Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate.

Bone degenerative pathologies like osteoporosis may be initiated by age-related shifts in anabolic and catabolic responses that control bone homeostasis. Here we show that sulforaphane (SFN), a naturally occurring isothiocyanate, promotes osteoblast differentiation by epigenetic mechanisms. SFN enhances active DNA demethylation viaTet1andTet2and promotes preosteoblast differentiation by enhancing extracellular matrix mineralization and the expression of osteoblastic markers (Runx2,Col1a1,Bglap2,Sp7,Atf4, andAlpl). SFN decreases the expression of the osteoclast activator receptor activator of nuclear factor-κB ligand (RANKL) in osteocytes and mouse calvarial explants and preferentially induces apoptosis in preosteoclastic cells via up-regulation of theTet1/Fas/Caspase 8 and Caspase 3/7 pathway. These mechanistic effects correlate with higher bone volume (∼20%) in both normal and ovariectomized mice treated with SFN for 5 weeks compared with untreated mice as determined by microcomputed tomography. This effect is due to a higher trabecular number in these mice. Importantly, no shifts in mineral density distribution are observed upon SFN treatment as measured by quantitative backscattered electron imaging. Our data indicate that the food-derived compound SFN epigenetically stimulates osteoblast activity and diminishes osteoclast bone resorption, shifting the balance of bone homeostasis and favoring bone acquisition and/or mitigation of bone resorptionin vivo Thus, SFN is a member of a new class of epigenetic compounds that could be considered for novel strategies to counteract osteoporosis.

Acute-phase protein serum amyloid A3 is a novel paracrine coupling factor that controls bone homeostasis.

Serum amyloid A (A-SAA/Saa3) was shown before to affect osteoblastic metabolism. Here, using RT-quantitative PCR and/or immunoblotting, we show that expression of mouse Saa3 and human SAA1 and SAA2 positively correlates with increased cellular maturation toward the osteocyte phenotype. Expression is not detected in C3H10T1/2 embryonic fibroblasts but is successively higher in preosteoblastic MC3T3-E1 cells, late osteoblastic MLO-A5 cells, and MLO-Y4 osteocytes, consistent with findings using primary bone cells from newborn mouse calvaria. Recombinant Saa3 protein functionally inhibits osteoblast differentiation as reflected by reductions in the expression of osteoblast markers and decreased mineralization in newborn mouse calvaria. Yet, Saa3 protein enhances osteoclastogenesis in mouse macrophages/monocytes based on the number of multinucleated and tartrate-resistant alkaline phosphatase-positive cells and Calcr mRNA expression. Depletion of Saa3 in MLO osteocytes results in the loss of the mature osteocyte phenotype. Recombinant osteocalcin, which is reciprocally regulated with Saa3 at the osteoblast/osteocyte transition, attenuates Saa3 expression in MLO-Y4 osteocytes. Mechanistically, Saa3 produced by MLO-Y4 osteocytes is integrated into the extracellular matrix of MC3T3-E1 osteoblasts, where it associates with the P2 purinergic receptor P2rx7 to stimulate Mmp13 expression via the P2rx7/MAPK/ERK/activator protein 1 axis. Our data suggest that Saa3 may function as an important coupling factor in bone development and homeostasis.

Homocysteine induces serum amyloid A3 in osteoblasts via unlocking RGD-motifs in collagen.

