E.P. Paschalis

Extracellular Matrix Mineralization and Osteoblast Gene Expression by Human Adipose Tissue–Derived Stromal Cells

Human adipose tissue represents an abundant reservoir of stromal cells with potential utility for tissue engineering. The current study demonstrates the ability of human adipose tissue-derived stromal cells to display some of the hallmarks of osteoblast differentiation in vitro. Following treatment with ascorbate, beta-glycerophosphate, dexamethasone, and 1,25 dihydroxy vitamin D(3), adipose tissue-derived stromal cells mineralize their extracellular matrix based on detection of calcium phosphate deposits using Alizarin Red and von Kossa histochemical stains. Fourier transform infrared analysis demonstrates the apatitic nature of these crystals. Mineralization is accompanied by increased expression or activity of the osteoblast-associated proteins osteocalcin and alkaline phosphatase. These and other osteoblast-associated gene markers are detected based on polymerase chain reaction. In contrast, the adipocyte gene markers--leptin, lipoprotein lipase, and peroxisome proliferator activated receptor gamma2--are reduced under mineralization conditions, consistent with the reciprocal relationship postulated to exist between adipocytes and osteoblasts. The current work supports the presence of a multipotent stromal cell population within human extramedullary adipose tissue. These findings have potential implications for human bone tissue bioengineering.

Spectroscopic characterization of collagen cross-links in bone

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross‐linking pattern. The intermolecular cross‐linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross‐linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6‐deficient chick homogenized bone and their age‐ and sex‐matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at ∼1660 cm−1 and ∼1690 cm−1 was related to collagen cross‐links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH‐DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross‐links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (∼5 μm) calcified tissue sections with a spatial resolution of ∼7 μm.

FTIR microspectroscopic analysis of human osteonal bone

Fourier Transform Infrared Microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of non-osteoporotic human osteonal bone. Spectral maps in four orthogonal directions (in 10 μm steps) from the centers towards the peripheries of individual osteons were obtained from iliac crest biopsies of two necropsy cases. Mineral to matrix ratios, calculated from the ratio of integrated areas of the phosphate v1,v3 band at 900–1200 cm-1 to the amide I band at 1585–1725 cm-1, increased from the center to the periphery of the osteon. The total carbonate (based on the v2 band at ≈850–900 cm-1) to phosphate v1,v3 ratio decreased as the mineral to matrix ratio increased. Analysis of the v2 CO32- band with a combination of second-derivative spectroscopy and curve fitting revealed a decrease in “labile” carbonate, a slight decrease in Type A and a slight increase in Type B carbonate from the center to the periphery of the osteon. Similar analysis of the components of the v1,v3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. These components were assigned by comparison with published frequencies for apatite and acid-phosphate containing calcium phosphates. The most consistent variations were alterations in the relative percent areas of bands at ≈1020 and ≈1030 cm-1, which had previously been assigned to nonstoichiometric and stoichiometric apatites, respectively. This ratio was used as an index of variation in crystal perfection throughout the osteon. This ratio decreased as the mineral to matrix ratio increased. The reproducibility of these parameters at multiple sites in multiple biopsies suggests their applicability for the analysis of mineral changes in disease.

Osteopontin Deficiency Increases Mineral Content and Mineral Crystallinity in Mouse Bone

Fourier transform infrared microspectroscopy (FTIRM) and infrared imaging (FTIRI) were used to characterize the mineral in bones of two different lines of Opn-deficient (Opn-/-) mice and their background-matched wild-type controls (Opn+/+). Sections of tibia and femur from 12-week-old and 16-week-old mice were evaluated with a spatial resolution between 10 microm (FTIRM) and 7 microm (FTIRI). FTIRI was used to examine 400 microm x 400 microm areas in cortical bone and trabecular bone and FTIRM examined selected 20 microm x 20 microm areas at sites within these anatomically defined areas. Despite the absence of an obvious phenotype in Opn-deficient mice, being undetectable by radiographic and histological methods, FTIRM analyses revealed that the relative amount of mineral in the more mature areas of the bone (central cortical bone) of Opn-knockout mice was significantly increased. Moreover, mineral maturity (mineral crystal size and perfection) throughout all anatomic regions of the Opn-deficient bone was significantly increased. The 2-dimensional, color-coded data (images) produced by FTIRI showed similar increases in mineral maturity in the Opn-/- bone, however, the crystallinity parameters were less sensitive, and significance was not achieved in all areas analyzed. Nonetheless, the findings of increased mineral content and increased crystal size/perfection in both lines of Opn-deficient mice at both ages are consistent with in vitro data indicating that Opn is a potent inhibitor of mineral formation and mineral crystal growth and proliferation, and also support a role for Opn in osteoclast recruitment and function.

