Human bone material characterization: integrated imaging surface investigation of male fragility fractures.

UNLABELLED: The interrelation of calcium and phosphorus was evaluated as a function of bone material quality in femoral heads from male fragility fracture patients via surface analytical imaging as well as scanning microscopy techniques. A link between fragility fractures and increased calcium to phosphorus ratio was observed despite normal mineralization density distribution. INTRODUCTION: Bone fragility in men has been recently recognized as a public health issue, but little attention has been devoted to bone material quality and the possible efficacy in fracture risk prevention. Clinical routine fracture risk estimations do not consider the quality of the mineralized matrix and the critical role played by the different chemical components that are present. This study uses a combination of different imaging and analytical techniques to gain insights into both the spatial distribution and the relationship of phosphorus and calcium in bone. METHODS: X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry imaging techniques were used to investigate the relationship between calcium and phosphorus in un-embedded human femoral head specimens from fragility fracture patients and non-fracture age-matched controls. The inclusion of the bone mineral density distribution via backscattered scanning electron microscopy provides information about the mineralization status between the groups. RESULTS: A link between fragility fracture and increased calcium and decreased phosphorus in the femoral head was observed despite normal mineralization density distribution. Results exhibited significantly increased calcium to phosphorus ratio in the fragility fracture group, whereas the non-fracture control group ratio was in agreement with the literature value of 1.66 M ratio in mature bone. CONCLUSIONS: Our results highlight the potential importance of the relationship between calcium and phosphorus, especially in areas of new bone formation, when estimating fracture risk of the femoral head. The determination of calcium and phosphorus fractions in bone mineral density measurements may hold the key to better fracture risk assessment as well as more targeted therapies.

Lathyrism-induced alterations in collagen cross-links influence the mechanical properties of bone material without affecting the mineral.

In the present study a rat animal model of lathyrism was employed to decipher whether anatomically confined alterations in collagen cross-links are sufficient to influence the mechanical properties of whole bone. Animal experiments were performed under an ethics committee approved protocol. Sixty-four female (47 day old) rats of equivalent weights were divided into four groups (16 per group): Controls were fed a semi-synthetic diet containing 0.6% calcium and 0.6% phosphorus for 2 or 4 weeks and ß-APN treated animals were fed additionally with ß-aminopropionitrile (0.1% dry weight). At the end of this period the rats in the four groups were sacrificed, and L2-L6 vertebra were collected. Collagen cross-links were determined by both biochemical and spectroscopic (Fourier transform infrared imaging (FTIRI)) analyses. Mineral content and distribution (BMDD) were determined by quantitative backscattered electron imaging (qBEI), and mineral maturity/crystallinity by FTIRI techniques. Micro-CT was used to describe the architectural properties. Mechanical performance of whole bone as well as of bone matrix material was tested by vertebral compression tests and by nano-indentation, respectively. The data of the present study indicate that ß-APN treatment changed whole vertebra properties compared to non-treated rats, including collagen cross-links pattern, trabecular bone volume to tissue ratio and trabecular thickness, which were all decreased (p<0.05). Further, compression tests revealed a significant negative impact of ß-APN treatment on maximal force to failure and energy to failure, while stiffness was not influenced. Bone mineral density distribution (BMDD) was not altered either. At the material level, ß-APN treated rats exhibited increased Pyd/Divalent cross-link ratios in areas confined to a newly formed bone. Moreover, nano-indentation experiments showed that the E-modulus and hardness were reduced only in newly formed bone areas under the influence of ß-APN, despite a similar mineral content. In conclusion the results emphasize the pivotal role of collagen cross-links in the determination of bone quality and mechanical integrity. However, in this rat animal model of lathyrism, the coupled alterations of tissue structural properties make it difficult to weigh the contribution of the anatomically confined material changes to the overall mechanical performance of whole bone. Interestingly, the collagen cross-link ratio in bone forming areas had the same profile as seen in actively bone forming trabecular surfaces in human iliac crest biopsies of osteoporotic patients.

T3 affects expression of collagen I and collagen cross-linking in bone cell cultures.

