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Calcific aortic valve disease (CAVD) is an inexorably degenerative pathology characterized by progressive calcific lesion formation on the valve leaflets. The interaction of valvular cells in advanced lesion environments is not well understood yet highly relevant as clinically detectable CAVD exhibits calcifications composed of non-stoichiometric hydroxyapatite (HA). In this study, Fourier transform infrared spectroscopic imaging was used to spatially analyze mineral properties as a function of disease progression. Crystallinity (size and perfection) increased with increased valve calcification. To study the relationship between crystallinity and cellular behavior in CAVD, valve cells were seeded into 3D mineral-rich collagen gels containing synthetic HA particles, which had varying crystallinities. Lower crystallinity HA drove myofibroblastic activation in both valve interstitial and endothelial cells, as well as osteoblastic differentiation in interstitial cells. Additionally, calcium accumulation within gels depended on crystallinity, and apoptosis was insufficient to explain differences in HA-driven cellular activity. The protective nature of endothelial cells against interstitial cell activation and calcium accumulation was completely inhibited in the presence of less crystalline HA particles. Elucidating valve cellular behavior post-calcification is of vital importance to better predict and treat clinical pathogenesis, and mineral-containing hydrogel models provide a unique 3D platform to evaluate valve cell responses to a later stage of valve disease., Statement of Significance: We implement a 3D in vitro platform with embedded hydroxyapatite (HA) nanoparticles to investigate the interaction between valve interstitial cells, valve endothelial cells, and a mineral-rich extracellular environment. HA nanoparticles were synthesized based on analysis of the mineral properties of calcific regions of diseased human aortic valves. Our findings indicate that crystallinity of HA drives activation and differentiation in interstitial and endothelial cells. We also show that a mineralized environment blocks endothelial protection against interstitial cell calcification. Our HA-containing hydrogel model provides a unique 3D platform to evaluate valve cell responses to a mineralized ECM. This study additionally lays the groundwork to capture the diversity of mineral properties in calcified valves, and link these properties to progression of the disease., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In vertebrates and invertebrates, biomineralization is controlled by the cell and the proteins they produce. A large number of these proteins are intrinsically disordered, gaining some secondary structure when they interact with their binding partners. These partners include the component ions of the mineral being deposited, the crystals themselves, the template on which the initial crystals form, and other intrinsically disordered proteins and peptides. This review speculates why intrinsically disordered proteins are so important for biomineralization, providing illustrations from the SIBLING (small integrin binding N-glycosylated) proteins and their peptides. It is concluded that the flexible structure, and the ability of the intrinsically disordered proteins to bind to a multitude of surfaces is crucial, but details on the precise-interactions, energetics and kinetics of binding remain to be determined., (Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Dentin sialophosphoprotein (DSPP) is one of the major non-collagenous proteins present in dentin, cementum and alveolar bone; it is also transiently expressed by ameloblasts. In humans many mutations have been found in DSPP and are associated with two autosomal-dominant genetic diseases - dentinogenesis imperfecta II (DGI-II) and dentin dysplasia (DD). Both disorders result in the development of hypomineralized and mechanically compromised teeth. The erupted mature molars of Dspp(-/-) mice have a severe hypomineralized dentin phenotype. Since dentin and enamel formations are interdependent, we decided to investigate the process of enamel onset mineralization in young Dspp(-/-) animals. We focused our analysis on the constantly erupting mouse incisor, to capture all of the stages of odontogenesis in one tooth, and the unerupted first molars. Using high-resolution microCT, we revealed that the onset of enamel matrix deposition occurs closer to the cervical loop and both secretion and maturation of enamel are accelerated in Dspp(-/-) incisors compared to the Dspp(+/-) control. Importantly, these differences did not translate into major phenotypic differences in mature enamel in terms of the structural organization, mineral density or hardness. The only observable difference was the reduction in thickness of the outer enamel layer, while the total enamel thickness remained unchanged. We also observed a compromised dentin-enamel junction, leading to delamination between the dentin and enamel layers. The odontoblast processes were widened and lacked branching near the DEJ. Finally, for the first time we demonstrate expression of Dspp mRNA in secretory ameloblasts. In summary, our data show that DSPP is important for normal mineralization of both dentin and enamel., (Copyright © 2016 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Cysteine (C)-X-C chemokine receptor-4 (CXCR4) is the primary transmembrane receptor for stromal cell-derived factor-1 (SDF-1). We previously reported in mouse or human bone marrow-derived mesenchymal stromal stem cells (BMSCs) that deleting or antagonizing CXCR4 inhibits bone morphogenetic protein-2 (BMP2)-induced osteogenic differentiation. The goal of this study was to determine whether CXCR4-deficiency in BMSCs is an age-related effect in association with impaired osteogenic differentiation potency of aged BMSCs. Using BMSCs derived from C57BL/6J wild type mice at ages ranging from 3 to 23 months old, we detected decreased CXCR4 mRNA and protein expression as well as SDF-1 secretion with advancing aging. Moreover, CXCR4-deficient BMSCs from elderly vs. young mice exhibited impaired osteogenic differentiation in response to BMP2 stimulation or when cultured in dexamethasone (Dex)-containing osteogenic medium, evidenced by decreased alkaline phosphatase activity, osteocalcin synthesis, and calcium deposition (markers for immature and mature osteoblasts). Mechanistically, impaired BMP2- or Dex-osteoinduction in BMSCs of elderly mice was mediated by inhibited phosphorylation of intracellular R-Smads and Erk1/2 or Erk1/2 and p38 proteins, and decreased Runx2 and Osx expression (osteogenesis "master" regulators) were also detected. Furthermore, adenovirus-mediated repair of CXCR4 expression in BMSCs of elderly mice restored their osteogenic differentiation potentials to both BMP2 treatment and osteogenic medium. Collectively, our results demonstrate for the first time that CXCR4 expression in mouse BMSCs declines with aging, and this CXCR4-deficiency impairs osteogenic differentiation potency of aged BMSCs. These findings provide novel insights into mechanisms underlying age-related changes in BMSC-osteogenesis, and will potentiate CXCR4 as a therapeutic target to improve BMSC-based bone repair and regeneration in broad orthopedic situations., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Chemokine stromal cell-derived factor-1 (SDF-1) signals via binding to its primary transmembrane receptor, cysteine (C)-X-C chemokine receptor-4 (CXCR4). We previously reported that SDF-1 regulates osteogenic differentiation of mesenchymal stem/progenitor cells (MPCs) induced by bone morphogenetic protein-2 (BMP2). Although BMP2 is also capable of inducing chondrogenic differentiation of MPCs, the involvement of SDF-1 signaling in this function of BMP2 remains unknown. In this study, we aimed to test the role of SDF-1 signaling involved in BMP2-induced chondrogenic differentiation, using ATDC5 chondroprogenitors and mouse bone marrow-derived mesenchymal stromal cells (BMSCs). Our data showed that blocking of the SDF-1/CXCR4 pathway inhibits the differentiation of these cells into the chondrocytic lineages in response to BMP2 stimulation, evidenced by the reduced expression of type II collagen α1 (Col2α1), aggrecan, and type X collagen α1 (Col10α1), markers for chondrogenic differentiation. This effect of blocking SDF-1 signaling on BMP2-chondrogenic differentiation is associated with suppressed Sox9 and Runx2 expression (key transcription factors required for early and late stages of chondrogenic differentiation, respectively) and mediated via inhibiting intracellular Smad and Erk activation. Moreover, we found that addition of exogenous SDF-1 protein synergistically enhances the BMP2-induced chondrogenic differentiation in a dose-dependent manner. Collectively, our results demonstrated a novel role of SDF-1 signaling in regulating BMP2-induced chondrogenic differentiation in vitro. These data provide new insights into molecular mechanisms underlying BMP2-osteo/chondrogenesis, and may lead to the identification of new therapeutic targets and strategies to improve cartilage repair and regeneration in broad orthopaedic situations., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Osteoporosis is a frequent problem in disorders characterized by iron overload, such as the thalassemias and hereditary hemochromatosis. The exact role of iron in the development of osteoporosis in these disorders is not established. To define the effect of iron excess in bone, we generated an iron-overloaded mouse by injecting iron dextran at 2 doses into C57/BL6 mice for 2 months. Compared with the placebo group, iron-overloaded mice exhibited dose-dependent increased tissue iron content, changes in bone composition, and trabecular and cortical thinning of bone accompanied by increased bone resorption. Iron-overloaded mice had increased reactive oxygen species and elevated serum tumor necrosis factor-α and interleukin-6 concentrations that correlated with severity of iron overload. Treatment of iron-overloaded mice with the antioxidant N-acetyl-L-cysteine prevented the development of trabecular but not cortical bone abnormalities. This is the first study to demonstrate that iron overload in mice results in increased bone resorption and oxidative stress, leading to changes in bone microarchitecture and material properties and thus bone loss.

