Activity Of Osteoblastic Cells Research Articles

Rutin nanosuspension for potential management of osteoporosis: effect of particle size reduction on oral bioavailability, in vitro and in vivo activity

Clinical significance of Rutin (RUT) is limited by poor dissolution rate and low oral bioavailability. The study was designed to improve the physicochemical and therapeutic potential of the drug by formulating nanosuspension (NS) for osteoporosis. Rutin nanosuspension (RUT-NS) was prepared after screening a range of stabilizers and their combinations at a different concentration by antisolvent precipitation technique. Effect of precipitation on crystallinity (differential scanning calorimetry DSC, X-ray diffraction studies XRD), morphology (scanning electron microscopy, SEM) and chemical interaction (attenuated total reflectance fourier-transform infrared spectroscopy ATR-FTIR) were studied through biophysical techniques. An optimized nanosuspension exhibited a minimum particle size of 122.85 ± 5.02 nm with higher dissolution of RUT-NS (87. 63 ± 2.29%) as compared to pure drug (39.77 ± 2.8 6%). The enhanced intestine absorption and apparent permeability were achieved due to the improved particle size, surface area and dissolution. RUT-NS displayed greater (3 folds) AUC0–24 h than pure drug. In vitro assays with RUT-NS depicted an increased cell proliferation, antioxidant (ROS) activity and osteocalcin production in MG-63 osteoblast cells. The augmented biochemical in vivo biomarkers and bone quality proved the protective effect of RUT-NS. The results supported RUT-NS as a potential therapy for maintaining bone health.

MiR-873-3p targets HDAC4 to stimulate matrix metalloproteinase-13 expression upon parathyroid hormone exposure in rat osteoblasts.

Matrix metalloproteinase-13 (MMP-13) plays a predominant role in endochondral bone formation and bone remodeling. Parathyroid hormone (PTH) stimulates the expression of MMP-13 via Runx2, a bone transcription factor in rat osteoblastic cells (UMR106-01), and histone deacetylase 4 (HDAC4) acts as a corepressor of Runx2. Moreover, microRNAs (miRNAs) play an important role in regulating genes posttranscriptionally. Here, we hypothesized that PTH upregulates the miRNAs targeting HDAC4, which could lead to increased Runx2 activity and MMP-13 expression in rat osteoblastic cells. We identified several miRNAs that putatively target rat HDAC4 using bioinformatics tools. miR-873-3p was significantly upregulated by PTH in rat osteoblasts. miR-873-3p overexpression downregulated HDAC4 protein expression, increased Runx2 binding at the MMP-13 promoter, and increased MMP-13 messenger RNA expression in UMR106-01 cells. A luciferase reporter assay identified the direct targeting of miR-873-3p at the 3'-untranslated region of HDAC4. Thus, miR-873-3p targeted HDAC4 and relieved the corepressor effect of HDAC4 on Runx2 for MMP-13 expression in rat osteoblasts. This study advances our knowledge of posttranscriptional gene regulation occurring in bone and bone-related diseases and clarifies the role of miRNAs as diagnostic biomarkers.

Catalpol protects against 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytotoxicity in osteoblastic MC3T3-E1 cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental contaminant that produces a wide variety of adverse effects in humans. Catalpol, a major bioactive compound enriched in the dried root of Rehmannia glutinosa, is a major iridoid glycoside that alleviates bone loss. However, the detailed mechanisms underlying the effects of catalpol remain unclear. The present study evaluated the effects of catalpol on TCDD-induced cytotoxicity in osteoblastic MC3T3-E1 cells. Catalpol inhibited TCDD-induced reduction in cell viability and increases in apoptosis and autophagic activity in osteoblastic MC3T3-E1 cells. Additionally, pretreatment with catalpol significantly decreased the nitric oxide and nitrite levels compared with a control in TCDD-treated cells and significantly inhibited TCDD-induced increases in the levels of cytochrome P450 1A1 and extracellular signal-regulated kinase. Pretreatment with catalpol also effectively restored the expression of superoxide dismutase and extracellular signal-regulated kinase 1 and significantly enhanced the expression of glutathione peroxidase 4 and osteoblast differentiation markers, including alkaline phosphatase and osterix. Taken together, these findings demonstrate that catalpol has preventive effects against TCDD-induced damage in MC3T3-E1 osteoblastic cells.

Suppressive effects of N-bisphosphonate in osteoblastic cells mitigated by non-N-bisphosphonate but not by sodium-dependent phosphate cotransporter inhibitor.

