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1.  Fourier Transform Infrared Spectroscopic Imaging of Fracture Healing in the Normal Mouse 
Fourier transform infrared spectroscopic imaging (FTIRI) was used to study bone healing with spatial analysis of various callus tissues in wild type mice. Femoral fractures were produced in 28 male C57BL mice by osteotomy. Animals were sacrificed at 1, 2, 4, and 8 weeks to obtain callus tissue at well-defined healing stages. Following microcomputerized tomography, bone samples were cut in consecutive sections for FTIRI and histology, allowing for spatial correlation of both imaging methods in different callus areas (early calcified cartilage, woven bone, areas of intramembranous and endochondral bone formation). Based on FTIRI, mineral/matrix ratio increased significantly during the first 4 weeks of fracture healing in all callus areas and correlated with bone mineral density measured by micro-CT. Carbonate/phosphate ratio was elevated in newly formed calcified tissue and at week 2 attained values comparable to cortical bone. Collagen maturity and mineral crystallinity increased during weeks 1–8 in most tissues while acid phosphate substitution decreased. Temporal and callus area dependent changes were detected throughout the healing period. These data assert the usefulness of FTIRI for evaluation of fracture healing in the mouse and its potential to evaluate pathologic fracture healing and the effects of therapeutic interventions.
doi:10.1155/2015/659473
PMCID: PMC4448139  PMID: 26034749
2.  Mineral and Matrix Changes in Brtl/+ Teeth Provide Insights into Mineralization Mechanisms 
BioMed Research International  2013;2013:295812.
The Brtl/+ mouse is a knock-in model for osteogenesis imperfecta type IV in which a Gly349Cys substitution was introduced into one COL1A1 allele. To gain insight into the changes in dentin structure and mineral composition in these transgenic mice, the objective of this study was to use microcomputed tomography (micro-CT), scanning electron microscopy (SEM), and Fourier transform infrared imaging (FTIRI) to analyze these structures at 2 and 6 months of age. Results, consistent with the dental phenotype in humans with type IV OI, showed decreased molar volume and reduced mineralized tissue volume in the teeth without changes in enamel properties. Increased acid phosphate content was noted at 2 and 6 months by FTIRI, and a trend towards altered collagen structure was noted at 2 but not 6 months in the Brtl/+ teeth. The increase in acid phosphate content suggests a delay in the mineralization process, most likely associated with the defect in the collagen structure. It appears that in the Brtl/+ teeth slow maturation of the mineralized structures allows correction of altered mineral content and acid phosphate distribution.
doi:10.1155/2013/295812
PMCID: PMC3681234  PMID: 23802117
3.  Are Changes in Composition in Response to Treatment of a Mouse Model of Osteogenesis Imperfecta Sex-dependent? 
Background
Osteogenesis imperfecta (OI) is a genetic disease characterized by skeletal fragility and deformity. There is extensive debate regarding treatment options in adults with OI. Antiresorptive treatment reduces the number of fractures in growing oim/oim mice, an animal model that reproducibly mimics the moderate-to-severe form of OI in humans. Effects of long-term treatments with antiresorptive agents, considered for treatment of older patients with OI with similar presentation (moderate-to-severe OI) are, to date, unknown.
Questions/purposes
Fourier transform infrared (FTIR) imaging, which produces a map of the spatial variation in chemical composition in thin sections of bone, was used to address the following questions: (1) do oim/oim mice show a sex dependence in compositional properties at 6.5 months of age; (2) is there a sex-dependent response to treatment with antiresorptive agents used in the treatment of OI in humans; and (3) are any compositional parameters in oim/oim mice corrected to wild-type (WT) values after treatment?
Methods
FTIR imaging data were collected from femurs from four to five mice per sex per genotype per treatment. Treatments were 24 weeks of saline, alendronate, or RANK-Fc; and 12 weeks of saline + 12 weeks RANK-Fc and 12 weeks of alendronate + RANK-Fc. FTIR imaging compositional parameters measured in cortical and cancellous bones were mineral-to-matrix ratio, carbonate-to-mineral ratio, crystal size/perfection, acid phosphate substitution, collagen maturity, and their respective distributions (heterogeneities). Because of the small sample size, nonparametric statistics (Mann-Whitney U- and Kruskal-Wallis tests with Bonferroni correction) were used to compare saline-treated male and female mice of different genotypes and treatment effects by sex and genotype, respectively. Statistical significance was defined as p < 0.05.
