Abnormal craniofacial and spinal bone development with col2a1a depletion in a zebrafish model of CHARGE syndrome

CHARGE syndrome patients commonly display craniofacial abnormalities. Furthermore, most patients show features of idiopathic scoliosis, reduced bone mineral density and in a few cases osteopenia. While several clinical cases and studies have documented the skeletal deformities in CHARGE syndrome bearing CHD7 mutations, the underlying mechanisms of the disorder remain elusive. Here, we detect and quantitatively analyze skeletal abnormalities in larval and adult chd7-/- zebrafish.We show that young chd7-/- larvae present with abnormal craniofacial development, especially related to cartilage. We also observe scoliosis-like spinal deformations at 9 dpf. Gene expression analysis confirmed the reduction of osteoblast markers and Pparγ targets. MicroCT analyses identified abnormal craniofacial structures, Weberian apparatus and vertebral body morphology in chd7-/- mutants, with highly mineralized inclusions, along with significant variances in bone mineral density and bone volume. Notably, we detect a specific depletion of Col2a1a in the cartilage of craniofacial regions and vertebrae, in line with a significantly reduced number of chondrocytes.Our study is the first to elucidate the mechanisms underlying morphological changes in craniofacial structure and vertebrae of adult chd7-/- zebrafish. The chd7-/- mutant zebrafish will be beneficial in future investigations of the underlying pathways of both craniofacial and spinal deformities commonly seen in CHARGE syndrome.

Effects of the Sintering Process on Nacre-Derived Hydroxyapatite Scaffolds for Bone Engineering

A hydroxyapatite scaffold is a suitable biomaterial for bone tissue engineering due to its chemical component which mimics native bone. Electronic states which present on the surface of hydroxyapatite have the potential to be used to promote the adsorption or transduction of biomolecules such as protein or DNA. This study aimed to compare the morphology and bioactivity of sinter and nonsinter marine-based hydroxyapatite scaffolds. Field emission scanning electron microscopy (FESEM) and micro-computed tomography (microCT) were used to characterize the morphology of both scaffolds. Scaffolds were co-cultured with 5 × 104/cm2 of MC3T3-E1 preosteoblast cells for 7, 14, and 21 days. FESEM was used to observe the cell morphology, and MTT and alkaline phosphatase (ALP) assays were conducted to determine the cell viability and differentiation capacity of cells on both scaffolds. Real-time polymerase chain reaction (rtPCR) was used to identify the expression of osteoblast markers. The sinter scaffold had a porous microstructure with the presence of interconnected pores as compared with the nonsinter scaffold. This sinter scaffold also significantly supported viability and differentiation of the MC3T3-E1 preosteoblast cells (p < 0.05). The marked expression of Col1α1 and osteocalcin (OCN) osteoblast markers were also observed after 14 days of incubation (p < 0.05). The sinter scaffold supported attachment, viability, and differentiation of preosteoblast cells. Hence, sinter hydroxyapatite scaffold from nacreous layer is a promising biomaterial for bone tissue engineering.

Sonic Hedgehog Regulates Bone Fracture Healing

Bone fracture healing involves the combination of intramembranous and endochondral ossification. It is known that Indian hedgehog (Ihh) promotes chondrogenesis during fracture healing. Meanwhile, Sonic hedgehog (Shh), which is involved in ontogeny, has been reported to be involved in fracture healing, but the details had not been clarified. In this study, we demonstrated that Shh participated in fracture healing. Six-week-old Sprague–Dawley rats and Gli-CreERT2; tdTomato mice were used in this study. The right rib bones of experimental animals were fractured. The localization of Shh and Gli1 during fracture healing was examined. The localization of Gli1 progeny cells and osterix (Osx)-positive cells was similar during fracture healing. Runt-related transcription factor 2 (Runx2) and Osx, both of which are osteoblast markers, were observed on the surface of the new bone matrix and chondrocytes on day seven after fracture. Shh and Gli1 were co-localized with Runx2 and Osx. These findings suggest that Shh is involved in intramembranous and endochondral ossification during fracture healing.

