Osteotoxicity of 3-methylcholanthrene in fish.

Many chemicals produced by human activities end up in the aquatic ecosystem causing adverse developmental and reproductive effects in aquatic organisms. There is evidence that some anthropogenic chemicals disturb bone formation and skeletal development but the lack of suitable in vitro and in vivo systems for testing has hindered the identification of underlying mechanisms of osteotoxicity. Several fish systems - an in vitro cell system to study extracellular matrix mineralization and in vivo systems to evaluate bone formation and skeletogenesis - were combined to collect data on the osteotoxic activity of 3-methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon. Anti-mineralogenic effects, increased incidence of skeletal deformities and reduced bone formation and regeneration were observed in zebrafish upon exposure to 3-MC. Pathway reporter array revealed the role of the aryl hydrocarbon receptor 2 (Ahr2) in the mechanisms underlying 3-MC osteotoxicity in mineralogenic cell lines. Analysis of gene expression in zebrafish larvae confirmed the role of Ahr2 in the signaling of 3-MC toxicity. It also indicated a possible complementary action of the pregnane X receptor (Pxr) in the regulation of genes involved in bone cell activity and differentiation but also in xenobiotic metabolism. Data reported here demonstrated the osteotoxicity of 3-MC but also confirmed the suitability of fish systems to gain insights into the toxic mechanisms of compounds affecting skeletal and bone formation.

MiR-29a is an enhancer of mineral deposition in bone-derived systems.

MicroRNAs (miRNAs) provide a mechanism for fine-tuning of intricate cellular processes through post-transcriptional regulation. Emerging evidences indicate that miRNAs play key roles in regulation of osteogenesis. The miR-29 family was previously implicated in mammalian osteoblast differentiation by targeting extracellular matrix molecules and modulating Wnt signaling. Nevertheless, the function of miR-29 in bone formation and homeostasis is not completely understood. Here, we provide novel insights into the biological effect of miR-29a overexpression in a mineralogenic cell system (ABSa15). MiR-29a gain-of-function resulted in significant increase of extracellular matrix mineralization, probably due to accelerated differentiation. We also demonstrated for the first time that miR-29a induced ß-catenin protein levels, implying a stimulation of canonical Wnt signaling. Our data also suggests that SPARC is a conserved target of miR-29a, and may contribute to the phenotype observed in ABSa15 cells. Finally, we provide evidences for miR-29a conservation throughout evolution based on sequence homology, synteny analysis and expression patterns. Concluding, miR-29a is a key player in osteogenic differentiation, leading to increased mineralization in vitro, and this function seems to be conserved throughout vertebrate evolution by interaction with canonical Wnt signaling and conservation of targets.

AnnexinA6: a potential therapeutic target gene for extracellular matrix mineralization.

The mineralization of the extracellular matrix (ECM) is an essential and crucial process for physiological bone formation and pathological calcification. The abnormal function of ECM mineralization contributes to the worldwide risk of developing mineralization-related diseases; for instance, vascular calcification is attributed to the hyperfunction of ECM mineralization, while osteoporosis is due to hypofunction. AnnexinA6 (AnxA6), a Ca2+-dependent phospholipid-binding protein, has been extensively reported as an essential target in mineralization-related diseases such as osteoporosis, osteoarthritis, atherosclerosis, osteosarcoma, and calcific aortic valve disease. To date, AnxA6, as the largest member of the Annexin family, has attracted much attention due to its significant contribution to matrix vesicles (MVs) production and release, MVs-ECM interaction, cytoplasmic Ca2+ influx, and maturation of hydroxyapatite, making it an essential target in ECM mineralization. In this review, we outlined the recent advancements in the role of AnxA6 in mineralization-related diseases and the potential mechanisms of AnxA6 under normal and mineralization-related pathological conditions. AnxA6 could promote ECM mineralization for bone regeneration in the manner described previously. Therefore, AnxA6 may be a potential osteogenic target for ECM mineralization.

Design of a custom-made device for real-time optical measurement of differential mineral concentrations in three-dimensional scaffolds for bone tissue engineering.