Fourier transform infrared spectroscopy of the solution-mediated conversion of amorphous calcium phosphate to hydroxyapatite: New correlations between X-ray diffraction and infrared data

Fourier Transform infrared spectroscopic analysis of maturing, poorly crystalline hydroxyapatite (HA) formed from the conversion of amorphous calcium phosphate (ACP) at constant pH or variable pH show only subtle changes in the ν1, ν3 phosphate absorption region (900 cm−1−1200 cm−1). This region is of interest because it can ve detected by analysis of mineralized tissue sections using FT-IR microscopy. To evaluate the subtle spectral changes occurring during the maturation, second derivatives of the spectra were calculated. HA formed at constant pH showed little or no variation in the second derivative peak positions with bands occurring at 960 cm−1, 985 cm−1, 1030 cm−1, 1055 cm−1, 1075 cm−1, 1096 cm−1, 1116 cm−1, and 1145 cm−1. These bands can be assigned to molecular vibrations of the phosphate (PO43−) moiety in an apatitic/stoichiometric environment of HA. In contrast, during the early stages of maturation of the HA formed at variable pH, second derivative peak positions occurring at 958 cm−1, 985 cm−1, 1020 cm−1, 1038 cm−1, 1112 cm−1, and 1127 cm−1 shifted in position with maturation, indicating, that the environment of the phosphate species is changing as the crystals mature. Peaks at 1020 cm−1, 1038 cm−1, 1112 cm−1, and 1127 cm−1 were attributable to nonstoichiometry and/or the presence of acid phosphate-containing species. This concept was supported by the lower Ca:P molar ratios measured by chemical analysis of the synthetic material made at variable pH. Using the second derivative peak positions as initial input parameters, the ν1, ν3 phosphate region of the synthetic HAs prepared at constant pH were curve fit. X-ray diffraction patterns of these same materials were also curve fit to calculate the changes in crystallinty (size/perfection) in the c-axis 002 reflection as well as the 102, 210, 211, 112, 300, 200, and 301 planes. Linear regression analysis showed that the changes in the percent area of the underlying bands at 982 cm−1, 999 cm−1, 1030 cm−1, 1075 cm−1, 1096 cm−1, 1116 cm−1, and 1145 cm−1 were correlated with changes in crystallinity in one or more of the reflection planes. It is suggested that a combination of second-derivative and curve-fitting analysis of the ν1, ν3 phosphate contour allows the most reproducible evaluation of these spectra.

Osteoblast-mediated mineral deposition in culture is dependent on surface microtopography

Osteoblast phenotypic expression in monolayer culture depends on surface microtopography. Here we tested the hypothesis that mineralized bone nodule formation in response to osteotropic agents such as bone morphogenetic protein-2 (BMP-2) and dexamethasone is also influenced by surface microtopography. Fetal rat calvarial (FRC) cells were cultured on Ti implant materials (PT [pretreated], Ra = 0.6 mm; SLA [course grit blasted and acid etched], Ra = 4.0 mm; TPS [Ti plasma sprayed], Ra = 5.2 mm) in the presence of either BMP-2 (20 ng/ml) or 10?8 M dexamethasone (Dex). At 14 days post-confluence, a homogenous layer of cells covered the surfaces, and stacks of cells that appeared to be nodules emerging from the culture surface were present in some areas on all three Ti surfaces. Cell proliferation decreased while alkaline phosphatase specific activity (ALPase) and nodule number generally increased with increasing surface roughness in both control and treated cultures. There was no difference in cell number between the control and Dex-treated cultures for a particular surface, but BMP-2 significantly reduced cell number compared with control or Dex-treated cultures. Treatment with Dex or BMP-2 further increased ALPase on all surfaces except for PT cultures with Dex. Dex had no effect on nodule area in cultures grown on PT or SLA disks, yet increased nodule number by more than 100% in cultures on PT disks. Though the effect of BMP-2 on nodule number was the same as Dex, BMP-2 increased nodule area on all surfaces except TPS, where area was decreased. Ca and P content of the cell layers in control cultures did not vary with surface roughness. However, cultures treated with Dex had increased Ca content on all surfaces, but the greatest increase was seen on SLA and TPS. BMP-2 increased Ca content in cultures on all surfaces, with the greatest increase on the PT surface. BMP-2 treatment increased P content on all surfaces, whereas Dex only increased P on rough surfaces. Of all cultures examined, the Ca/P weight ratio was 2:1 only on rough surfaces with BMP-2, indicating the presence of bone-like apatite. This was further validated by Fourier transform infrared (FTIR) imaging showing a close association between mineral and matrix on TPS and SLA surfaces with BMP-2-treated cells, and individual spectra indicated the presence of an apatitic mineral phase comparable to bone. In contrast, mineral on the smooth surface of BMP-2-treated cultures and on all surfaces where cultures were treated with Dex was not associated with the matrix and the spectra, not typical of bone apatite, implying dystrophic mineralization. This demonstrates that interactions between growth factor or hormone and surface microtopography can modulate bone cell differentiation and mineralization.