LncRNA ODIR1 inhibits osteogenic differentiation of hUC-MSCs through the FBXO25/H2BK120ub/H3K4me3/OSX axis

Long noncoding RNAs (lncRNAs) have been demonstrated to be important regulators during the osteogenic differentiation of mesenchymal stem cells (MSCs). We analyzed the lncRNA expression profile during osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and identified a significantly downregulated lncRNA RP11-527N22.2, named osteogenic differentiation inhibitory lncRNA 1, ODIR1. In hUC-MSCs, ODIR1 knockdown significantly promoted osteogenic differentiation, whereas overexpression inhibited osteogenic differentiation in vitro and in vivo. Mechanistically, ODIR1 interacts with F-box protein 25 (FBXO25) and facilitates the proteasome-dependent degradation of FBXO25 by recruiting Cullin 3 (CUL3). FBXO25 increases the mono-ubiquitination of H2BK120 (H2BK120ub) which subsequently promotes the trimethylation of H3K4 (H3K4me3). Both H2BK120ub and H3K4me3 form a loose chromatin structure, inducing the transcription of the key transcription factor osterix (OSX) and increasing the expression of the downstream osteoblast markers, osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP). In summary, ODIR1 acts as a key negative regulator during the osteogenic differentiation of hUC-MSCs through the FBXO25/H2BK120ub/H3K4me3/OSX axis, which may provide a novel understanding of lncRNAs that regulate the osteogenesis of MSCs and a potential therapeutic strategy for the regeneration of bone defects.

Hyperphosphatemia is required for initiation but not propagation of kidney failure-induced calcific aortic valve disease

High serum levels of phosphate are associated with uremia-induced calcific aortic valve disease (CAVD). However, it is not clear whether hyperphosphatemia is required in all phases of the process. Our aim was to determine the effects of phosphate and phosphate depletion at different phases of valve disease. The experimental design consisted of administering a uremia-inducing diet, with or without phosphate enrichment, to rats for 7 wk. Forty-two rats were fed with a phosphate-enriched uremic regimen that caused renal insufficiency and hyperphosphatemia. Another 42 rats were fed with a phosphate-depleted uremic regimen, which induces similar severity of renal insufficiency, but without its related mineral disorder. Aortic valves were evaluated at several points during the time of diet administration. In the second part, additional 54 rats were fed a phosphate-enriched diet for various time periods and were then switched to a phosphate-depleted diet to complete 7 wk of uremic diet. Osteoblast-like phenotype, inflammation, and eventually valve calcification were observed only in rats that were fed with a phosphate-enriched regimen. Significant valve calcification was observed only in rats that were fed a phosphate-enriched diet for at least 4 wk. Valve calcification was observed only when the switch to a phosphate-depleted regimen occurred after osteoblast markers and activation of Akt and ERK intracellular signaling pathways had already been found in the valve. Phosphate is essential for the initiation of the calcification process. However, when osteoblast markers are already expressed in valve tissue, phosphate depletion will not halt the disease. NEW & NOTEWORTHY High serum levels of phosphate are associated with uremia-induced calcific aortic valve disease. However, it is not clear whether hyperphosphatemia is required in all phases of the process. Our aim was to determine the effects of phosphate and phosphate depletion at different phases of valve disease. Our findings indicated that phosphate is essential for the initiation of the process that includes macrophage accumulation and osteoblast phenotype. Furthermore, hyperphosphatemia is dispensable beyond a certain phase of the process, a point of “no return” after which phosphate depletion does not prevent calcification. This point is relatively early in the course of calcification, when no calcification is apparent, but the inflammation, osteoblast markers, and activation of ERK and Akt pathways have already been identified. Our findings emphasize the complexity of the calcification process and suggest that different mediators might be required during different phases and that the role of phosphate precedes the actual calcification.

MiR-128 inhibits the osteogenic differentiation in osteoporosis by down-regulating SIRT6 expression

Background: MicroRNAs (miRNAs) are involved in the regulation of osteogenic differentiation and chondrification in vivo. The purpose of the present study was to explore the potential mechanism of miR-128 in osteoporosis (OP). Methods: Quantitative real-time PCR (qRT-PCR) was used to determine the expression of miR-128 in femoral neck trabecular bones of OP patients (n=40) and non-OP patients (n=40). C2C12 cells were transfected with miR-128 mimic or inhibitor to determine the effect of miR-128 on osteoblastic differentiation of C2C12 cells. Bioinformatics and luciferase reporter genes were used to determine the molecular mechanism of miR-128 in osteoblastic differentiation of C2C12 cells. Results: The qRT-PCR results showed that the expression level of miR-128 in bone samples of OP patients was significantly higher than that of non-OP patients, while miR-128 was significantly down-regulated during the osteogenic differentiation of C2C12 cells. In addition, the results showed that overexpression of miR-128 significantly inhibited the mRNA and protein expression levels of osteocalcin (OC), alkaline phosphatase (ALP) and collagen I type-α1 (COL1A1) in C2C12 cells, while miR-128 inhibitor could reverse this effect. Bioinformatics analysis and dual-luciferase reporter assay found that silencing information regulatory protein 6 (SIRT6) was a direct target of miR-128. The qRT-PCR and Western Blot results found that miR-128 significantly down-regulated the mRNA and protein expressions of SIRT6. Furthermore, silencing SIRT6 significantly inhibited the promoting effect of the miR-128 inhibitor on the expression of osteoblast markers. Conclusion: The above results confirmed that miR-128 inhibited osteoblast differentiation in OP by down-regulating SIRT6 expression, thus accelerating the development of OP.