Results
At 6.5 months, saline-treated male cortical oim/oim bone had increased mineral-to-matrix ratio (p = 0.016), increased acid phosphate substitution (p = 0.032), and decreased carbonate-to-mineral ratio (p = 0.016) relative to WT. Cancellous bone in male oim/oim also had increased mineral-to-matrix ratio (p = 0.016) relative to male WT. Female oim/oim mouse bone composition for all cortical and cancellous bone parameters was comparable to WT (p > 0.05). Only the female WT mice showed a response of mean compositional properties to treatment, increasing mineral-to-matrix after RANK-Fc treatment in cancellous bone (p = 0.036) compared with saline-treated mice. Male oim/oim increased mineral-to-matrix cortical and cancellous bone heterogeneity in response to all long-term treatments except for saline + RANK-Fc (p < 0.04); female oim/oim cortical mineral-to-matrix bone heterogeneity increased with ALN + RANK-Fc and all treatments increased cancellous female oim/oim bone acid phosphate substitution heterogeneity (p < 0.04).
Conclusions
Both oim/oim and WT mice, which demonstrate sex-dependent differences in composition with saline treatment, showed few responses to long-term treatment with antiresorptive agents. Female WT mice appeared to be more responsive; male oim/oim mice showed more changes in compositional heterogeneity. Changes in bone composition caused by these agents may contribute to improved bone quality in oim/oim mice, because the treatments are known to reduce fracture incidence.
Clinical Relevance
The optimal drug therapy for long-term treatment of patients with moderate-to-severe OI is unknown. Based on bone compositional changes in mice, antiresorptive treatments are useful for continued treatment in OI. There is a reported sexual dimorphism in fracture incidence in adults with OI, but to date, no one has reported differences in response to pharmaceutical intervention. This study suggests that such an investigation is warranted.
doi:10.1007/s11999-015-4268-z
PMCID: PMC4488219  PMID: 25903941
4.  FTIR-I Compositional Mapping of the Cartilage-to-Bone Interface as a Function of Tissue Region and Age 
Soft tissue-to-bone transitions, such as the osteochondral interface, are complex junctions that connect multiple tissue types and are critical for musculoskeletal function. The osteochondral interface enables pressurization of articular cartilage, facilitates load transfer between cartilage and bone, and serves as a barrier between these two distinct tissues. Presently, there is a lack of quantitative understanding of the matrix and mineral distribution across this multitissue transition. Moreover, age-related changes at the interface with the onset of skeletal maturity are also not well understood. Therefore, the objective of this study is to characterize the cartilage-to-bone transition as a function of age, using Fourier transform infrared spectroscopic imaging (FTIR-I) analysis to map region-dependent changes in collagen, proteoglycan, and mineral distribution, as well as collagen organization. Both tissue-dependent and age-related changes were observed, underscoring the role of postnatal physiological loading in matrix remodeling. It was observed that the relative collagen content increased continuously from cartilage to bone, whereas proteoglycan peaked within the deep zone of cartilage. With age, collagen content across the interface increased, accompanied by a higher degree of collagen alignment in both the surface and deep zone cartilage. Interestingly, regardless of age, mineral content increased exponentially across the calcified cartilage interface. These observations reveal new insights into both region- and age-dependent changes across the cartilage-to-bone junction and will serve as critical benchmark parameters for current efforts in integrative cartilage repair.
doi:10.1002/jbmr.2284
PMCID: PMC4963234  PMID: 24839262
COLLAGEN; MATRIX MINERALIZATION; OSTEOARTHRITIS; BIOENGINEERING; AGING
5.  Bone composition: relationship to bone fragility and antiosteoporotic drug effects 
BoneKEy Reports  2015;4:710.
doi:10.1038/bonekey.2015.79
PMCID: PMC4455689  PMID: 26069736
6.  The Effect of Osteoporosis Treatments on Fatigue Properties of Cortical Bone Tissue 
Bone reports  2015;2:8-13.
Bisphosphonates are commonly prescribed for treatment of osteoporosis. Long-term use of bisphosphonates has been correlated to atypical femoral fractures (AFF). AFFs arise from fatigue damage to bone tissue that cannot be repaired due to pharmacologic treatments. Despite fatigue being the primary damage mechanism of AFFs, the effects of osteoporosis treatments on fatigue properties of cortical bone are unknown. To examine if fatigue-life differences occur in bone tissue after different pharmacologic treatments for osteoporosis, we tested bone tissue from the femurs of sheep given a metabolic acidosis diet to induce osteoporosis, followed by treatment with a selective estrogen reception modulator (raloxifene), a bisphosphonate (alendronate or zoledronate), or parathyroid hormone (teriparatide, PTH). Beams of cortical bone tissue were created and tested in four-point bending fatigue to failure. Tissues treated with alendronate had reduced fatigue life and less modulus loss at failure compared to other treatments, while tissue treated with PTH had a prolonged fatigue life. No loss of fatigue life occurred with zoledronate treatment despite its greater binding affinity and potency compared to alendronate. Tissue mineralization measured by microCT did not explain the differences seen in fatigue behavior. Increased fatigue life with PTH suggests that current treatment methods for AFF could have beneficial effects for restoring fatigue life. These results indicate that fatigue life differs with each type of osteoporosis treatment.