In situ analysis of mineral content and crystallinity in bone using infrared micro-spectroscopy of the ν4 PO43- vibration

Measurements of bone mineral content and composition in situ provide insight into the chemistry of bone mineral deposition. Infrared (IR) micro-spectroscopy is well suited for this purpose. To date, IR microscopic (including imaging) analyses of bone apatite have centered on the nu(1),nu(3) PO(4)(3-) contour. The nu(4) PO(4)(3-) contour (500-650 cm(-1)), which has been extensively used to monitor the crystallinity of hydroxyapatite in homogenized bone samples, falls in a frequency region below the cutoff of the mercury-cadmium-telluride detectors used in commercial IR microscopes, thereby rendering this vibration inaccessible for imaging studies. The current study reports the first IR micro-spectroscopy spectra of human iliac crest cross sections in the nu(4) PO(4)(3-) spectral regions, obtained with a synchrotron radiation source and a Cu-doped Ge detector coupled to an IR microscope. The acid phosphate (HPO(4)(2-)) content and mineral crystallite perfection (crystallinity) of a human osteon were mapped. To develop spectra-structure correlations, a combination of X-ray powder diffraction data and conventional Fourier transform IR spectra have been obtained from a series of synthetic hydroxyapatite crystals and natural bone powders of various species and ages. X-ray powder diffraction data demonstrate that there is an increase in average crystal size as bone matures, which correlates with an increase in the nu(4) PO(4)(3-) FTIR absorption peak ratio of two peaks (603/563 cm(-1)) within the nu(4) PO(4)(3-) contour. Additionally, the IR results reveal that a band near 540 cm(-1) may be assigned to acid phosphate. This band is present at high concentrations in new bone, and decreases as bone matures. Correlation of the nu(4) PO(4)(3-) contour with the nu(2) CO (3)(2-) contour also reveals that when acid phosphate content is high, type A carbonate content (i.e., carbonate occupying OH(-) sites in the hydroxyapatite lattice) is high. As crystallinity increases and acid phosphate content decreases, carbonate substitution shifts toward occupation of PO(4)(3-) sites in the hydroxyapatite lattice. Thus, IR microscopic analysis of the nu(4) PO(4)(3-) contour provides a straightforward index of both relative mineral crystallinity and acid phosphate concentration that can be applied to in situ IR micro-spectroscopic analysis of bone samples, which are of interest for understanding the chemical mechanisms of bone deposition in normal and pathological states.

In Vivo RANK Signaling Blockade Using the Receptor Activator of NF‐κB:Fc Effectively Prevents and Ameliorates Wear Debris‐Induced Osteolysis via Osteoclast Depletion Without Inhibiting Osteogenesis

Prosthesis failure due to wear debris‐induced osteolysis remains a major clinical problem and the greatest limitation for total joint arthroplasty. Based on our knowledge of osteoclast involvement in this process and the requirements of receptor activator of NF‐κB (RANK) signaling in osteoclastogenesis and bone resorption, we investigated the efficacy of RANK blockade in preventing and ameliorating titanium (Ti)‐induced osteolysis in a mouse calvaria model. Compared with placebo controls we found that all doses of RANK:Fc above 1 mg/kg intraperitoneally (ip) per 48 h significantly inhibited osteoclastogenesis and bone resorption in response to Ti implanted locally. Complete inhibition occurred at 10 mg/kg ip per 48 h, yielding results that were statistically equivalent to data obtained with Ti‐treated RANK−/− mice. We also evaluated the effects of a single injection of RANK:Fc on day 5 on established osteolysis and found that Ti‐treated were still depleted for multinucleated tartrate‐resistant acid phosphatase‐positive (TRAP+) cells 16 days later. More importantly, this osteoclast depletion did not affect bone formation because the bone lost from the osteolysis on day 5 was restored by day 21. An assessment of the quantity and quality of the newly formed bone in these calvariae by calcein labeling and infrared (IR) microscopy, respectively, showed no significant negative effect of RANK:Fc treatment. These studies indicate that osteoclast depletion via RANK blockade is an effective method to prevent and reverse wear debris‐induced osteolysis without jeopardizing osteogenesis.