Hyperhomocysteinemia is a risk factor for osteoporotic fractures. Homocysteine (Hcys) inhibits collagen cross-linking and consequently decreases bone extracellular matrix (ECM) quality. Serum amyloid A (A-SAA), an acute-phase protein family, plays an important role in chronic and inflammatory diseases and up-regulates MMP13, which plays an important role in bone development and remodeling. Here, we investigate the effect of Hcys on expression of SAA3, a member of the A-SAA gene family, in osteoblasts characterizing underlying mechanisms and possible consequences on bone metabolism. MC3T3-E1 osteoblast-like cells were cultured up to 21 d with Hcys (low millimolar range) or reseeded onto ECM resulting from untreated or Hcys-treated MC3T3-E1 cells. Fourier-transformed infrared spectroscopy and a discriminative antibody were used to characterize the resulting ECM. Gene expression and signaling pathways were analyzed by gene chip, quantitative RT-PCR, and immunoblotting. Transcriptional regulation of Saa3 was studied by promoter transfection assays, chromatin immunoprecipitation, and immunofluorescence microscopy. Hcys treatment resulted in reduced collagen cross-linking, uncovering of RGD-motifs, and activation of the PTK2-PXN-CTNNB1 pathway followed by RELA activation. These signaling events led to increased SAA3 expression followed by the production of MMP13 and several chemokines, including Ccl5, Ccl2, Cxcl10, and Il6. Our data suggest Saa3 as link between hyperhomocysteinemia and development of osteoporosis.

Mitogen-activated protein kinase 2 regulates physiological and pathological bone turnover.

The objective of this study was to investigate the role of the serine-threonine kinase mitogen-activated protein kinase 2 (MK2) in bone homeostasis. Primary bone cell cultures from MK2(+/+) and MK2(-/-) mice were assessed for osteoclast and osteoblast differentiation, bone resorption, and gene expression. Bone architecture of MK2(+/+) and MK2(-/-) mice was investigated by micro-computed tomography and histomorphometry. Ovariectomy was performed in MK2(+/+) and MK2(-/-) mice to assess the role of MK2 in postmenopausal bone loss. Osteoclastogenesis, bone resorption, and osteoclast gene expression were significantly impaired in monocytes from MK2(-/-) compared to MK2(+/+) mice. Mechanistically, loss of MK2 causes impaired DNA binding of c-fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) to tartrate-resistant acid phosphatase (TRAP) and the calcitonin receptor gene promoter. In addition, MK2(-/-) mice showed an age-dependent increase in trabecular bone mass and cortical thickness, fewer osteoclasts, and lower markers of bone resorption than MK2(+/+) mice. Furthermore, MK2(-/-) mice were protected from ovariectomy-induced bone loss. Osteoblastogenesis and bone formation were unchanged in MK2(-/-) mice, whereas osteoblast expression of osteoprotegerin (OPG) and serum levels of OPG were higher in MK2(-/-) than in MK2(+/+) mice. Loss of MK2 effectively blocks bone resorption and prevents the development of postmenopausal bone loss. Small-molecule inhibitors of MK2 could thus emerge as highly effective tools to block bone resorption and to treat postmenopausal bone loss.

Geometry as a factor for tissue growth: towards shape optimization of tissue engineering scaffolds.

Scaffolds for tissue engineering are usually designed to support cell viability with large adhesion surfaces and high permeability to nutrients and oxygen. Recent experiments support the idea that, in addition to surface roughness, elasticity and chemistry, the macroscopic geometry of the substrate also contributes to control the kinetics of tissue deposition. In this study, a previously proposed model for the behavior of osteoblasts on curved surfaces is used to predict the growth of bone matrix tissue in pores of different shapes. These predictions are compared to in vitro experiments with MC3T3-E1 pre-osteoblast cells cultivated in two-millimeter thick hydroxyapatite plates containing prismatic pores with square- or cross-shaped sections. The amount and shape of the tissue formed in the pores measured by phase contrast microscopy confirms the predictions of the model. In cross-shaped pores, the initial overall tissue deposition is twice as fast as in square-shaped pores. These results suggest that the optimization of pore shapes may improve the speed of ingrowth of bone tissue into porous scaffolds.

How linear tension converts to curvature: geometric control of bone tissue growth.