doi:10.1016/j.bonr.2014.10.004
PMCID: PMC4306187  PMID: 25642445
7.  The Kidney Sodium-Phosphate Co-Transporter Alters Bone Quality in an Age and Gender Specific Manner 
Bone  2013;53(2):546-553.
Mutations in the kidney NaPiIIa co-transporter are clinically associated with hypophosphatemia, hyperphosphaturia (phosphate wasting), hypercalcemia, nephrolithiasis and bone demineralization. The mouse lacking this co-transporter system was reported to recover its skeletal defects with age, but the “quality” of the bones was not considered. To assess changes in bone quality we examined both male and female NaPiIIa knockout (KO) mice at 1 and 7 months of age using micro-computed tomography (micro-CT) and Fourier transform infrared imaging (FT-IRI). KO cancellous bones at both ages had greater bone volume fraction, trabecular thickness and lesser structure model index based on micro-CT values relative to age- and sex-matched wildtype animals. There was a sexual-dimorphism in the micro-CT parameters, with differences at 7 months seen principally in males. Cortical bone at 1 month showed an increase in bone volume fraction, but this was not seen at 7 months. Cortical thickness which was elevated in the male and female KO at 1 month was lower in the male KO at 7 months.. FTIRI showed a reduced mineral and acid phosphate content in the male and female KO’s bones at 1 month with no change in acid phosphate content at 7 months. Collagen maturity was reduced in KO cancellous bone at 1 month. The observed sexual dimorphism in the micro-CT data may be related to altered phosphate homeostasis, differences in animal growth rates and other factors. These data indicate that the bone quality of the KO mice at both ages differs from the normal and suggests that these bone quality differences may contribute to skeletal phenotype in humans with mutations in this co-transporter.
doi:10.1016/j.bone.2013.01.011
PMCID: PMC3593750  PMID: 23333524
Bone quality; FTIR imaging; micro-computed tomography; NaPi IIa; knockout mice; sexual dimorphism
8.  Fourier Transformed Infra-Red Imaging of Femoral Neck Bone: Reduced Heterogeneity of Mineral-to-Matrix and Carbonate-to-Phosphate and more Variable Crystallinity in Treatment-Naïve Fracture Cases compared to Fracture-Free Controls 
After age 60 hip fracture risk strongly increases, but only a fifth of this increase is attributable to reduced mineral density (BMD, measured clinically). Changes in bone quality, specifically bone composition as measured by Fourier Transform Infrared spectroscopic imaging (FTIRI), also contribute to fracture risk. Here, FTIRI was applied to study the femoral neck and provide spatially derived information on its mineral and matrix properties in age-matched fractured and non-fractured bones. Whole femoral neck cross sections, divided into quadrants along the neck’s axis, from 10 women with hip fracture and 10 cadaveric controls were studied using FTIRI and micro-computed Tomography. Although 3-dimensional micro-CT bone mineral densities were similar, the mineral-to-matrix ratio was reduced in the cases of hip fracture, confirming previous reports. New findings were that the FTIRI microscopic variation (heterogeneity) of the mineral-to-matrix ratio was substantially reduced in the fracture group as was the heterogeneity of the carbonate-to-phosphate ratio. Conversely, the heterogeneity of crystallinity was increased. Increased variation of crystallinity was statistically associated with reduced variation of the carbonate-to-phosphate ratio. Anatomical variation in these properties between the different femoral neck quadrants was reduced in the fracture group compared to controls. While our treatment-naïve patients had reduced rather than increased bending resistance, these changes in heterogeneity associated with hip fracture are in another way comparable to the effects of experimental bisphosphonate therapy, which decreases heterogeneity and other indicators of bone’s toughness as a material.
