In situ examination of the time-course for secondary mineralization of Haversian bone using synchrotron Fourier transform infrared microspectroscopy

Abstract

At the tissue level it is well established that the rate of remodeling is related to the degree of mineralization. However, it is unknown how long it takes for an individual bone structural unit (BSU) to become fully mineralized during secondary mineralization. Using synchrotron Fourier transform infrared microspectroscopy (FTIRM) we examined the time required for newly formed bone matrix to reach a physiological mineralization limit. Twenty-six, four-month old female New Zealand white rabbits were administered up to four different fluorochrome labels at specific time points to evaluate the chemical composition of labeled osteons from the tibial diaphysis that had mineralized for 1, 8, 18, 35, 70, 105, 140, 175, 210, 245, 280, 315, 350, and 385 days. Interstitial bone from 505 day old rabbits was used as a reference value for the physiological limit to which bone mineralizes. Using synchrotron FTIRM, area integrations were carried out on protein (Amide I: 1688–1623 cm^− 1), carbonate (v₂CO₃²⁻: 905–825 cm^− 1), and phosphate (v₄PO₄³⁻: 650–500 cm^− 1) IR bands. IR spectral data are presented as ratios of phosphate/protein (overall matrix mineralization) and carbonate/protein. The rate of mineralization of osteonal bone proceeded rapidly between day 1 and 18, reaching 67% of interstitial bone levels. This was followed by a slower, more progressive accumulation of mineral up to day 350. By 350 days the rate of increase plateaued. The ratio of carbonate/protein also increased rapidly during the first 18 days, reaching 73% of interstitial bone levels. The ratio of carbonate/protein plateaued by day 315, reaching levels not significantly different to interstitial bone levels. In conclusion, our data demonstrate that bone accumulates mineral rapidly during the first 18 days (primary mineralization), followed by a more gradual increase in the accumulation of mineral (secondary mineralization) which we found to be completed in 350 days.

Introduction

Osteons, also referred to as Haversian systems, are cylindrical basic structural units (BSU) that represent the main structural unit of cortical bone (Marotti, 1996, Jee, 2001). At a microscopic level, cortical bone is a heterogeneous tissue comprised of newly formed and mature osteons that vary in their degree of mineralization (Strandh, 1960, Portigliatti Barbos et al., 1983, Grynpas, 1993, Boivin and Meunier, 2002a, Boivin and Meunier, 2002b). Consequently, neighboring osteons may have different degrees of mineralization depending on when they were formed. The degree to which an individual BSU mineralizes depends on the rate of remodeling. In situations of increased turnover a BSU may be resorbed before it reaches its full mineralization potential. In contrast, reductions in turnover, as found with bisphosphonate therapy, allow a greater number of BSU's to achieve a physiological mineralization limit (Boivin and Meunier, 2002a, Boivin and Meunier, 2002b, Fratzl et al., 2004, Ruffoni et al., 2007).

Bone is formed by the deposition of osteoid (unmineralized bone matrix) followed by mineralization approximately 5–10 days later (Parfitt, 1987). Osteoid accumulates mineral in a series of two sequential, continuous phases (Marotti et al., 1972, Wergedal and Baylink, 1974), which were operationalized by Marroti et al. as primary and secondary mineralization (Marotti et al., 1972, Marotti, 1976). This terminology refers to the length of time required for an individual BSU to mineralize, and does not represent the length of time required for bone tissue at the organ level to mineralize. During primary mineralization newly formed osteoid rapidly accumulates mineral, becoming ∼ 65–70% mineralized when compared to the final mineralization value attained during the phase of secondary mineralization (Amprino and Engstrom, 1952, Marotti et al., 1972). The bone matrix continues to accumulate mineral at a slower, more progressive rate during the secondary phase of mineralization until the amount of mineral reaches a physiological limit. Estimates for the completion of secondary mineralization have ranged from a few days for periosteal bone which represents primary bone (Amprino and Engstrom, 1952, Wergedal and Baylink, 1974, Marotti, 1976, Busa et al., 2005), to several months for secondary bone which represents the replacement of previously existing bone (Marotti et al., 1972, Akkus et al., 2003, Huang et al., 2003).

The objective of this study was to determine the length of time required for newly formed bone matrix to reach a physiological mineralization limit using fluorescence-assisted synchrotron Fourier transform infrared microspectroscopy (FTIRM). In this study the physiological limit for mineralization is defined as interstitial bone which contains space for tissue fluid, osteocyte lacunae and canaliculae which have not filled in with mineral (Frost, 1960, Jowsey, 1964, Martin, 1984, Parfitt, 1987). FTIRM is commonly used to investigate the structural features of organic molecules found in bone by examining the frequency at which molecular bonds vibrate (i.e. stretching and bending) (Miller et al., 2001, Boskey and Mendelsohn, 2005). Using this technique, we evaluated the chemical composition of fluorescently labeled regions of osteonal bone that had mineralized for 1, 8, 18, 35, 70, 105, 140, 175, 210, 245, 280, 315, 350, and 385 days. Interstitial bone from animals that were 505 days old was used as a reference value for the physiological limit to which bone mineralizes. For each time point we examined inorganic (phosphate and carbonate) and organic (protein) components of bone.