This study investigated how substrate geometry influences in-vitro tissue formation at length scales much larger than a single cell. Two-millimetre thick hydroxyapatite plates containing circular pores and semi-circular channels of 0.5 mm radius, mimicking osteons and hemi-osteons respectively, were incubated with MC3T3-E1 cells for 4 weeks. The amount and shape of the tissue formed in the pores, as measured using phase contrast microscopy, depended on the substrate geometry. It was further demonstrated, using a simple geometric model, that the observed curvature-controlled growth can be derived from the assembly of tensile elements on a curved substrate. These tensile elements are cells anchored on distant points of the curved surface, thus creating an actin "chord" by generating tension between the adhesion sites. Such a chord model was used to link the shape of the substrate to cell organisation and tissue patterning. In a pore with a circular cross-section, tissue growth increases the average curvature of the surface, whereas a semi-circular channel tends to be flattened out. Thereby, a single mechanism could describe new tissue growth in both cortical and trabecular bone after resorption due to remodelling. These similarities between in-vitro and in-vivo patterns suggest geometry as an important signal for bone remodelling.

Microcracks and osteoclast resorption activity in vitro.

During bone remodeling osteoclasts resorb bone, thus removing material, e.g., damaged by microcracks, which arises as a result of physiological loading and could reduce bone strength. Such a process needs targeted bone resorption exactly at damaged sites. Osteocytic signaling plays a key role in this process, but it is not excluded that osteoclasts per se may possess toposensitivity to recognize and resorb damaged bone since it has been shown that resorption spaces are associated with microcracks. To address this question, we used an in vitro setup of a pure osteoclast culture and mineralized substrates with artificially introduced microcracks and microscratches. Histomorphometric analyses and statistical evaluation clearly showed that these defects had no effect on osteoclast resorption behavior. Osteoclasts did not resorb along microcracks, even when resorption started right beside these damages. Furthermore, quantification of resorption on three different mineralized substrates, cortical bone, bleached bone (bone after partial removal of the organic matrix), and dentin, revealed lowest resorption on bone, significantly higher resorption on bleached bone, and highest resorption on dentin. The difference between native and bleached bone may be interpreted as an inhibitory impact of the organic matrix. However, the collagen-based matrix could not be the responsible part as resorption was highest on dentin, which contains collagen. It seems that osteocytic proteins, stored in bone but not present in dentin, affect osteoclastic action. This demonstrates that osteoclasts per se do not possess a toposensitivity to remove microcracks but may be influenced by components of the organic bone matrix.

Two stages in three-dimensional in vitro growth of tissue generated by osteoblastlike cells.

Bone regeneration is controlled by a variety of biochemical, biomechanical, cellular, and hormonal mechanisms. In particular, physical properties of the substrate such as stiffness and architecture highly influence the proliferation and differentiation of cells. The aim of this work is to understand the influence of scaffold stiffness and cell seeding densities on the formation of tissue by osteoblast cells within polyether urethane scaffolds containing pores of different sizes. MC3T3-E1 preosteoblast cells were seeded on the scaffold, and the amount of tissue formed within the pores was analyzed for culture times up to 49 days by phase contrast microscopy. The authors show that the kinetics of three-dimensional tissue growth in these scaffolds follows two stages and can be described by a universal growth law. The first stage is dominated by cell-material interactions with cell adherence and differentiation being strongly dependent on the polymer material. After a delay time of a few weeks, cells begin to grow within their own matrix, the delay being strongly dependent on substrate stiffness and seeding protocols. In this later stage of growth, three-dimensional tissue amplification is controlled rather by the pore geometry than the scaffold material properties. This emphasizes how geometric constraints may guide tissue formation in vitro and shows that optimizing scaffold architectures may improve tissue formation independent of the scaffold material used.

Differential effects of homocysteine and beta aminopropionitrile on preosteoblastic MC3T3-E1 cells.

