Yohann Bala

Time sequence of secondary mineralization and microhardness in cortical and cancellous bone from ewes

Bone mineral is a major determinant of the mechanical resistance of bones. In bone structural units (BSUs), mineralization of osteoid tissue begins with a rapid primary mineralization followed by a secondary mineralization phase, i.e., a slow and gradual maturation of the mineral component leading to complete mineralization during an unknown period. The aim of this study was to determine the chronology of secondary bone mineralization in ewes, an animal model with a remodeling activity close to humans. Eighteen ewes received different fluorescent labels every 6 months to date the age of each labeled BSU. The degree of mineralization of bone (DMB) and Vickers microhardness were measured in labeled BSUs, while mineralization at the crystal level was assessed by Fourier transform infrared microspectroscopy (FTIRM). During the first 6 months of mineralization, degree of mineralization and microhardness significantly increased. They then increased more slowly until at 30 months they reach their maximal values. This progression during secondary mineralization was associated with an improvement of both the maturation and the crystal perfection of the mineral part of bone matrix. Finally, secondary mineralization in BSUs is completed after a period of 30 months. This observation should be taken into account for understanding the effects of long-term treatments of bone diseases.

Bone micromechanical properties are compromised during long-term alendronate therapy independently of mineralization.

In the treatment of postmenopausal osteoporosis (PMOP), the use of alendronate (ALN) leads to a decrease in the risk of vertebral and nonvertebral fractures. To explore the possible adverse effects of prolonged ALN therapy, we studied the effects of 8u2009±u20092 years (6–10 years) of ALN treatment on the iliac cortical bone mineral and collagen quality and micromechanical properties; by design, our study examined these parameters, independent of the degree of mineralization. From six ALN‐treated and five age‐matched untreated PMOP women, 153 bone structural units have been chosen according their degree of mineralization to obtain the same distribution in each group. In those bone structural units, Fourier transform infrared spectroscopy, quantitative microradiography, and nanoindentation were used to assess bone quality. Irrespective of the degree of mineralization, ALN treatment was associated with higher collagen maturity (+7%, pu2009<u20090.001, c.v.u2009=u200913% and 16% in treated and untreated women, respectively) and lower mineral crystallinity than that observed in the untreated PMOP group (−2%, pu2009<u20090.0001, c.v.u2009=u20093% in both groups). Bone matrix from ALN‐treated women also had lower elastic modulus (−12%, pu2009<u20090.0001, c.v.u2009=u200914% in both groups) and, contact hardness (−6%, pu2009<u20090.05, c.v.u2009=u200914% in both groups) than that of untreated women. Crystallinity (which reflects the size and perfection of crystals) was associated with both elastic modulus and contact hardness in treated women exclusively (ru2009=u20090.43 and ru2009=u20090.54, pu2009<u20090.0001, respectively), even after adjustment for the amount of mineral. We infer that long‐term ALN treatment compromises micromechanical properties of the bone matrix as assessed ex vivo. The strength deficits are in part related to difference in crystallinity, irrespective of the mineral amount and mineral maturity. These novel findings at local levels of bone structure will have to be taken into account in the study of the pathophysiology of bone fragilities associated with prolonged ALN treatment.

Respective roles of organic and mineral components of human cortical bone matrix in micromechanical behavior: an instrumented indentation study.

Bone is a multiscale composite material made of both a type I collagen matrix and a poorly crystalline apatite mineral phase. Due to remodeling activity, cortical bone is made of Bone Structural Units (BSUs) called osteons. Since osteon represents a fundamental level of structural hierarchy, it is important to investigate the relationship between mechanical behavior and tissue composition at this scale for a better understanding of the mechanisms of bone fragility. The aim of this study is to analyze the links between ultrastructural properties and the mechanical behavior of bone tissue at the scale of osteon. Iliac bone biopsies were taken from untreated postmenopausal osteoporotic women, embedded, sectioned and microradiographed to assess the degree of mineralization of bone (DMB). On each section, BSUs of known DMB were indented with relatively high load (~500 mN) to determine local elastic modulus (E), contact hardness (H(c)) and true hardness (H) of several bone lamellae. Crystallinity and collagen maturity were measured by Fourier Transform InfraRed Microspectroscopy (FTIRM) on the same BSUs. Inter-relationships between mechanical properties and ultrastructural components were analyzed using multiple regression analysis. This study showed that elastic deformation was only explained by DMB whereas plastic deformation was more correlated with collagen maturity. Contact hardness, reflecting both elastic and plastic behaviors, was correlated with both DMB and collagen maturity. No relationship was found between crystallinity and mechanical properties at the osteon level.