Monitoring bone tissue engineered (TEed) constructs during their maturation is important to ensure the quality of applied protocols. Several destructive, mainly histochemical, methods are conventionally used to this aim, requiring the sacrifice of the investigated samples. This implies (i) to plan several scaffold replicates, (ii) expensive and time consuming procedures and (iii) to infer the maturity level of a given tissue construct from a cognate replica. To solve these issues, non-destructive techniques such as light spectroscopy-based methods have been reported to be useful. Here, a miniaturized and inexpensive custom-made spectrometer device is proposed to enable the non-destructive analysis of hydrogel scaffolds. Testing involved samples with a differential amount of calcium salt. When compared to a reference standard device, this custom-made spectrometer demonstrates the ability to perform measurements without requiring elaborate sample preparation and/or a complex instrumentation. This preliminary study shows the feasibility of light spectroscopy-based methods as useful for the non-destructive analysis of TEed constructs. Based on these results, this custom-made spectrometer device appears as a useful option to perform real-time/in-line analysis. Finally, this device can be considered as a component that can be easily integrated on board of recently prototyped bioreactor systems, for the monitoring of TEed constructs during their conditioning.

Changes in scaffold porosity during bone tissue engineering in perfusion bioreactors considerably affect cellular mechanical stimulation for mineralization.

Bone tissue engineering (BTE) experiments in vitro have shown that fluid-induced wall shear stress (WSS) can stimulate cells to produce mineralized extracellular matrix (ECM). The application of WSS on seeded cells can be achieved through bioreactors that perfuse medium through porous scaffolds. In BTE experiments in vitro, commonly a constant flow rate is used. Previous studies have found that tissue growth within the scaffold will result in an increase of the WSS over time. To keep the WSS in a reported optimal range of 10-30 mPa, the applied external flow rate can be decreased over time. To investigate what reduction of the external flow rate during culturing is needed to keep the WSS in the optimal range, we here conducted a computational study, which simulated the formation of ECM, and in which we investigated the effect of constant fluid flow and different fluid flow reduction scenarios on the WSS. It was found that for both constant and reduced fluid flow scenarios, the WSS did not exceed a critical value, which was set to 60 mPa. However, the constant flow velocity resulted in a reduction of the cell/ECM surface being exposed to a WSS in the optimal range from 50% at the start of culture to 18.6% at day 21. Reducing the fluid flow over time could avoid much of this effect, leaving the WSS in the optimal range for 40.9% of the surface at 21 days. Therefore, for achieving more mineralized tissue, the conventional manner of loading the perfusion bioreactors (i.e. constant flow rate/velocity) should be changed to a decreasing flow over time in BTE experiments. This study provides an in silico tool for finding the best fluid flow reduction strategy.

3D Superparamagnetic Scaffolds for Bone Mineralization under Static Magnetic Field Stimulation.

We reported on three-dimensional (3D) superparamagnetic scaffolds that enhanced the mineralization of magnetic nanoparticle-free osteoblast cells. The scaffolds were fabricated with submicronic resolution by laser direct writing via two photons polymerization of Ormocore/magnetic nanoparticles (MNPs) composites and possessed complex and reproducible architectures. MNPs with a diameter of 4.9 ± 1.5 nm and saturation magnetization of 30 emu/g were added to Ormocore, in concentrations of 0, 2 and 4 mg/mL. The homogenous distribution and the concentration of the MNPs from the unpolymerized Ormocore/MNPs composite were preserved after the photopolymerization process. The MNPs in the scaffolds retained their superparamagnetic behavior. The specific magnetizations of the scaffolds with 2 and 4 mg/mL MNPs concentrations were of 14 emu/g and 17 emu/g, respectively. The MNPs reduced the shrinkage of the structures from 80.2 ± 5.3% for scaffolds without MNPs to 20.7 ± 4.7% for scaffolds with 4 mg/mL MNPs. Osteoblast cells seeded on scaffolds exposed to static magnetic field of 1.3 T deformed the regular architecture of the scaffolds and evoked faster mineralization in comparison to unstimulated samples. Scaffolds deformation and extracellular matrix mineralization under static magnetic field (SMF) exposure increased with increasing MNPs concentration. The results are discussed in the frame of gradient magnetic fields of ~3 × 10-4 T/m generated by MNPs over the cells bodies.

Identification of a fish short-chain dehydrogenase/reductase associated with bone metabolism.