Stromal derived factor-1 (SDF-1) is a chemokine signaling molecule that binds to its transmembrane receptor CXC chemokine receptor-4 (CXCR4). While we previously detected that SDF-1 was co-required with bone morphogenetic protein 2 (BMP2) for differentiating mesenchymal C2C12 cells into osteoblastic cells, it is unknown whether SDF-1 is similarly involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). Therefore, here we examined the role of SDF-1 signaling during BMP2-induced osteogenic differentiation of primary MSCs that were derived from human and mouse bone marrow. Our data showed that blocking of the SDF-1/CXCR4 signal axis or adding SDF-1 protein to MSCs significantly affected BMP2-induced alkaline phosphatase (ALP) activity and osteocalcin (OCN) synthesis, markers of preosteoblasts and mature osteoblasts, respectively. Moreover, disrupting the SDF-1 signaling impaired bone nodule mineralization during terminal differentiation of MSCs. Furthermore, we detected that blocking of the SDF-1 signaling inhibited the BMP2-induced early expression of Runt-related factor-2 (Runx2) and osterix (Osx), two "master" regulators of osteogenesis, and the SDF-1 effect was mediated via intracellular Smad and Erk activation. In conclusion, our results demonstrated a regulatory role of SDF-1 in BMP2-induced osteogenic differentiation of MSCs, as perturbing the SDF-1 signaling affected the differentiation of MSCs towards osteoblastic cells in response to BMP2 stimulation. These data provide novel insights into molecular mechanisms underlying MSC osteogenesis, and will contribute to the development of MSC therapies for enhancing bone formation and regeneration in broad orthopaedic situations., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. (45)Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of (45)Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.

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.

Vibrational spectroscopy (Infrared and Raman), and in particular micro-spectroscopy and micro-spectroscopic imaging has been used to characterize developmental changes in bone and other mineralized tissues, to monitor these changes in cell cultures, and to detect disease and drug-induced modifications. Examples of the use of infrared micro-spectroscopy and micro-spectroscopic imaging are discussed in this review.

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 deposition of calcium-containing crystals in tissues is due to a combination of factors: elevation in the concentrations of precipitating ions, formation of specific nucleators, and removal of macromolecules that inhibit crystal deposition. This study tested the hypothesis that calcium acidic phospholipid phosphate complexes, which promote hydroxyapatite deposition both in vitro and in vivo, are associated only with hydroxyapatite deposits, and furthermore, that the presence of these complexes is associated with all such hydroxyapatite deposits. Lipid analysis of 76 surgical specimens containing evidence of pathologic calcification (35 hydroxyapatite, 35 calcium pyrophosphate dihydrate, and 6 containing other crystalline materials) had mean complexed acidic phospholipid contents of 8.7, 1, and 0.012, (microgram/mg demineralized dry weight) respectively. Tissues that contained larger, more perfect hydroxyapatite crystals based on x-ray diffraction analyses, had a higher complexed acidic phospholipid content (7.5 +/- 4 micrograms/mg demineralized dry weight, N = 16) than tissues with poorly crystallized hydroxyapatite (3.9 +/- 2 micrograms/mg, N = 11). Histologically, tissues containing larger crystals were characterized by cell or tissue necrosis. Poorly crystalline deposits were found in tissues showing little or no evidence of cell necrosis or tissue degeneration.