Compounds, like beta-aminopropionitrile (bAPN) and homocysteine (hcys), are known to inhibit a stable matrix formation. Osteoblast-synthesized collagen matrix regulates the differentiation of precursor cells into mature osteoblasts. They express lysyl oxidase, an enzyme involved in the collagen cross-linking process. Lately, plasma hcys levels have recently been strongly correlated with fracture in humans. We have previously shown that bAPN not only disturbs collagen cross-links but also affects osteoblastic differentiation in a cell culture system. The aim of the present study was to investigate the effects of bAPN and hcys on collagen cross-links and gene expression at the mRNA level by FTIR and quantitative RT-PCR, respectively. We found that bAPN and hcys down-regulated cell multiplication. While bAPN also down-regulated the metabolic activity of MC3T3-E1 cells, hcys down-regulated it by lower concentrations but up-regulated it by higher; both substances up-regulated alkaline phosphatase activity. The substances increased the ratio of pyr/divalent cross-links of collagen, and down-regulated mRNA expression of lysyl hydroxylase (Plod2) and lysyl oxidase (Lox), genes which play an important role in the formation of a stable matrix. Furthermore, we demonstrate that both substances stimulated the expression of Runx2, an indispensable regulator of osteoblastic differentiation. However, analysis of genome wide mRNA expression suggests that hcys and bAPN have differential effects on genes involved in osteoblastic differentiation and phenotype regulation. The results indicate that although both bAPN and hcys affect collagen cross-link post-translational modifications in a similar manner as far as pyr and divalent cross-links are concerned, they have differential effects on the monitored genes expression at the mRNA level, with hcys exerting a broader effect on the genome wide mRNA expression.

Osteocalcin attenuates T3- and increases vitamin D3-induced expression of MMP-13 in mouse osteoblasts.

Osteocalcin (OCN), the most abundant non-collagenous protein of the bone matrix, whose function is not fully understood, was recently suggested to act as endocrine factor regulating energy metabolism. Besides OCN, osteoblasts also express MMP-13, a matrix metallo-proteinase important for bone development and remodeling. Although differentially, both genes are regulated by 1,25-dihydroxy vitamin D3 (1,25D3) and T3, important hormones for bone metabolism. In mouse osteoblasts with a distinct differentiation status, T3 increases the expression of both proteins. By contrast, 1,25D3 stimulates the expression of MMP-13 but inhibits the expression of OCN in these cells. In humans, however, 1,25D3 upregulates both genes while T3 inhibits the OCN expression. Using northern blot hybridization we studied gene expression in the mouse osteoblastic cell line MC3T3-E1. We show that MMP-13 expression was strongly increased by T3 when the stimulation of OCN was low and, inversely, that the MMP-13 increase was low when T3 strongly stimulated the OCN expression. These findings suggest an interrelationship between OCN and MMP-13 expression. In fact, we observed that externally added OCN attenuated the T3 induced MMP-13 expression dose dependently and, furthermore, increased the 1,25D3 stimulated MMP-13 expression. Using a protein kinase A inhibitor we were able to show that this inhibitor mimics the effect of OCN suggesting a PKA dependent pathway to be involved in this regulatory process. We therefore hypothesize that OCN is a modulator of the hormonally regulated MMP-13 expression.

Congenital contractures and distinctive phenotypic features consistent with Stuve-Wiedmann syndrome in a male infant.

INTRODUCTION: Expressionless face associated with multiple contractures has been encountered in an infant. There is a wide range of misconception regarding the categorization of children with multiple contractures among different pediatric disciplines. The fundamental element in categorizing children with multiple contractures is "the etiological understanding". In the absence of concomitant neuromuscular disease, however, the search for other reasons is mandatory. Our present paper signifies the necessity of proper interpretations of unusual clinical and radiographic features. CASE PRESENTATION: We describe a 3-months-old-infant presented with the phenotypic and the radiographic features consistent with the diagnosis of Stüve-Wiedemann syndrome. We report what might be the first clinical report of Stüve-Wiedemann syndrome from a consanguineous family in Austria. CONCLUSION: Congenital limitations of the hips in a newborn infant raise the possibility of " Congenital Hip Dislocation". As congenital hip dislocation is a dysplastic process. Here further knowledge by the pediatrician and the orthopaedic surgeon is needed. Our present patient appears to constitute a distinct pathological entity consistent with Stüve-Wiedemann syndrome (SWS). Superti-Furga et al, and Cormier-Daire et al, also suggest that Stüve-Wiedemann syndrome and Schwartz-Jampel syndrome type 2 are allelic conditions. We wish to stress that, given the rarity of syndromic malformation complex, our impression is that it is more common than it is reported.