Complementary information on bone ultrastructure from scanning small angle X-ray scattering and Fourier-transform infrared microspectroscopy

Scanning small angle X-ray scattering (scanning SAXS) and Fourier-transform infrared microspectroscopy (FT-IRM) have previously been utilized independently to characterize the structural properties of bone in an anatomical position-resolved fashion. Whereas SAXS provides a direct measure of the physical characteristics of apatitic crystals, FT-IRM assesses structure of both mineral and organic matrix at the molecular level. In the present study both methods were applied to examine the same developing bone tissue from the L-4 vertebra of a 14-month-old (accidental death). A 200-microm-thick section was processed for examination by scanning electron microscopy and SAXS. Spectra were collected at 200 microm spatial resolution at specific locations in cortical and cancellous bone. Parameters determined included total SAXS intensity, crystal thickness (T), and degree and direction of predominant crystal orientation. For FT-IRM analysis, a section 4 microm thick was cut longitudinally from the top of the sample. Spectra of regions 100 x 100 microm2 were acquired from the same locations as the SAXS spectra. Integrated areas of the phosphate nu(1,3) collagen amide I, and carbonate nu2 absorbances, were calculated to obtain mineral: matrix and carbonate:mineral ratios. The relative quantities of types A, B, and labile carbonate (substituted for apatite hydroxyl, phosphate, and surface positions, respectively) were also evaluated. Polarized FT-IRM data were collected to determine molecular orientation of the apatite and collagen components. The results of this study show that the information obtained from the two techniques is complementary. Both SAXS and FT-IRM data revealed that the crystals were significantly larger in the cancellous region compared with the cortical region, that mineralization was greater in the cortex, and that the crystals were oriented to a larger degree in the cancellous compared with the cortical bone. The scanning SAXS measure of crystal thickness was significantly correlated to the FT-IRM measures of crystallinity, type A carbonate substitution, and crystal orientation. In conclusion, it was found that the combined use of SAXS and FT-IRM provides valuable, unique information on structural changes in bone at both the microstructural and ultrastructural level. Although each method can be used individually, the combination of techniques provides additional insights into the mechanism of bone crystal maturation.

Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation

Fourier-transform infrared microspectroscopy (FTIRM) allows analysis of mineral content, mineral crystal maturity and mineral composition at ~10-μ spatial resolution. Previous FTIRM analyses comparing 4-μ thick sections from non-decalcified iliac crest biopsies from women with post-menopausal osteoporosis, as contrasted with iliac crest tissue from individuals without evidence of metabolic bone disease, demonstrated significant differences in average mineral content (decreased in osteoporosis) and mineral crystal size/perfection (increased in osteoporosis). More importantly, these parameters, which vary throughout the tissue in relation to the tissue age in healthy bone, showed no such variation in bone biopsies from patients with osteoporosis. The present study compares the spatial and temporal variation in mineral quantity and properties in trabecular bone in high- and low-turnover osteoporosis. Specifically, six biopsies from women (n=5) and one man with high-turnover osteoporosis (age range 39–77) and four women and two men with low turnover osteoporosis (age range 37–63) were compared to ten “normal” biopsies from three men and seven woman (age range: 27–69). “High turnover” was defined as the presence of increased resorptive surface, higher than normal numbers of osteoclasts and greater than or equal to normal osteoblastic activity. “Low turnover” was defined as lower than normal resorptive surface, decreased osteoclast number and less than normal osteoblastic activity. Comparing variations in FTIR-derived values for each of the parameters measured at the surfaces of the trabecular bone to the maximum value observed in multiple trabeculae from each person, the high-turnover samples showed little change in the mineral: matrix ratio, carbonate: amide I ratio, crystallinity and acid phosphate content. The low-turnover samples also showed little change in these parameters, but in contrast to the high-turnover samples, the low-turnover samples showed a slight increase in these parameters, indicative of retarded, but existent resorption and formation. These data indicate that FTIR microspectroscopy can provide quantitative information on mineral changes in osteoporosis that are consistent with proposed mechanisms of bone loss.