There are two types of bisphosphonates (BPs), nitrogen-containing (N-BPs) and those free from nitrogen (non-N-BPs). Although N-BPs show greater inhibition of bone resorption than non-N-BPs, their effects are likely accompanied with inflammation, which non-N-BPs mitigate. We examined the competitive effects of zoledronate (ZOL), an N-BP, and etidronate (ETI), a non-N-BP, in osteoblasts. ZOL, but not ETI, markedly reduced alkaline phosphatase activity and cell viability in osteoblastic MC3T3-E1 and Saos2 cells, while that inhibition was relieved by simultaneous administration of ETI, possibly because of competition with ZOL for cellular uptake. However, phosphonoformate, an inhibitor of the phosphonate transporters SLC20A and SLC34A, did not mitigate the reducing effects of ZOL, suggesting that those transporters are not involved in BP uptake in osteoblastic cells. Additionally, ZOL reduced fibroblastic NIH3T3 and C3H10T1/2 cell viability, which was relieved by administration of both ETI and phosphonoformate. Transporter gene expression levels were significantly lower in osteoblasts as compared with fibroblasts, which may account for the distinct effects of phosphonoformate with different cell types. Together, our results suggest existence of a common uptake route of N-BPs and non-N-BPs into osteoblastic cells that is unrelated to the SLC20A and SLC34A families. SIGNIFICANCE OF THE STUDY: N-BP ZOL was shown to suppress differentiation and viability of osteoblasts. ZOL-induced cell viability suppression was also observed in fibroblasts, which was markedly relieved by addition of the non-N-BP ETI. Additionally, mitigation of the effects of ZOL was achieved with phosphonoformate, a sodium-phosphate cotransporter inhibitor, in fibroblastic cells but not osteoblasts. Expression levels of SLC20A and SLC34A family genes were significantly lower in osteoblasts as compared with fibroblasts. These observations suggest that incorporation of N-BPs and non-N-BPs in osteoblasts is mediated via common transporters that appear to be distinct from SLC20A and 34A, which operate in fibroblasts.

Scopoletin and Coumarin Attenuate Aberrant Bone Remodeling in Glucose-exposed Osteoblasts and Osteoclasts (P06-062-19)

ObjectivesDiabetes mellitus is a complex disease with harmful effects on the osteoblast and osteoclast activity. Type 2 diabetic patients have normal or high bone mineral density but associated with an increased risk of fractures. Coumarins (1-benzopyran-2-one) are chemical compounds in the benzopyrone class of organic compounds found in many plants. The purpose of this study was to identify that hyperglycemia resulted in impaired bone remodeling and to examine whether coumarins were capable of preventing diabetic osteoporosis. MethodsThe in vitro study employed osteoblastic MC3T3-E1 cells that were exposed to 33 mM glucose for 6 days in the presence of 20 μM coumarins of scopoletin, aesculetin and coumarin. Alkaline phosphatase (ALP) activity was quantitatively determined in osteoblastic MC3T3-E1 cells by using stable p-nitrophenyl phosphate. In addition, murine macrophage Raw 264.7 cells were differentiated with receptor activator of nuclear factor-κΒ ligand (RANKL) in 33 mM glucose and 20 μM coumarins for 5 days. Tartrate-resistant acid phosphatase (TRAP) activity was measured using an assay kit. ResultsHigh glucose attenuated the ALP activity of osteoblastic MC3T3-E1 cells, which was enhanced by treating scopoletin, aesculetin and coumarin to cells. In addition, 33 mM glucose diminished TRAP activity in RANKL-differentiated Raw 264.7 macrophages, indicating that bone remodeling was impaired in diabetic osteoclasts. In contrast, scopoletin and coumarin elevated osteoclastic differentiation and activation. Interestingly, coumarin was highly effective in balancing bone turnover of osteoblasts and osteoclasts. ConclusionsThese results demonstrate that glucose evoked abnormal bone remodeling leading to diabetic bone disorder. In addition, coumarins such as scopoletin and coumarin ameliorated bone remodeling impaired in diabetic osteoblasts and osteoclasts. These findings suggest the possibility that coumarins might be a potential agent for the treatment of diabetic osteoporosis. Funding SourcesThis work (Grants No. C0501612) was supported by project for Cooperative R&D between Industry, Academy, and Research Institute funded Korea Ministry of SMEs and Startups in 20.

Toward Defining the Pharmacophore for Positive Allosteric Modulation of PTH1 Receptor Signaling by Extracellular Nucleotides.