Achondroplasia manifesting as enchondromatosis and ossification of the spinal ligaments: a case report.

INTRODUCTION: A girl presented with achondroplasia manifested as mild knee pain associated with stiffness of her back. A skeletal survey showed enchondroma-like metaphyseal dysplasia and ossification of the spinal ligaments. Magnetic resonance imaging of the spine further clarified the pathological composites. CASE PRESENTATION: A 7-year-old girl presented with the classical phenotypic features of achondroplasia. Radiographic documentation showed the co-existence of metaphyseal enchondromatosis and development of spinal bony ankylosis. Magnetic resonance imaging showed extensive ossification of the anterior and posterior spinal ligaments. Additional features revealed by magnetic resonance imaging included calcification of the peripheral vertebral bodies associated with anterior end-plate irregularities. CONCLUSION: Enchondromas are metabolically active and may continue to grow and evolve throughout the patient's lifetime; thus, progressive calcification over a period of years is not unusual. Ossification of the spinal ligaments has a specific site of predilection and often occurs in combination with senile ankylosing vertebral hyperostosis. Nevertheless, ossification of the spinal ligaments has been encountered in children with syndromic malformation complex. It is a multifactorial disease in which complex genetic and environmental factors interact, potentially leading to chronic pressure on the spinal cord and nerve roots with subsequent development of myeloradiculopathy. Our patient presented with a combination of achondroplasia, enchondroma-like metaphyseal dysplasia and calcification of the spinal ligaments. We suggest that the development of heterotopic bone formation along the spinal ligaments had occurred through an abnormal ossified enchondral mechanism. We postulate that ossification of the spinal ligaments and metaphyseal enchondromatous changes are related to each other and represent impaired terminal differentiation of chondrocytes in this particular case. Standard radiographic examination showed spinal bony ankylosis only. The pathological composites of the vertebrae have been clarified using scanning technology. Extensive spinal ligament ossification associated with calcification of the peripheral vertebral bodies and anterior end-plate irregularities were notable. We report what may be a novel spinal and extraspinal malformation complex in a girl with achondroplasia.

The effect of geometry on three-dimensional tissue growth.

Tissue formation is determined by uncountable biochemical signals between cells; in addition, physical parameters have been shown to exhibit significant effects on the level of the single cell. Beyond the cell, however, there is still no quantitative understanding of how geometry affects tissue growth, which is of much significance for bone healing and tissue engineering. In this paper, it is shown that the local growth rate of tissue formed by osteoblasts is strongly influenced by the geometrical features of channels in an artificial three-dimensional matrix. Curvature-driven effects and mechanical forces within the tissue may explain the growth patterns as demonstrated by numerical simulation and confocal laser scanning microscopy. This implies that cells within the tissue surface are able to sense and react to radii of curvature much larger than the size of the cells themselves. This has important implications towards the understanding of bone remodelling and defect healing as well as towards scaffold design in bone tissue engineering.

Live cell adhesion assay with attenuated total reflection infrared spectroscopy.

Living confluent fish fibroblast cells RTG-P1 from rainbow trout adherent on diamond were examined by attenuated total reflection (ATR) infrared (IR) spectroscopy. In particular, IR spectra were recorded dynamically during the adsorption of the cells onto the diamond and during their biochemically induced structural responses to the subsequent addition of trypsin and cytochalasin D. It is shown that changes in the IR spectra result from changes in cell morphology and surface coverage. The results demonstrate the potential and the applicability of ATR IR spectroscopy for live cell adhesion assays.

Biomimetic mineral-organic composite scaffolds with controlled internal architecture.