doi:10.1002/jbmr.1724
PMCID: PMC3515703  PMID: 22865771
Hip fracture; Bone fragility; Mineral/Matrix ratio; Bone heterogeneity; Collagen cross-links maturity; quadrant analysis
9.  POST-TRANSLATIONAL MODIFICATION OF OSTEOPONTIN: EFFECTS on in vitro HYDROXYAPATITE FORMATION and GROWTH 
The manuscript tests the hypothesis that posttranslational modification of the SIBLING family of proteins in general and osteopontin in particular modify the abilities of these proteins to regulate in vitro hydroxyapatite (HA) formation. Osteopontin has diverse effects on hydroxyapatite (HA) mineral crystallite formation and growth depending on the extent of phosphorylation. We hypothesized that different regions of full-length OPN would also have distinct effects on the mineralization process. Thrombin fragmentation of milk OPN (mOPN) was used to test this hypothesis. Three fragments were tested in a de novo HA formation assay; an N-terminal fragment (aa 1–147), a central fragment (aa 148–204) denoted SKK-fragment and a C-terminal fragment (aa 205–262). Compared to intact mOPN the C- and N-terminal fragments behaved comparably, promoting HA formation and growth, but the central SKK-fragment acted as a mineralization inhibitor. In a seeded growth experiment all fragments inhibited mineral proliferation, but the SKK-fragment was the most effective inhibitor. These effects, seen in HA-formation and seeded growth assays in a gelatin gel system and in a pH-stat experiment were lost when the protein or fragments were dephosphorylated. Effects of the fully phosphorylated protein and fragments were also altered in the presence of fibrillar collagen. The diverse effects can be explained in terms of the intrinsically disordered nature of OPN and its fragments which enable them to interact with their multiple partners.
doi:10.1016/j.bbrc.2012.02.024
PMCID: PMC3299831  PMID: 22342723
Osteopontin; hydroxyapatite; SIBLING proteins; mineralization mechanisms
10.  Bone Quality: From Bench to Bedside: Opening Editorial Comment 
doi:10.1007/s11999-011-1785-2
PMCID: PMC3126974  PMID: 21344274
11.  Changes in Matrix Protein Gene Expression Associated With Mineralization in the Differentiating Chick Limb-bud Micromass Culture System 
Journal of Cellular Biochemistry  2011;112(2):607-613.
Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed.
doi:10.1002/jcb.22951
PMCID: PMC3346962  PMID: 21268082
CHICK LIMB-BUD; MICROMASS CULTURE; MINERALIZATION; MICROARRAY; GENE EXPRESSION
12.  Research Perspectives: The 2013 AAOS/ORS Research Symposium on Bone Quality and Fracture Prevention 
Bone fracture resistance is determined by the amount of bone present (“bone quantity”) and by a number of other geometric and material factors grouped under the term “bone quality.” In May 2013, a workshop was convened among a group of clinicians and basic science investigators to review the current state of the art in Bone Quality and Fracture Prevention and to make recommendations for future directions for research. The AAOS/ORS/OREF workshop was attended by 64 participants, including two representatives of the National Institutes of Arthritis and Musculoskeletal and Skin Diseases and 13 new investigators whose posters stimulated additional interest. A key outcome of the workshop was a set of recommendations regarding clinically relevant aspects of both bone quality and quantity that clinicians can use to inform decisions about patient care and management. The common theme of these recommendations was the need for more education of clinicians in areas of bone quality and for basic science studies to address specific topics of pathophysiology, diagnosis, prevention, and treatment of altered bone quality. In this report, the organizers with the assistance of the speakers and other attendees highlight the major findings of the meeting that justify the recommendations and needs for this field.
doi:10.1002/jor.22626
PMCID: PMC4716655  PMID: 24700449
bone quality; fragility fracture; fracture prevention; workshop
13.  Contribution of mineral to bone structural behavior and tissue mechanical properties 
Calcified tissue international  2010;87(5):450-460.
Bone geometry and tissue material properties jointly govern whole-bone structural behavior. While the role of geometry in structural behavior is well characterized, the contribution of the tissue material properties is less clear, partially due to the multiple tissue constituents and hierarchical levels at which these properties can be characterized. Our objective was to elucidate the contribution of the mineral phase to bone mechanical properties across multiple length scales, from the tissue material level to the structural level. Vitamin D and calcium deficiency in 6-week-old male rats was employed as a model of reduced mineral content with minimal collagen changes. The structural properties of the humeri were measured in three-point bending and related to the mineral content and geometry from microcomputed tomography. Whole-cortex and local bone tissue properties were examined with infrared (IR) spectroscopy, Raman spectroscopy, and nanoindentation, to understand the role of altered mineral content on the constituent material behavior. Structural stiffness (-47%) and strength (-50%) were reduced in vitamin D-deficient (-D) humeri relative to controls. Moment of inertia (-38%), tissue mineral density (TMD, -9%), periosteal mineralization (-28%), and IR mineral:matrix ratio (-19%) were reduced in -D cortices. Thus, both decreased tissue mineral content and changes in cortical geometry contributed to impaired skeletal load bearing function. In fact, 97% of the variability in humeral strength was explained by moment of inertia, TMD, and IR mineral:matrix ratio. The strong relationships between structural properties and cortical material composition demonstrate a critical role of the microscale material behavior in skeletal load-bearing performance.