NURR1 Downregulation Favors Osteoblastic Differentiation of MSCs

Mesenchymal stem cells (MSCs) have been identified in human dental tissues. Dental pulp stem cells (DPSCs) were classified within MSC family, are multipotent, can be isolated from adult teeth, and have been shown to differentiate, under particular conditions, into various cell types including osteoblasts. In this work, we investigated how the differentiation process of DPSCs toward osteoblasts is controlled. Recent literature data attributed to the nuclear receptor related 1 (NURR1), a still unclarified role in osteoblast differentiation, while NURR1 is primarily involved in dopaminergic neuron differentiation and activity. Thus, in order to verify if NURR1 had a role in DPSC osteoblastic differentiation, we silenced it during all the processes and compared the expression of the main osteoblastic markers with control cultures. Our results showed that the inhibition of NURR1 significantly increased the expression of osteoblast markers collagen I and alkaline phosphatase. Further, in long time cultures, the mineral matrix deposition was strongly enhanced in NURR1-silenced cultures. These results suggest that NURR1 plays a key role in switching DPSC differentiation toward osteoblasts rather than neuronal or even other cell lines. In conclusion, DPSCs represent a source of osteoblast-like cells and downregulation of NURR1 strongly prompted their differentiation toward the osteoblastogenesis process.

Effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 osteoblast-like cells

Background:Evidence shows that oxidative stress induced by nicotine plays an important role in bone loss. Vitamin E with its antioxidative properties may be able to reverse the effects of nicotine on bone. This study aimed to assess the effects of nicotine in the presence and absence of vitamin E on morphology, viability and osteogenic gene expression in MG-63 (osteosarcoma) human osteoblast-like cells.Methods:We treated the cells with 5 mM nicotine. The viability and morphology of cells were evaluated respectively using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) and crystal violet assays. The effect of nicotine on osteogenic gene expression in MG-63 cells was assessed by real-time reverse-transcription polymerase chain reaction of osteoblast markers, namely, alkaline phosphatase, osteocalcin and bone sialoprotein.Results:The results revealed that survival and proliferation of MG-63 cells were suppressed following exposure to nicotine, and cytoplasm vacuolization occurred in the cells. Nicotine significantly down-regulated the expression of osteogenic marker genes. Such adverse effects on morphology, viability and osteogenic gene expression of MG-63 cells were reversed by vitamin E therapy.Conclusions:In conclusion, vitamin E supplementation may play a role in proliferation and differentiation of osteoblasts, and vitamin E can be considered as an anabolic agent to treat nicotine-induced bone loss.

In Vitro Osteogenic Properties of Two Dental Implant Surfaces

Current dental implant research aims at understanding the biological basis for successful implant therapy. The aim of the study was to perform a full characterization of the effect of two commercial titanium (Ti) surfaces, OsseoSpeed and TiOblast, on the behaviour of mouse preosteoblast MC3T3-E1 cells. The effect of these Ti surfaces was compared with tissue culture plastic (TCP). In vitro experiments were performed to evaluate cytotoxicity, cell morphology and proliferation, alkaline phosphatase activity, gene expression, and release of a wide array of osteoblast markers. No differences were observed on cell viability and cell proliferation. However, changes were observed in cell shape after 2 days, with a more branched morphology on OsseoSpeed compared to TiOblast. Moreover, OsseoSpeed surface increased BMP-2 secretion after 2 days, and this was followed by increased IGF-I, BSP, and osterix gene expression and mineralization compared to TiOblast after 14 days. As compared to the gold standard TCP, both Ti surfaces induced higher osteocalcin and OPG release than TCP and differential temporal gene expression of osteogenic markers. The results demonstrate that the gain of using OsseoSpeed surface is an improved osteoblast differentiation and mineralization, without additional effects on cell viability or proliferation.