The parathyroid hormone 1 receptor (PTH1R) is a Class B G-protein-coupled receptor that is a target for osteoporosis therapeutics. Activated PTH1R couples through Gs to the stimulation of adenylyl cyclase. As well, β-arrestin is recruited to PTH1R leading to receptor internalization and MAPK/ERK signaling. Previously, we reported that the agonist potency of PTH1R is increased in the presence of extracellular ATP, which acts as a positive allosteric modulator of PTH signaling. Another nucleotide, cytidine 5'-monophosphate (CMP), also enhances PTH1R signaling, suggesting that ATP and CMP share a moiety responsible for positive allostery, possibly ribose-5-phosphate. Therefore, we examined the effect of extracellular sugar phosphates on PTH1R signaling. cAMP levels and β-arrestin recruitment were monitored using luminescence-based assays. Alone, ribose-5-phosphate had no detectable effect on adenylyl cyclase activity in UMR-106 rat osteoblastic cells, which endogenously express PTH1R. However, ribose-5-phosphate markedly enhanced the activation of adenylyl cyclase induced by PTH. Other sugar phosphates, including glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-bisphosphate, also potentiated PTH-induced adenylyl cyclase activation. As well, some sugar phosphates enhanced PTH-induced β-arrestin recruitment to human PTH1R heterologously expressed in HEK293H cells. Interestingly, the effects of glucose-1-phosphate were greater than those of its isomer glucose-6-phosphate. Our results suggest that phosphorylated monosaccharides such as ribose-5-phosphate contain the pharmacophore for positive allosteric modulation of PTH1R. At least in some cases, the extent of modulation depends on the position of the phosphate group. Knowledge of the pharmacophore may permit future development of positive allosteric modulators to increase the therapeutic efficacy of PTH1R agonists.

Ti6Al4V Surface Modification by Hydroxyapatite Powder Mixed Electrical Discharge Machining for Medical Applications

Titanium surface modification by the Hydroxyapatite (HA) mixed Electrical Discharge Machining (EDM) is an alternative and promising technique to enhance the biocompatibility and to promote the biological performance in bone, which is dependent on surface properties, such as surface roughness, chemistry, and wettability. HA powder is used for the first time with electrical discharge machining to improve osteoblastic cell activity on the developed surfaces for Ti6Al4V. Different HA concentrations in deionized water were tested as an experimental variable during EDM. Abrasive polishing and electrical discharge machined control surfaces without powder addition also analyzed to compare the results. The surface characteristics of analyzed samples were evaluated by Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS), X-Ray Diffractometry (XRD), white light interferometry, and contact angle measurements. The wettability tests suggest that the hydroxyapatite powder mixed EDM’ed surfaces shows highly hydrophilic characteristics compared the other surfaces, abrasive polished and EDM’ed without powder addition in the dielectric. The results from the MTT assay revealed that those surfaces modified using HA powder addition in distilled water dielectric liquid promoted the most significant cell attachment/growth. The results indicate that HA powder mixed EDM offers a promising method for the surface modification of biomaterials such as titanium alloys

Icariin-Functionalized Coating on TiO2 Nanotubes Surface to Improve Osteoblast Activity In Vitro and Osteogenesis Ability In Vivo

Surface modification of titanium is encouraged to facilitate early osseointegration in dental and orthopedic fields. Icariin is the main active constituents of Herba Epimedii, which has good bone-promoting ability. We established an icariin-functionalized coating composed of icariin and poly (lactic-co-glycolic acid) (PLGA) on TiO2 nanotubes surface (NT-ICA-PLGA) to promote osteoblast cell activity and early osseointegration. Surface topography, wettability and drug release pattern of the established NT-ICA-PLGA surface were characterized by scanning electron microscopy (SEM), contact angle test and drug release test. MC3T3-E1 osteoblast cell activity tests were performed using SEM, immunofluorescent staining, cell counting kit-8 and alkaline phosphatase assays. The osteogenic effects of different surfaces were observed using a rat model. Surface characterization proved the successful fabrication of the icariin-functionalized coating on the TiO2 nanotube structure, with increased wettability. The NT-ICA-PLGA substrate showed sustained release of icariin until two weeks. Osteoblast cells grown on the NT-ICA-PLGA substrate displayed improved cell adhesion, proliferation and differentiation ability than the control Ti surface. The in vivo experiment also revealed superior bone forming ability on the NT-ICA-PLGA surface, compared to the pure Ti control. These results imply that the developed NT-ICA-PLGA substrate has a promising future use as functionalized coating for implant surface modification.

Bacteria Death and Osteoblast Metabolic Activity Correlated to Hydrothermally Synthesised TiO₂ Surface Properties.

Orthopaedic surgery comes with an inherent risk of bacterial infection, prolonged antibiotic therapy and revision surgery. Recent research has focused on nanostructured surfaces to improve the bactericidal and osseointegrational properties of implants. However, an understanding of the mechanical properties of bactericidal materials is lacking. In this work, the surface properties of hydrothermal TiO2 nanostructured surfaces are investigated for their effect on bactericidal efficiency and cellular metabolic activity of human osteoblast cells. TiO2 nanostructures, approximately 307 nm in height and 14 GPa stiffness, were the most effective structures against both gram-positive (Staphylococcus aureus) and gram-negative (Pseudomonas aeruginosa) bacteria. Statistical analysis significantly correlated structure height to the death of both bacteria strains. In addition, the surface contact angle and Young’s modulus were correlated to osteoblast metabolic activity. Hydrophilic surfaces with a contact angle between 35 and 50° produced the highest cellular metabolic activity rates after 24 h of incubation. The mechanical tests showed that nanostructures retain their mechanical stability and integrity over a long time-period, reaffirming the surfaces’ applicability for implants. This work provides a thorough examination of the surface, mechanical and wettability properties of multifunctional hydrothermally synthesised nanostructured materials, capable of killing bacteria whilst improving osteoblast metabolic rates, leading to improved osseointegration and antibacterial properties of orthopaedic implants.