doi:10.1007/s00223-010-9404-x
PMCID: PMC2965269  PMID: 20730582
bone strength; material properties; mineral; rat; Fourier transform infrared spectroscopy
14.  Bisphosphonate Treatment Modifies Canine Bone Mineral and Matrix Properties and their Heterogeneity 
Bone  2009;46(3):666-672.
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 one-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 thirty normal beagles that had been treated daily for one 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.
doi:10.1016/j.bone.2009.11.011
PMCID: PMC2823979  PMID: 19925895
Bisphosphonate; bone remodeling; FTIR imaging; bone heterogeneity; osteoporosis
15.  DSPP Effects on in vivo Bone Mineralization 
Bone  2008;43(6):983-990.
Dentin sialophosphoprotein has been implicated in the mineralization process based on the defective dentin formation in Dspp null mice (Dspp-/-). Dspp is expressed at low levels in bone and Dspp-/- femurs assessed by quantitative micro-computed tomography (microCT) and Fourier transform infrared spectroscopic imaging (FTIRI) exhibit some mineral and matrix property differences from wildtype femurs in both developing and mature mice. Compared to wildtype, Dspp-/- mice initially (5 weeks) and at 7 months had significantly higher trabecular bone volume fractions and lower trabecular separation, while at 9 months, bone volume fraction and trabecular number were lower. Cortical bone mineral density, area, and moments of inertia in Dspp-/- were reduced at 9 months. By FTIRI, Dspp-/- animals initially (5 months) contained more stoichiometric bone apatite with higher crystallinity (crystal size/perfection) and lower carbonate substitution. This difference progressively reversed with age (significantly decreased crystallinity and increased acid phosphate content in Dspp-/- cortical bone by 9 months of age). Mineral density as determined in 3D micro-CT and mineral-to-matrix ratios as determined by 2D FTIRI in individual cortical and trabecular bones were correlated (r2=0.6, p<0.04). From the matrix analysis, the collagen maturity of both cortical and trabecular bone was greater in Dspp-/- than controls at 5 weeks; by 9 months this difference in cross-linking pattern did not exist. Variations in mineral and matrix properties observed at different ages are attributable, in part, to the ability of the Dspp gene products to regulate both initial mineralization and remodeling, implying an effect of Dspp on bone turnover.
doi:10.1016/j.bone.2008.08.110
PMCID: PMC2621360  PMID: 18789408
Dentin sialophosphoprotein; FTIR spectroscopic imaging; bone; micro-computed tomography; FTIRI
16.  Modulation of Extracellular Matrix Protein Phosphorylation Alters Mineralization in Differentiating Chick Limb-bud Mesenchymal Cell Micromass Cultures 
Bone  2008;42(6):1061-1071.
Protein phosphorylation and dephosphorylation are important regulators of cellular and extracellular events. The purpose of this study was to define how these events regulate cartilage matrix calcification in a cell culture system that mimics endochondral ossification. The presence of casein kinase II (CK2), an enzyme known to phosphorylate matrix proteins, was confirmed by immunohistochemistry. The importance of phosphoprotein phosphorylation and dephosphorylation was examined by comparing effects of inhibiting CK2 or phosphoprotein phosphatases on mineral accretion relative to untreated mineralizing controls. Specific inhibitors were added to differentiating chick limb bud mesenchymal cell micromass cultures during the development of a mineralized matrix at the times of cell differentiation, proliferation, formation of the mineralized matrix, or proliferation of the mineral crystals. The mineralizing media for these cultures contained 4mM inorganic phosphate and no organic-phosphate esters; control cultures had 1mM inorganic phosphate. Mineralization was monitored based on 45Ca uptake and infrared characterization of the mineral; cell viability was assessed by three independent methods. Treatments that caused cell toxicity were excluded from the analysis.
Inhibition of CK2 activity with apigenin or CK2 inhibitor II reduced the rate of mineral deposition, but did not block mineral accretion. Effects were greatest during the time of mineralized matrix formation. Inhibition of phosphoprotein phosphatase activities with okadaic acid, calyculin A, and microcystin LR, at early time points also markedly inhibited mineral accretion. Inhibition after mineralization had commenced increased the mineral yield. Levamisole, an alkaline phosphatase inhibitor, had no effect on mineral accretion in this system, suggesting the involvement of other phosphatases. Adding additional inorganic phosphate to the inhibited cultures after mineralization had started, but not earlier, reversed the inhibition indicating the phosphatases were, in part, providing a source of inorganic phosphate.
To characterize the roles of specific phosphoproteins blocking studies were performed. Blocking with anti-osteopontin antibody confirmed osteopontin’s previously reported role as a mineralization inhibitor. Blocking antibodies to bone sialoprotein added from day 9 or on days 9 and 11 retarded mineralization, supporting its role as a mineralization nucleator. Antibodies to osteonectin slightly stimulated early mineralization, but had no effect after the time that initial mineral deposition occurs. Taken together, the results of this study demonstrate the importance of the phosphorylation state of extracellular matrix proteins in regulating mineralization in this culture system.
doi:10.1016/j.bone.2008.01.016
PMCID: PMC2442476  PMID: 18396125
micromass culture; avian; mineralization; casein kinase II; phosphoprotein phosphatase
18.  Studying Variations in Bone Composition at Nano-Scale Resolutions: A Preliminary Report 
Calcified tissue international  2014;95(5):413-418.
Bone has a hierarchical structure extending from the micrometer to the nanometer scale. We report here the first analysis of non-human primate osteonal bone obtained using a spectrometer coupled to an AFM-microscope (AFM-IR), with a resolution of 50–100 nm. Average spectra correspond to those observed with conventional FTIR spectroscopy. The following validated FTIR parameters were calculated based on intensities observed in scans covering ~60 µm from the osteon center: mineral content (1030 cm−1/1660 cm−1); crystallinity (1030 cm−1)/1020 cm−1), collagen maturity (1660 cm−1/1690 cm−1) and acid phosphate content (1128 cm−1/1096 cm−1). A repeating pattern was found in most of these calculated IR parameters corresponding to the reported inter- and intra-lamellar spacing in human bone, indicating that AFM-IR measurements will be able to provide novel compositional information on the variation in bone at the nanometer level.
doi:10.1007/s00223-014-9909-9
PMCID: PMC4192085  PMID: 25155443
AFM-IR; nano-IR; bone composition; osteonal bone; bone nano-structure
19.  Chondrocyte Apoptosis Is Not Essential for Cartilage Calcification: Evidence From an In Vitro Avian Model 
Journal of cellular biochemistry  2007;100(1):43-57.
The calcification of cartilage is an essential step in the process of normal bone growth through endochondral ossification. Chondrocyte apoptosis is generally observed prior to the transition of calcified cartilage to bone. There are, however, contradictory reports in the literature as to whether chondrocyte apoptosis is a precursor to cartilage calcification, a co-event, or occurs after calcification. The purpose of this study was to test the hypothesis that chondrocyte apoptosis is not a requirement for initial calcification using a cell culture system that mimics endochondral ossification. Mesenchymal stem cells harvested from Stages 21-23 chick limb buds were plated as micro-mass cultures in the presence of 4 mM inorganic phosphate (mineralizing conditions). The cultures were treated with either an apoptosis inhibitor or stimulator and compared to un-treated controls before the start of calcification on day 7. Inhibition of apoptosis with the caspase inhibitor Z-Val-Ala-Asp (O-Me)-fluoromethylketone (Z-VAD-fmk) caused no decreases in calcification as indicated by radioactive calcium uptake or Fourier transform infrared (FT-IR) analysis of mineral properties. When apoptosis was inhibited, the cultures showed more robust histological features (including more intense staining for proteoglycans, and more intact cells within the nodules as well as along the periphery of the cells as compared to untreated controls), more proliferation as noted by bromo-deoxyuridine (BrdU) labeling, decreases in terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick-end labeling (TUNEL) staining, and fewer apoptotic bodies in electron microscopy. Stimulation of apoptosis with 40-120 nM staurosporine prior to the onset of calcification resulted in inhibition of calcium accretion, with the extent of total calcium uptake significantly decreased, the amount of matrix deposition impaired, and the formation of abnormal mineral crystals. These results indicate that chondrocyte apoptosis is not a pre-requisite for calcification in this culture system.
doi:10.1002/jcb.20977
PMCID: PMC1751482  PMID: 16888817
micro-mass; apoptosis; calcification; staurosporine; caspase-inhibition; hydroxyapatite
20.  DMP1 Depletion Decreases Bone Mineralization In Vivo: An FTIR Imaging Analysis 
The role of DMP1 in mineralization was analyzed by comparing bone mineral and matrix properties in dmp1-null female mice to heterozygous and wildtype controls by FTIR imaging spectroscopy. The observed decreased mineral content in dmp1 null mice indicates a key role for dmp1 in bone mineralization. Indirect effects of DMP1 on other systems also determine the KO phenotype.