Bone and cartilage generation by three-dimensional scaffolds is one of the promising techniques in tissue engineering. One approach is to generate histologically and functionally normal tissue by delivering healthy cells in biocompatible scaffolds. These scaffolds provide the necessary support for cells to proliferate and maintain their differentiated function, and their architecture defines the ultimate shape. Rapid prototyping (RP) is a technology by which a complex 3-dimensional (3D) structure can be produced indirectly from computer aided design (CAD). The present study aims at developing a 3D organic-inorganic composite scaffold with defined internal architecture by a RP method utilizing a 3D printer to produce wax molds. The composite scaffolds consisting of chitosan and hydroxyapatite were prepared using soluble wax molds. The behaviour and response of MC3T3-E1 pre-osteoblast cells on the scaffolds was studied. During a culture period of two and three weeks, cell proliferation and in-growth were observed by phase contrast light microscopy, histological staining and electron microscopy. The Giemsa and Gömöri staining of the cells cultured on scaffolds showed that the cells proliferated not only on the surface, but also filled the micro pores of the scaffolds and produced extracellular matrix within the pores. The electron micrographs showed that the cells covering the surface of the struts were flattened and grew from the periphery into the middle region of the pores.

The expression of matrix metalloproteinase-13 and osteocalcin in mouse osteoblasts is related to osteoblastic differentiation and is modulated by 1,25-dihydroxyvitamin D3 and thyroid hormones.

Matrix metalloproteinase-13 (MMP-13), is a key protein of bone matrix degradation, and is highly expressed by osteoblasts. We used the osteoblast-like MC3T3-E1 cell line and compared the stimulatory effects of the bone resorptive agents 1,25-dihydroxyvitamin D3 (1,25-(OH)(2)D(3)) 3,3',5-triido-L-thyronine (T3) on the expression of MMP-13 mRNA. We showed that the stimulatory effects were time and dose dependent, and were also transduced to the protein level, with 1,25-(OH)(2)D(3)being more potent.MMP-13 expression in different mouse cells and its localization within developing bone from the onset of osteogenesis were also investigated. 1,25-(OH)(2)D(3)- and T3-regulated osteocalcin (Osc) expression in mouse osteoblasts was compared to hormonal effects on MMP-13 expression and activity. Here we show divergent and common roles of 1,25-(OH)(2)D(3)and T3 action on the expression of these marker proteins, depending on the stage of cell differentiation. In addition, we propose a role for MMP-13 in the bone collar of developing long bones. The results could help to more precisely characterize hormonal regulation in the developmental sequence of osteoblasts.

A novel cDNA transcript inversely regulated to osteocalcin in differentiating osteoblast-like cells.

In vitro MC3T3-E1 osteoblastic cells differentiate from a preosteoblastic to a mature osteoblastic phenotype constitutively expressing osteocalcin. For this process cell contacts seem to be an essential prerequisite. Using differential display of mRNA from subconfluent and confluent MC3T3-E1 cultures, we isolated and cloned a novel cDNA transcript, named Mc2, exhibiting an open reading frame of 162 bp and without extensive homologies to sequences in the EMBL database. The presence of the Mc2 mRNA was verified in primary mouse osteoblasts. Mc2 mRNA was upregulated during the transition of MC3T3-E1 cell cultures from subconfluence to confluence. In long-term cultures, Mc2 mRNA expression reached a maximum between days 8 and 12, and decreased again on day 21, when osteocalcin expression significantly increased. Treatment of these cells with by 3,3',5-triiodo-l-thyronine resulted in an inhibition of the culture time-dependent upregulation of Mc2 mRNA, whereby osteocalcin mRNA was highly expressed. This inverse regulation of Mc2 and osteocalcin mRNAs was also found in ROS 17/2.8 cells and in mouse bone marrow stromal cells. Transfection experiments showed that uncontrolled expression of a Mc2-GFP vector led to increased cell death in MC3T3-E1 cells. The transient upregulation of Mc2 mRNA in osteoblast-like cells and its interesting inverse regulation to osteocalcin suggest an important role in osteoblastic differentiation.