Experimental Stimulation of Bone Healing with Teriparatide: Histomorphometric and Microhardness Analysis in a Mouse Model of Closed Fracture

Fracture consolidation is a crucial goal to achieve as early as possible, but pharmacological stimulation has been neglected so far. Teriparatide has been considered for this purpose for its anabolic properties. We set up a murine model of closed tibial fracture on which different doses of teriparatide were tested. Closed fracture treatment avoids any bias introduced by surgical manipulations. Teriparatide’s effect on callus formation was monitored during the first 4xa0weeks from fracture. Callus evolution was determined by histomorphometric and microhardness assessment. Daily administration of 40xa0μg/kg of teriparatide accelerated callus mineralization from day 9 onward without significant increase of sizes, and at day 15 the microhardness properties of treated callus were similar to those of bone tissue. Teriparatide considerably improved callus consolidation in the very early phases of bone healing.

Determinants of microdamage in elderly human vertebral trabecular bone.

Previous studies have shown that microdamage accumulates in bone as a result of physiological loading and occurs naturally in human trabecular bone. The purpose of this study was to determine the factors associated with pre-existing microdamage in human vertebral trabecular bone, namely age, architecture, hardness, mineral and organic matrix. Trabecular bone cores were collected from human L2 vertebrae (nu200a=u200a53) from donors 54–95 years of age (22 men and 30 women, 1 unknown) and previous cited parameters were evaluated. Collagen cross-link content (PYD, DPD, PEN and % of collagen) was measured on surrounding trabecular bone. We found that determinants of microdamage were mostly the age of donors, architecture, mineral characteristics and mature enzymatic cross-links. Moreover, linear microcracks were mostly associated with the bone matrix characteristics whereas diffuse damage was associated with architecture. We conclude that linear and diffuse types of microdamage seemed to have different determinants, with age being critical for both types.

Strontium does not affect the intrinsic bone quality at tissue and BSU levels in iliac samples from Macaca fascicularis monkeys

Our purpose was to evaluate the impact of strontium ranelate (SrRan) on bone mineral quality at both tissue and bone structural unit (BSU) levels. Thirty iliac bone samples (dehydrated then embedded) were taken from monkeys who received 0 (controls), 200, 500 or 1250 mg/kg/day of SrRan for 52 weeks and were sacrificed either at the end of administration (treated animals, n=16) or 10 weeks later (reverse animals, n=14). Degree of mineralization (DMB), heterogeneity index of mineralization (HI), Vickers microhardness (Hv) and focal bone strontium content (BSC) were measured globally at tissue level and focally on the same 923 BSUs. Mineral and collagen characteristics, as well as chemometric analyses were performed on younger and older tissues in cortical bone and cancellous bone in 737 other BSUs. At tissue level, SrRan preserved material properties. At BSU level, BSC increased (significant) dose dependently in treated and reverse animals. DMB and Hv were greater in older than in younger bone in controls and treated animals. In treated animals, DMB was positively correlated with Hv and inversely correlated with the BSC. Thus, younger BSUs were less mineralized and less hard than older BSUs independently from the presence of strontium. Mineral maturity, crystallinity index, mineralization index, carbonation and collagen maturity were not modified by SrRan. Chemometry confirmed the absence of a direct effect of strontium on mineralization. Thus, surrogates of micro- and nano-structural mineral properties were not altered by SrRan and remained at a physiological level.

Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy

Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization.