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Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) are techniques utilized in the analysis of bone mineral and matrix properties in health and disease. Since the spatial arrangement of bone tissue is conserved using FTIRM and FTIRI, quantitative data can be obtained on bone mineral (hydroxyapatite) crystalline size and composition, and on matrix structure and composition at discrete anatomic locations with a spatial resolution from approximately 7 mm (FTIRI) to 10 mm (FTIRM). To section bone for FTIRM and FTIRI, it must be preserved ("fixed") to maintain its properties, and embedded in a hard supportive material. Since most of the embedding media have components that spectrally overlap the components of mineralized tissues, it is critical to define optimal embedding and fixation protocols that have the least effect on mineral and matrix spectra. In the current study, the spectra of mouse calvaria in seven different fixatives and six different commonly used embedding media were assessed by FTIRM and FTIRI. The fixatives evaluated were absolute ethanol, 70% ethanol, glycerol, formaldehyde, EM fixative, and formalin in cacodylate or phosphate-buffered saline. The embedding media tested were Araldite, Epon, JB-4, LR White, PMMA, and Spurr. Comparisons were made to FTIR spectra obtained from unprocessed ground calvaria and to spectra of cryosections of unfixed tissue, fast-frozen in polyvinyl alcohol (5% PVA). Non-aqueous fixatives and embedding in LR White, Spurr, Araldite, and PMMA had the least effect on the spectral parameters measured (mineral to matrix ratio, mineral crystallinity, and collagen maturity) compared with cryo-sectioned calvaria and non-fixed, non-embedded calvaria in KBr pellets.

Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate in vitro to form a cartilaginous structure analogous to the epiphyseal growth plate. When inorganic phosphate, Pi, is included in the medium such that the total Pi concentration is 4 mM, apatite mineral precipitates around the "hypertrophic" chondrocytes. These hypertrophic chondrocytes are characterized by their increased expression of type X collagen, alkaline phosphatase activity, and apoptosis, as well as by the ability of their extracellular matrices to support mineral deposition. Under standard mineralizing conditions (0.8 x 10(6)cells/micromass; 4 mM Pi, 1.3 mM Ca(2+), 10% FCS, and antibiotics) mineralization does not commence until day 14-16. Based on the ability of bone morphogenic protein 6 (BMP-6) to stimulate chondrocyte maturation in other systems, 100 ng/ml BMP-6 was added to chick limb-bud mesenchymal cell cultures 2 and 5 days after plating, and the effects of this addition on mineral accretion and the characteristics of the mineral and matrix determined. Addition of BMP-6 accelerated the differentiation of the mesenchymal cells to hypertrophic chondrocytes. In the presence of BMP-6 added on both days 2 and 5, mineralization (assessed on basis of (45)Ca uptake) commenced by day 12. Fourier transform infrared imaging (FTIRI) was used to monitor the mineral content and mineral crystallinity as a function of time from day 9 to 21 in cultures with and without exogenous BMP-6. While BMP-6 accelerated the rate of mineral accretion, and the crystals that were formed in the BMP-6 cultures were initially more mature, by day 21 the crystal size distribution in experimental and control cultures were not significantly different. This study, the first to report the detailed application of FTIRI to cell cultures, indicates the importance of the extracellular matrix in the control of crystal maturation., (Copyright 2001 Wiley-Liss, Inc.)

Differentiating chick limb-bud mesenchymal cell micro-mass cultures routinely mineralize in the presence of 10% fetal calf serum, antibiotics, 4 mM inorganic phosphate (or 2.5 mM beta-glycerophosphate), 0.3 mg/ml glutamine and either 25 microg/ml vitamin C or 5-12 microg/ml vitamin C-sulfate. The failure of these cultures to produce a mineralized matrix (assessed by electron microscopy, 45Ca uptake and Fourier transform infrared microscopy) led to the evaluation of each of these additives. We report here that the "stable" vitamin C-sulfate (ascorbic acid-2-sulfate) causes increased sulfate incorporation into the cartilage matrix. Furthermore, the release of sulfate from the vitamin C derivative appears to be responsible for the inhibition of mineral deposition, as demonstrated in cultures with equimolar amounts of vitamin C and sodium sulfate.

The differentiating chick limb-bud mesenchymal cell micro-mass culture system has been used as a model for monitoring the effects of matrix modification on cell-mediated calcification. In this study, we show that treating these micro-mass cultures with homocysteine (Hcys) impairs cartilage calcification. Cultures were treated from day 2 to day 7 with two nonphysiological concentrations of Hcys equivalent to 100x and 1000x avian serum levels (0.36 and 3.6 mmol/L), and from days 9-13 with one tenth the concentration. Mineralization assays were done at days 16, 19, and 21, and matrix and cell properties were examined between days 5 and 21. Mineral accretion, based on differential (45)Ca uptake (mineralizing minus control cultures), was significantly reduced in the high-Hcys-concentration group, and slightly reduced in the low-Hcys-concentration group. Electron microscopy at culture day 21 showed that the collagen matrix was less abundant and its banding pattern less obvious in the Hcys-treated groups than in the untreated cultures. Pyridinoline (Pyr) and deoxypyridinoline (d-Pyr) contents were not detectable in day 21 cultures with either 0.36 or 3.6 mmol/L homocysteine, whereas values in mineralizing and nonmineralizing controls ranged from 0.06 to 0.08 and 0.03 to 0.06 (moles/mole collagen) for Pyr and d-Pyr, respectively. Fourier transform infrared (FTIR) imaging also indicated a decreased content of pyridinoline cross-links. Hcys caused other matrix changes as well. Whereas at culture day 5 there was no significant difference in the number of chondrocyte nodules formed, by day 11 the proteoglycan content (measured by Alcian blue dye binding at 595 nm) was significantly reduced in both mineralizing and control cultures in the high- and low-Hcys groups. In contrast, there were no detectable differences in type X collagen and alkaline phosphatase staining in the mineralizing cultures with or without Hcys supplements. Because vital dye stains and electron microscopy studies indicated that cells in the control and experimental groups did not differ in terms of viability, the observed differences cannot be attributed to toxicity. Thus, Hcys treatment, which causes matrix disorganization, decreases the ability of the matrix to support mineralization.

Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage. In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage. In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules. During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen. However, type I collagen persists in the matrix. Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system. Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures. Nonspecific antibody added before day 7 blocked development of the cultures. Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth. Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral. There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times. Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies. The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain). In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits. The data demonstrate that type I collagen is important for the mineralization of these cultures., (Copyright 2000 Wiley-Liss, Inc.)

In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10(-10) M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-1R microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on 45Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization.

It is a common belief that chondrocyte death must precede calcification in the growth plate. To challenge this dogma, cell devitalization was induced in an in vitro model that mimics in situ cartilage calcification. Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate to form a cartilaginous matrix which mineralizes in the presence of inorganic or organic phosphate. The mineral formed resembles physiologic mineral in crystal size, composition, and distribution. Killing cells by water lysis, ethanol fixation, freeze-thawing, trypsinization, or impairing their function by oligomycin treatment prior to the time at which mineralization commenced, prevented mineral deposition. In contrast, devitalizing cells by any of these techniques after mineralization commenced resulted in dystrophic calcification (excessive, randomly distributed mineral of larger than physiologic crystal size). Based on analyses of 45Ca uptake, FT-IR microscopy, X-ray diffraction, and transmission electron microscopy, it is concluded that the presence of viable cells is obligatory for physiologic cartilage calcification in the differentiating chick limb-bud mesenchymal cell culture system.

Loss of bone mass during periods of skeletal unloading remains an important clinical problem. To determine the extent to which resorption contributes to the relative loss of bone during skeletal unloading of the growing rat and to explore potential means of preventing such bone loss, 0.1 mg P/kg alendronate was administered to rats before unloading of the hindquarters. Skeletal unloading markedly reduced the normal increase in tibial mass and calcium content during the 9 day period of observation, primarily by decreasing bone formation, although bone resorption was also modestly stimulated. Alendronate not only prevented the relative loss of skeletal mass during unloading but led to a dramatic increase in calcified tissue in the proximal tibia compared with the vehicle-treated unloaded or normally loaded controls. Bone formation, however, assessed both by tetracycline labeling and by [3H]proline and 45Ca incorporation, was suppressed by alendronate treatment and further decreased by skeletal unloading. Total osteoclast number increased in alendronate-treated animals, but values were similar to those in controls when corrected for the increased bone area. However, the osteoclasts had poorly developed brush borders and appeared not to engage the bone surface when examined at the ultrastructural level. We conclude that alendronate prevents the relative loss of mineralized tissue in growing rats subjected to skeletal unloading, but it does so primarily by inhibiting the resorption of the primary and secondary spongiosa, leading to altered bone modeling in the metaphysis.

When chick limb-bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix. Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3-4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo. When the high-energy phosphates adenosine 5'-triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo. This is in marked contrast to the mineralized matrix formed in the presence of 2.5-5 mM beta-glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger. This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells. In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H-leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate. However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is presented. It is suggested that ATP promotes mineral deposition by providing a high-energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes.

Chick limb bud mesenchymal cells differentiate into chondrocytes and form a cartilaginous matrix in culture. In this study, the mineral formed in different areas within cultures supplemented with 4 mM inorganic phosphate, or 2.5, 5.0, and 10 mM beta-glycerophosphate (beta GP), was characterized by Fourier-transform infrared (FT-IR) microscopy. The relative mineral-to-matrix ratios, and distribution of crystal sizes at specific locations throughout the matrix were measured from day 14 to day 30. The only mineral phase detected was a poorly crystalline apatite. Cultures receiving 4 mM inorganic phosphate had smaller crystals which were less randomly distributed around the cartilage nodules than those in the beta GP-treated cultures. beta GP-induced mineral consisted of larger, more perfect apatite crystals. In cultures receiving 5 or 10 mM beta GP, the relative mineral-to-matrix ratios (calculated from the integrated intensities of the phosphate and amide I bands, respectively) were higher than in the cultures with 4 mM inorganic phosphate or in the in vivo calcified chick cartilage.

The optimal conditions for obtaining a calcified cartilage matrix approximating that which exists in situ were established in a differentiating chick limb bud mesenchymal cell culture system. Using cells from stage 21-24 embryos in a micro-mass culture, at an optimal density of 0.5 million cells/20 microliters spot, the deposition of small crystals of hydroxyapatite on a collagenous matrix and matrix vesicles was detected by day 21 using X-ray diffraction, FT-IR microscopy, and electron microscopy. Optimal media, containing 1.1 mM Ca, 4 mM P, 25 micrograms/ml vitamin C, 0.3 mg/ml glutamine, no Hepes buffer, and 10% fetal bovine serum, produced matrix resembling the calcifying cartilage matrix of fetal chick long bones. Interestingly, higher concentrations of fetal bovine serum had an inhibitory effect on calcification. The cartilage phenotype was confirmed based on the cellular expression of cartilage collagen and proteoglycan mRNAs, the presence of type II and type X collagen, and cartilage type proteoglycan at the light microscopic level, and the presence of chondrocytes and matrix vesicles at the EM level. The system is proposed as a model for evaluating the events in cell mediated cartilage calcification.

Mesenchymal cells isolated from stage 21-24 chick limb-buds plated in a micro-mass culture differentiate to form chondrocytes and synthesize a calcifiable matrix. In the presence of inorganic phosphate (4 mM), hydroxyapatite mineral deposits around cartilage nodules. Ascorbic acid is, in general, an essential co-factor for extracellular matrix synthesis in culture, since it is required for collagen synthesis. In this study we demonstrate that in the absence of ascorbic acid supplementation in the mesenchymal cell cultures, mineral deposition (indicated by X-ray diffraction, measurement of Ca:hydroxyproline ratio, and 45Ca uptake) does not occur. Concentrations of 10-50 micrograms/ml ascorbate were compared to find the "optimal" concentration for cell mediated mineralization; 25 micrograms/ml was selected as optimal based on matrix appearance at the EM level and the rate of 45Ca uptake. High concentrations of ascorbic acid (greater than 75 micrograms/ml), while increasing the amount of hydroxyproline in the matrix synthesized, caused some cell death and hence less cell-mediated mineralization. This study demonstrates both the need for viable cells and a proper matrix for in vitro cell-mediated mineralization, and shows that varying the concentration of L-ascorbate (vitamin C) in the medium can have a marked effect on mineralization in vitro.