Introduction
Dentin matrix protein 1 (DMP1), an acidic phosphorylated extracellular matrix protein, is highly expressed in mineralized tissues. In vitro, DMP1 peptides can promote or inhibit mineralization depending on the extent of phosphorylation, the peptide size, and concentration. To clarify the biological function of DMP1 protein on in vivo mineralization, this study analyzed bone properties of dmp1 knockout (KO) mice compared with heterozygous (HET) and wildtype (WT) controls.
Materials and Methods
Tibias from dmp1 KO and age-, sex-, and background-matched HET and WT mice at 4 and 16 weeks (Ntotal = 60) were examined by Fourier transform infrared imaging (FTIRI), histology (n = 6 per genotype and age; N = 36), and geometry by μCT (n = 4 per genotype and age; N = 24). Serum ionic calcium and phosphate concentrations were also determined.
Results
The mineral-to-matrix ratios (spectroscopic parameter of relative mineral content) were significantly lower in dmp1 KO mice tibias compared with WT and HET at 4 and 16 weeks. The mineral crystallinity (crystal size/perfection) was significantly increased in dmp1 KO and HET mice relative to WT. Collagen cross-link ratios (a spectroscopic parameter related to the relative amounts of nonreducible/reducible collagen cross-links) in dmp1 KO were not significantly different from WT and HET. Based on μCT, cortical bone cross-sectional areas at 16 but not 4 weeks were significantly reduced in the KO compared with controls. Maximum, minimum, and polar cross-sectional moments of inertia were significantly lower in dmp1 KO than in HET at 16 weeks but not at 4 weeks. Histological analysis and μCT 3-D images suggested that dmp1 KO mice had osteomalacia. Dmp1 KO mice had significantly lower ionic calcium and phosphate concentrations relative to WT, whereas in the HET, values for phosphate were equivalent, and calcium values were decreased relative to WT values.
Conclusions
The findings of decreased mineral-to-matrix ratio and increased crystal size in bones of dmp1 KO mice suggest that DMP1 has multiple roles (both direct and indirect) in the regulation of postnatal mineralization. We suggest that direct effects on mineral formation, crystal growth, and indirect effects on regulation of Ca × P concentrations and matrix turnover all contribute to the dominant phenotype in the dmp1 KO mouse.
doi:10.1359/JBMR.050815
PMCID: PMC1456072  PMID: 16294270
dentin matrix protein-1; Fourier transform infrared imaging; mineralization; osteomalacia model; mouse; bone geometry
21.  Mineral Changes in Osteoporosis A Review 
Bone mineral composition, crystallinity, and bone mineral content of osteoporotic patients are different from those of normal subjects. We review the evidence that these mineralization parameters contribute to the strength (fracture resistance) of bone and the methods that have been used to examine them. A specific example is provided from analysis of biopsies from the Multiple Outcomes in Raloxifene Evaluation trial. For the analyses, randomly selected biopsies from placebo, low-dose, and high-dose groups (n = 5 per group) obtained at time zero and 2 years after treatment were examined by infrared imaging spectroscopy. In all cases, comparable increases in mineral content were found, but there were no significant variations in mineral crystallinity.
doi:10.1097/01.blo.0000200241.14684.4e
PMCID: PMC1459416  PMID: 16462423
22.  Ultrastructural Organization of Dentin in Mice Lacking Dentin Sialo-phosphoprotein 
Connective tissue research  2014;55(0 1):92-96.
Dentin Sialophosphoprotein (DSPP) is the major noncollagenous protein of dentin and plays a significant role in dentin mineralization. Recently, animal models lacking DSPP have been developed and the DSPP KO phenotype has been characterized at the histological level. Little is known, however, about the DSPP KO dentin at nano- and meso-scale. Dentin is a hierarchical material spanning from nano- to macroscale, hence information on the effects of DSPP deficiency at the submicron scale is essential for understanding of its role in dentin biomineralization. To bridge this gap we have conducted ultrastructural studies of dentin from DSPP KO animals. Transmission electron microscopy (TEM) studies of DSPP KO dentin revealed that although the overall ultrastructural organization was similar to the WT, the mineral particles were less organized. Scanning electron microscopy in the back-scattered mode (BSS MA of the DSPP KO dentin revealed that circumpulpal dentin comprises large areas of nonmineralized matrix, with numerous spherulitic mineralized inclusions, while the mantle dentin appeared largely unaffected. Analysis of the mineral distribution in the circumpalpal dentin of the DSPP KO mice suggests a reduction in the number of mineral nucleation sites and an increase in the nucleation barrier in DSPP KO dentin. These preliminary results indicate that in addition to the reduction of mineralized and total dentin volume in DSPP KO animals significant changes in the ultrastructural organization exist. These changes are likely related to the role of DSPP in the regulation of mineral formation and organization in dentin.