Although human and mouse genetics have largely contributed to the better understanding of the mechanisms underlying skeletogenesis, much more remains to be uncovered. In this regard alternative and complementary systems have been sought and cell systems capable of in vitro calcification have been developed to study the mechanisms underlying bone formation. In gilthead seabream (Sparus aurata), a gene coding for an unknown protein that is strongly up-regulated during extracellular matrix (ECM) mineralization of a pre-osteoblast cell line was recently identified as a potentially important player in bone formation. In silico analysis of the deduced protein revealed the presence of domains typical of short-chain dehydrogenase/reductases (SDR). Closely related to carbonyl reductase 1, seabream protein belongs to a novel subfamily of SDR proteins with no orthologs in mammals. Analysis of gene expression by qPCR confirmed the strong up-regulation of sdr-like expression during in vitro mineralization but also revealed high expression levels in calcified tissues. A possible role for Sdr-like in osteoblast and bone metabolism was further evidenced through (i) the localization by in situ hybridization of sdr-like transcript in pre-osteoblasts of the operculum and (ii) the regulation of sdr-like gene transcription by Runx2 and retinoic acid receptor, two regulators of osteoblast differentiation and mineralization. Expression data also indicated a role for Sdr-like in gastrointestinal tract homeostasis and during gilthead seabream development at gastrulation and metamorphosis. This study reports a new subfamily of short-chain dehydrogenases/reductases in vertebrates and, for the first time, provides evidence of a role for SDRs in bone metabolism, osteoblast differentiation and/or tissue mineralization.

Development of a synthetic tissue engineered three-dimensional printed bioceramic-based bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro.

Over the last decade there have been increasing efforts to develop three-dimensional (3D) scaffolds for bone tissue engineering from bioactive ceramics with 3D printing emerging as a promising technology. The overall objective of the present study was to generate a tissue engineered synthetic bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro, thereby mimicking the advantageous properties of autogenous bone grafts and facilitating usage for reconstructing segmental discontinuity defects in vivo. To this end, 3D scaffolds were developed from a silica-containing calcium alkali orthophosphate, using, first, a replica technique - the Schwartzwalder-Somers method - and, second, 3D printing, (i.e. rapid prototyping). The mechanical and physical scaffold properties and their potential to facilitate homogenous colonization by osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture were examined. Osteoblastic cells were dynamically cultured for 7 days on both scaffold types with two different concentrations of 1.5 and 3 × 109 cells/l. The amount of cells and bone matrix formed and osteogenic marker expression were evaluated using hard tissue histology, immunohistochemical and histomorphometric analysis. 3D-printed scaffolds (RPS) exhibited more micropores, greater compressive strength and silica release. RPS seeded with 3 × 109 cells/l displayed greatest cell and extracellular matrix formation, mineralization and osteocalcin expression. In conclusion, RPS displayed superior mechanical and biological properties and facilitated generating a tissue engineered synthetic bone graft in vitro, which mimics the advantageous properties of autogenous bone grafts, by containing homogenously distributed terminally differentiated osteoblasts and mineralizing bone matrix and therefore is suitable for subsequent in vivo implantation for regenerating segmental discontinuity bone defects. Copyright © 2016 John Wiley & Sons, Ltd.

Concise Review: In Vitro Formation of Bone-Like Nodules Sheds Light on the Application of Stem Cells for Bone Regeneration.

: Harnessing the differentiation of stem cells into bone-forming cells represents an intriguing avenue for the creation of functional skeletal tissues. Therefore, a profound understanding of bone development and morphogenesis sheds light on the regenerative application of stem cells in orthopedics and dentistry. In this concise review, we summarize the studies deciphering the mechanisms that govern osteoblast differentiation in the context of in vitro formation of bone-like nodules, including morphologic and molecular events as well as cellular contributions to mineral nucleation, occurring during osteogenic differentiation of stem cells. This article also highlights the limitations of current translational applications of stem cells and opportunities to use the bone-like nodule model for bone regenerative therapies. SIGNIFICANCE: Harnessing the differentiation of stem cells into bone-forming cells represents an intriguing avenue for the creation of functional skeletal tissues. Therefore, a profound understanding of bone development and morphogenesis sheds light on the regenerative application of stem cells in orthopedics and dentistry. In this concise review, studies deciphering the mechanisms that govern osteoblast commitment and differentiation are summarized. This article highlights the limitations of current translational applications of stem cells and the opportunities to use the bone-like nodule model for bone regenerative therapies.