Infrared imaging microscopy of bone: Illustrations from a mouse model of Fabry disease

AbstractBone is a complex tissue whose composition and properties vary with age, sex, diet, tissue type, health and disease. In this review, we demonstrate how infrared spectroscopy and infrared spectroscopic imaging can be applied to the study of these variations. A specific example of mice with Fabry disease (a lipid storage disease) is presented in which it is demonstrated that the bones of these young animals, while showing typical spatial variation in mineral content, mineral crystal size, and collagen maturity, do not differ from the bones of age- and sex-matched wild type animals

Applications of Fourier Transform Infrared (FT-IR) Microscopy to the Study of Mineralization in Bone and Cartilage

Knowledge of the phase, composition, and crystallite size and perfection of the mineral in normal and abnormally calcified tissues provides insight into the mechanism by which this mineral was deposited. These data also can be used to develop rational therapies for pathological conditions characterized by abnormal mineral deposition. As illustrated in this review, coupling of an optical microscope with a Fourier transform infrared (FT-IR) spectrophotometer permits the mapping at 20 μm spatial resolution of changes in mineral characteristics (content, particle size, composition) in the growth plate, in bone biopsies, in mineralizing cell culture systems, and in soft tissue calcifications. Based on the infrared properties of apatitic compounds, and comparisons with x-ray diffraction data, correlations have been established from which mineral parameters can be determined. The validity of these spectral correlations has been demonstrated by independent measurements of mineral content (ash weight), and crystal particle size (dark field electron microscopy)

Vitamin B6 deficiency experimentally-induced bone and joint disorder: microscopic, radiographic and biochemical evidence

In the present study the effect of pyridoxine deficiency on the utrastructure and morphology of bone and its metabolism was examined in the rapidly growing chick. Pyridoxine-deficient animals had tibias of reduced dry weight and cortical thickness. Histomorphometry demonstrated a disproportionately high eroded surface, lower amount of osteoid tissue and reduced mineralized trabecular width. Anterior-posterior radiographs of the tibiotarsometatarsal joint showed reduced secondary ossification centres and coarse trabeculation. Decalcified metaphyseal cartilage showed irregular trabeculas and a markedly reduced amount of Fast-green counterstain matrix suggesting that there is less collagen present and in turn less availability for matrix to be laid down for later calcification. Plasma activity of the bone alkaline phosphatase isoenzyme (EC) 3. 1.3.1) was decreased. Plasma Ca and PO4 levels did not vary. The present bone study referring to a pseudo-lathyritic state in which collagen maturation is not completely achieved supports the hypothesis that pyridoxine is an essential nutrient for the connective tissue matri

Bisphosphonate Treatment Modifies Canine Bone Mineral and Matrix Properties and their Heterogeneity

Bone loss and alterations in bone quality are major causes leading to bone fragility in postmenopausal women. Although bisphosphonates are well known to reduce bone turnover and prevent bone loss in postmenopausal osteoporosis, their effects on other bone properties are not fully characterized. Changes in bone mineral and matrix properties may contribute to the anti-fracture efficacy observed with bisphosphonate treatments. The aim of this work was to analyze the effect of a 1-year treatment with either alendronate or risedronate, at low and high doses, on spatially resolved bone material and compositional properties that could contribute to the fracture efficacy of these agents. Distal tibias from 30 normal beagles that had been treated daily for 1 year with oral doses of vehicle (Veh), alendronate (Aln) at 0.2 or 1 mg/kg, and risedronate (Ris) at 0.1 or 0.5 mg/kg were analyzed by Fourier Transform Infrared imaging (FTIRI) to assess the changes in both mineral and matrix properties in discrete bone areas. The widths at half maximum of the pixel histograms for each FTIRI parameter were used to assess the heterogeneity of the bone tissue. Aln and Ris increased the mineral content and the collagen maturity mainly in cancellous bone and at the endocortical surface. Significant differences were observed in the mineral content and in the hydroxyapatite crystallinity distribution in bone tissue, which can contribute to reduced ductility and micro-crack accumulation. No significant differences were observed between low and high dose nor between Aln and Ris treatments. These results show that pharmacologic suppression of bone turnover increases the mineral and matrix bone tissue maturity in normal cancellous and endocortical bone areas where bone turnover is higher. These positive effects for decreased fracture risk are also associated with a loss of bone heterogeneity that could be one factor contributing to increased bone tissue brittleness and micro-crack accumulation

Imaging of Alkaline Phosphatase Activity in Bone Tissue

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with 19Flourine magnetic resonance spectroscopic imaging (19FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using 19Fluorine magnetic resonance spectroscopy (19FMRS) and 19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. 19FMRS and 19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. 19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized 19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of 19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, 19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications

Regulating in vivo calcification of alginate microbeads

Alginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs). Variables included the delivery method, site of delivery, sex of the animal, time in vivo, crosslinking solution, and method of storage prior to delivery. Calciumcrosslinked alginate microbeads mineralized when injected subcutaneously or implanted intramuscularly after 1e6 months. More extensive analysis with histology, microCT, FTIR, XRD, and EDS showed calcium phosphate deposits throughout the microbeads with surface mineralization that closely matched hydroxyapatite found in bone. Incorporating 25 mM bisphosphonate reduced alginate calcification whereas using barium chloride eliminated mineralization. Buffering the crosslinking solution with HEPES at pH 7.3 while washing and storing samples in basal media prior to implantation also eliminated calcification in vivo. This study shows that alginate processing prior to implantation can significantly influence bulk hydroxyapatite formation and presents a method to regulate alginate calcificationAlginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs). Variables included the delivery method, site of delivery, sex of the animal, time in vivo, crosslinking solution, and method of storage prior to delivery. Calciumcrosslinked alginate microbeads mineralized when injected subcutaneously or implanted intramuscularly after 1e6 months. More extensive analysis with histology, microCT, FTIR, XRD, and EDS showed calcium phosphate deposits throughout the microbeads with surface mineralization that closely matched hydroxyapatite found in bone. Incorporating 25 mM bisphosphonate reduced alginate calcification whereas using barium chloride eliminated mineralization. Buffering the crosslinking solution with HEPES at pH 7.3 while washing and storing samples in basal media prior to implantation also eliminated calcification in vivo. This study shows that alginate processing prior to implantation can significantly influence bulk hydroxyapatite formation and presents a method to regulate alginate calcificatio

Decorin modulates collagen matrix assembly and mineralization

Decorin (DCN) is one of the major matrix proteoglycans in bone. To investigate the role of DCN in matrix mineralization, the expression of DCN in MC3T3-E1 (MC) cell cultures and the phenotypes of MC-derived clones expressing higher (sense; S-DCN) or lower (antisense; AS-DCN) levels of DCN were characterized. DCN expression was significantly decreased as the mineralized nodules were formed and expanded in vitro. In S-DCN clones, in vitro matrix mineralization was inhibited, whereas in AS-DCN clones, mineralization was accelerated. At the microscopic level, collagen fibers in S-DCN clones were thinner while those of AS-DCN clones were thicker and lacked directionality compared to the controls. At the ultrastructural level, the collagen fibrils in S-DCN clones were markedly thinner, whereas those of AS-DCN clones were larger and irregular in shape. The results from Fourier transform infrared spectroscopy analysis demonstrated that in AS-DCN cultures the mineral content was greater but the crystallinity of mineral was poorer than that of the controls at early stage of mineralization. The in vivo transplantation assay demonstrated that no mineralized matrices were formed in S-DCN transplants, whereas they were readily detected in AS-DCN transplants at 3 weeks of transplantation. The areas of bone-like matrices in AS-DCN transplants were significantly greater than the controls at 3 weeks but became comparable at 5 weeks. The bone-like matrices in AS-DCN transplants exhibited woven bone-like non-lamellar structure while the lamellar bone-like structure was evident in the control transplants. These results suggest that DCN regulates matrix mineralization by modulating collagen assembly

Reduced Tissue-Level Stiffness and Mineralization in Osteoporotic Cancellous Bone

Osteoporosis alters bone mass and composition ultimately increasing the fragility of primarily cancellous skeletal sites; however, effects of osteoporosis on tissue-level mechanical properties of cancellous bone are unknown. Dual-energy x-ray absorptiometry (DXA) scans are the clinical standard for diagnosing osteoporosis though changes in cancellous bone mass and mineralization are difficult to separate using this method. The goal of this study was to investigate possible difference in tissue-level properties with osteoporosis as defined by donor T-scores. Spine segments from Caucasian female cadavers (58–92 yrs) were used. A T-score for each donor was calculated from DXA scans to determine osteoporotic status. Tissue level composition and mechanical properties of vertebrae adjacent to the scan region were measured using nanoindentation and Raman spectroscopy. Based on T-scores, six samples were in the Osteoporotic group (58–74 yrs) and four samples were in the Not Osteoporotic group (65–92 yrs). The indentation modulus and mineral to matrix ratio (mineral:matrix) were lower in the Osteoporotic group than the Not Osteoporotic group. Mineral:matrix ratio decreased with age (r2 = 0.35, p = 0.05), and the indentation modulus increased with a real bone mineral density (aBMD) (r2 = 0.41, p = 0.04)

Changes in matrix phosphorylation during bovine dentin development

Phosphorylation of the organic matrix proteins of dentin is important for the initiation of mineralization, but its relevance in later mineralization stages is controversial. The objective of this study was to analyze changes in the total matrix phosphate content during dentin development and to identify their origin. Amino acid and total matrix phosphate analyses of microdissected developing mantle and circumpulpal fetal bovine dentin specimens were performed. The amino acid composition showed few changes during mantle and circumpulpal dentin maturation. However, the total matrix phosphate content showed a significant, positive correlation with tissue maturation in both mantle and circumpulpal dentin, with a two- and a three-fold increase, respectively, being observed. The data indicate that changes occur in the pattern of phosphorylation of matrix proteins during dentin maturation, which we suggest may play a functional role in later stages of tooth mineralization