doi:10.3109/03008207.2014.923861
PMCID: PMC4338995  PMID: 25158189
biomineralization; calcium phosphate; dentinogenesis imperfecta; transmission electron microscopy; scanning electron microscopy; microCT
23.  Reduced tissue-level stiffness and mineralization in osteoporotic cancellous bone 
Calcified tissue international  2014;95(2):125-131.
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).
This study is the first to examine cancellous bone composition and mechanical properties from a fracture prone location with osteoporosis. We found differences in tissue composition and mechanical properties with osteoporosis that could contribute to increased fragility in addition to changes in trabecular architecture and bone volume.
doi:10.1007/s00223-014-9873-4
PMCID: PMC4104238  PMID: 24888692
Nanoindentation; Raman spectroscopy; Osteoporosis; Human trabecular bone
24.  Reduced Cortical Bone Compositional Heterogeneity with Bisphosphonate Treatment in Postmenopausal Women with Intertrochanteric and Subtrochanteric Fractures 
Reduction of bone turnover with bisphosphonate treatment alters bone mineral and matrix properties. Our objective was to investigate the effect of bisphosphonate treatment on bone tissue properties near fragility fracture sites in the proximal femur in postmenopausal women with osteoporosis. The mineral and collagen properties of cortico-cancellous biopsies from the proximal femur were compared in bisphosphonate-naive (-BIS, n=20) and bisphosphonate-treated (+BIS, n=20, duration 7 ± 5 y) patients with intertrochanteric (IT) and subtrochanteric (ST) fractures using Fourier transform infrared imaging (FTIRI). The mean values of the FTIRI parameter distributions were similar across groups, but the widths of the parameter distributions tended to be reduced in the +BIS group relative to the -BIS group. Specifically, the distribution widths of the cortical collagen maturity and crystallinity were reduced in the +BIS group relative to those of the -BIS group by 28% (+BIS 0.45 ± 0.18 vs. -BIS 0.63 ± 0.28, p=0.03) and 17% (+BIS 0.087 ± 0.012 vs. -BIS 0.104 ± 0.036, p=0.05), respectively. When the tissue properties were examined as a function of fracture morphology within the +BIS group, the FTIR parameters were generally similar regardless of fracture morphology. However, the cortical mineral:matrix ratio was 8% greater in tissue from patients with atypical ST fractures (n =6) than that of patients with typical (IT or spiral ST) fractures (n=14) (Atypical 5.6 ± 0.3 vs. Typical 5.2 ± 0.5, p=0.03). Thus, although the mean values of the FTIR properties were similar in both groups, the tissue in bisphosphonate-treated patients had a more uniform composition than that of bisphosphonate-naïve patients. The observed reductions in mineral and matrix heterogeneity may diminish tissue-level toughening mechanisms.
doi:10.1002/jbmr.560
PMCID: PMC4404705  PMID: 22072397
Fourier transform infrared imaging; cortical bone; material properties; hip fracture; atypical subtrochanteric fracture
25.  Bone composition: relationship to bone fragility and antiosteoporotic drug effects 
BoneKEy Reports  2013;2:447.
The composition of a bone can be described in terms of the mineral phase, hydroxyapatite, the organic phase, which consists of collagen type I, noncollagenous proteins, other components and water. The relative proportions of these various components vary with age, site, gender, disease and treatment. Any drug therapy could change the composition of a bone. This review, however, will only address those pharmaceuticals used to treat or prevent diseases of bone: fragility fractures in particular, and the way they can alter the composition. As bone is a heterogeneous tissue, its composition must be discussed in terms of the chemical makeup, properties of its chemical constituents and their distributions in the ever-changing bone matrix. Emphasis, in this review, is placed on changes in composition as a function of age and various diseases of bone, particularly osteoporosis. It is suggested that while some of the antiosteoporotic drugs can and do modify composition, their positive effects on bone strength may be balanced by negative ones.
doi:10.1038/bonekey.2013.181
PMCID: PMC3909232  PMID: 24501681

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