Thyroid hormones (T3,T4) have a broad range of effects on bone, however, its role in determining the quality of bone matrix is poorly understood. In-vitro, the immortalized mouse osteoblast-like cell line MC3T3-E1 forms a tissue like structure, consisting of several cell layers, whose formation is affected by T3 significantly. In this culture system, we investigated the effects of T3 on cell multiplication, collagen synthesis, expression of genes related to the collagen cross-linking process and on the formation of cross-links. T3 compared to controls modulated cell multiplication, up-regulated collagen synthesis time and dose dependently, and stimulated protein synthesis. T3 increased mRNA expressions of procollagen-lysine-1,2-oxoglutarate 5-dioxygenase 2 (Plod2) and of lysyloxidase (Lox), both genes involved in post-translational modification of collagen. Moreover, it stimulated mRNA expression of bone morphogenetic protein 1 (Bmp1), the processing enzyme of the lysyloxidase-precursor and of procollagen. An increase in the collagen cross-link-ratio Pyr/deDHLNL indicates, that T3 modulated cross-link maturation in the MC3T3-E1 culture system. These results demonstrate that T3 directly regulates collagen synthesis and collagen cross-linking by up-regulating gene expression of the specific cross-link related enzymes, and underlines the importance of a well-balanced concentration of thyroid hormones for maintenance of bone quality.

Bone matrix quality and plasma homocysteine levels.

It has recently been reported in the clinical literature that blood homocysteine levels correlate well with fracture risk, although a couple of reports exist to the opposite. Bone strength depends on both bone quantity and quality. The purpose of the present study was to investigate possible correlations between plasma homocysteine levels and bone material properties (Bone Mineral Density Distribution; BMDD, and collagen cross-link ratio). In the present study, femoral heads from subjects (N=19, females, age range 70-95 years old) with known homocysteine plasma levels were investigated. The bone material was collected during hemiarthroplasty surgery. We have determined collagen cross-link ratio and bone mineralization density distribution (BMDD) in bone tissue from patients with acute femoral neck fractures, by Fourier Transform Infrared Imaging (FTIRI) and quantitative Backscattered Electron Imaging (qBEI), respectively. The collagen cross-link ratio that was spectroscopically determined was pyridinoline/divalent cross-links (pyr/divalent). The BMDD variables quantified were: CaMean: the weighted mean calcium concentration; CaPeak: the most frequent Ca concentration; CaWidth: the width of the distribution, a measure of the mineralization homogeneity; CaLow: the percentage of bone area that is mineralized below the 5th percentile in the reference range; CaHigh: the percentage of bone area that is mineralized above the 95th percentile in the reference range. There was a significant correlation between plasma homocysteine levels and collagen cross-link ratio in areas of primary mineralized bone (p<0.0001), unlike the case of trabecular bone surfaces undergoing resorption (p>0.05). On the other hand there was no correlation in any of the BMDD parameters and plasma homocysteine levels (p>0.05). The results are consistent with the known effect of homocysteine on collagen post-translational modifications. These changes were independent of bone mineral characteristics. The results of the present study offer a mechanism by which homocysteine affects bone quality, but caution should be exercised since all patients examined had sustained fracture.

Collagen cross-linking influences osteoblastic differentiation.

Osteoblasts synthesize collagen matrix, which itself regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase (LOX), which is involved in the collagen cross-linking process. Lathyrogens, like ss-aminopropionitrile (ssAPN), inhibit the formation of a stable matrix. The aim of the present study was to investigate the influence of cross-linking on osteoblastic differentiation. MC3T3-E1 cells were seeded and treated with or without 400 muM ssAPN for 1 week. Thereafter, living cells were removed and, on this extracellular matrix, new MC3T3-E1 cells were seeded and cultured for 1 week without ssAPN. RNA was isolated, and expression of specific marker genes was determined by quantitative reverse transcription-polymerase chain reaction. Changes in specific cross-links after ssAPN treatment were measured with Fourier-transform infrared spectroscopy. The collagen matrix that formed showed a significant reduction of two major cross-links of bone collagen, deH-DHLNL and pyr, compared to control cultures. Gene expression studies showed an increase of collagen alpha1 (I) (COL1A1) to 150%. Expression of LOX and osteocalcin (OCN) mRNA was significantly downregulated to about 75%. When fresh MC3T3-E1 cells were seeded on this altered matrix without ssAPN, COL1A1 mRNA expression was upregulated (140%), OCN was downregulated (60%), and LOX mRNA expression remained unaffected. These results indicate that ssAPN treatment not only disrupts collagen cross-link formation but also affects osteoblastic activity and expression. In conclusion, the disrupted matrix produced in the presence of lathyrogen influences, even in its absence, the expression of osteoblastic genes.