of lipids in bone and other calcified matrices is difficult, and their ultrastructural investigation almost impossible. The possibility that these substances can regulate osteogenesis through specific molecules increases the difficulty of studying their role in the process. This chapter indirectly introduces the next, which deals with the role of matrix vesicles in calcification. More than twenty years after their recognition in epiphyseal cartilage, the role of these bodies of cellular origin is still debated. This is principally due to two reasons. First, they have variable frequency in different calcifying matrices; second, the mechanism of matrix vesicle calcification is not known. The observation that they have a strong alkaline phosphatase activity can offer a new explanation to their role in inducing calcium-phosphate precipitation. In vitro systems have great potentiality for studies of cells and matrix of calcified tissues. Cell phenotype, stimulation or inhibition of cell activities, effects of hormones, growth factors and other substances, early stages of calcification, and several other aspects of bone formation, calcification and resorption can all be studied in culture. The chapter on “Cell-mediated Cal­ cification In Vitro” offers a pertinent example of what can be attained with this important technique. The first six chapters deal with basic biological aspects of the calcification process. The seventh chapter introduces the study of calcification in cells and tissues. Of course, basic problems are treated and discussed also in this and following chapters; however, the second part of the volume chiefly aims at considering cell- and tissue-specific modifications during calcification rather than molecular mechanisms involved in the process. These chapters appear particularly interesting because they consider cells and tissues with very different characteristics. Thus, the subjects they deal with (unicellular organisms, spicules of sea urchin embryos, mollusc shells, epiphyseal cartilage, bone, dentine, enamel, urinary stones, and the soft tissues which can undergo ectopic calcification) represent different models of a process characterized in all cases by deposition of inorganic substance in organic matrix. They offer the opportunity of comparatively considering calcifying organic matrices with extremely variable structure, composition and complexity, stiffened by inorganic substance of various nature and degree of crystallinity. The possible finding of common characteristics in such dif­ ferent cells and tissues might be of remarkable importance to the discovery of fundamental aspects of the calcification process and might allow a unified calcification theory, if any, to be reached. Some conflicting concepts can be found in the various chapters; no attempt has been made to reduce them, because they can give rise to reflection and discussion. Moreover, because of the limited number of pages, many im­ portant subjects, including calcification in plants, have been omitted, although of great general interest. This shortcoming can perhaps be forgiven, consid­ ering that treatment of all aspects of the calcification process would require a book much more bulky than the present one. However, not only are the selected subjects the most prominent in the area of biological calcification, they are also contributed by outstanding Authors, whose competence in their research area is internationally acknowledged. For this reason, it is hoped that Calcification in Biological Systems, although necessarily limited to sub­ jects of greatest interest, can increase the knowledge on such a fundamental process and can stimulate the production of new ideas and investigations. In this connection, it is hoped that the volume is a valid tool both for those who are taking their first steps in the study of calcified tissues and for experienced investigators. Ermanno Bonucci THE EDITOR Ermanno Bonucci, M.D., is Professor of Pathological Anatomy at the University of Rome “La Sapienza”, Department of Human Biopathology. Dr. Bonucci received his M.D. degree in 1956 from the Faculty of Med­ icine at University of Perugia (summa cum laude). He began his scientific activity as a scholar of the Rockefeller Foundation in 1960. He served as Assistant Professor of Pathological Anatomy at the University of Pisa from 1962 to 1970, when he became Associate Professor of Histochemistry at the University of Rome ‘ ‘La Sapienza’ ’. He assumed his present position in 1980. He was Director of the Department of Human Biopathology from 1983 to 1988. Dr. Bonucci is a member of the Roman Academy of Medicine, Italian Society of Pathological Anatomy, International Academy of Pathology, Eu­ ropean Society for Calcified Tissues, Mineralized Tissue Group of the Amer­ ican Association for Dental Research, Italian Society of Electron Microscopy, Italian Society of Mineral and Electrolyte Metabolism, Italian Society of Osteoporosis, and the International Society for Bio-analoging Skeletal Sys­ tems. He is Associate Editor of the Italian Journal of Mineral and Electrolyte Metabolism and is a member of the Editorial Board of the European Journal of Basic and Applied Histochemistry. Dr. Bonucci has received the 1989 “Antonio Feltrinelli” award for Med­ icine from the National Academy of Lincei. He has been and is the recipient of many research grants from the Italian National Research Council, the Ministry of Public Education, and the Ministry of University and Scientific and Technological Research. He is the Italian coordinator of the Dental Re­ search branch of the program for United States-Italy Cooperation in Health and Biomedicine, and of the Integrated Action between the University of Rome and the University of Cadiz (Spain) on “Mechanisms of the osteo- cartilagineous calcification’’. Dr. Bonucci has published more than 200 scientific papers and is the author of the book Manual of Histochemistry (Lombardo, Rome, 1981, in Italian). He was co-editor of the book Ultrastructure of Bone and Cartilage. His current major research interests are in ultrastructure, histochemistry, and immunohistochemistry of normal and pathological calcified tissues.