Development of genipin-crosslinked and fucoidan-adsorbed nano-hydroxyapatite/hydroxypropyl chitosan composite scaffolds for bone tissue engineering

Hydroxypropyl chitosan (HPCS) has recently attracted increasing attention in biomedical applications because it has enhanced water solubility, excellent biocompatibility, and better antioxidant and antibacterial activities compared with chitosan. However, HPCS doesn't meet the mechanical strength requirement in bone tissue engineering and is not suitable for cell adhesion and growth because of its hydrophilic nature and low crystallinity. In this study, nano-scaled hydroxyapatite (n-HA) and HPCS were synthesized, respectively, and then n-HA/HPCS nanocomposite scaffolds were developed by incorporating n-HA into HPCS matrix accompanied with crosslinking of HPCS by a naturally occurring compound, genipin (GP), which in turn greatly altered the hydrophilicity and mechanical properties. The nanocomposite scaffolds showed an open structure with interconnected pores and a rough morphology with n-HA inserted in the GP-crosslinked HPCS matrix. The porosity, swelling capacity, compressive strength, fluorescence emission and degradation rate can be regulated by varying GP concentrations and n-HA contents. An osteoconductive and osteogenic marine algae polysaccharide, fucoidan, was further adsorbed to the composite scaffolds via electrostatic interactions. Incorporation of n-HA and adsorption of FD into the composite scaffolds increased ALP activity in 7F2 osteoblast cells and promoted their mineralization. The FD-adsorbed n-HA/HPCS composite scaffolds can be a potential biomaterial for BTE applications.

Advanced glycation end-product 2 and Porphyromonas gingivalis lipopolysaccharide increase sclerostin expression in mouse osteocyte-like cells

Sclerostin is a secreted glycoprotein that is mainly expressed in osteocytes, exerts negative effects on bone formation, and is present at elevated levels in diabetes mellitus (DM). Periodontitis is an infectious disease caused by periodontopathic bacteria, a complication of DM, and sometimes associated with severe inflammation and alveolar bone resorption. Advanced glycation end-products (AGEs) are a major pathogen in DM complications and adversely influence periodontitis in DM patients. In the present study, the effects of AGE2 and Porphyromonas gingivalis lipopolysaccharide (P-LPS) on the expression of sclerostin in mouse osteocyte-like cells (MLO-Y4-A2 cells) and its function in osteoblast differentiation were investigated. AGE2 and P-LPS up-regulated the expressions of receptor of AGE (RAGE) and Toll-like receptor 2 (TLR2), respectively, and significantly up-regulated that of sclerostin and interleukin 6 (IL-6) in osteocytes. Sclerostin, RAGE and TLR2 levels were synergistically increased by AGE2 and P-LPS. The siRNAs of RAGE and TLR2 significantly inhibited AGE2- and P-LPS-induced sclerostin expression. AGE2 up-regulated sclerostin expression in osteocyte-like cells via the RAGE, ERK and JNK, and NF-κB signal pathways. On the other hand, P-LPS elevated sclerostin levels via the TLR2, JNK and p38, and NF-κB signal pathways. When osteocytes pre-treated with AGE2 and P-LPS and osteoblastic cells (MC3T3-E1) were co-cultured in the medium with a sclerostin-neutralizing antibody, AGE2- and P-LPS-induced decreases in alkaline phosphatase activity and Runx2 expression in osteoblastic cells were significantly inhibited by the sclerostin-neutralizing antibody. These results suggest that AGE2 and P-LPS influence bone metabolism and inflammation through the regulation of sclerostin expression, and may aggravate periodontitis with DM.

Surface modification of chitosan films with a fibronectin fragment-DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior.

RationaleProtein dynamics are fundamental for biological activity. Direct surface functionalization with these biomolecules often creates regions presenting non‐homogeneous and altered protein functionality. Aptamers have been shown to be an effective method to enhance the biocompatibility of biomaterials by acting as docking points for biomolecules capable of improving cell responses and the colonization/proliferation processes. Here mass spectrometry (MS) was successfully applied as an analytical approach to study the interactions between a fibronectin fragment (FN) and anti‐fibronectin aptamers in solution and on a chitosan film.MethodsA 30 kDa N‐terminal fibronectin fragment, a ssDNA anti‐fibronectin 40 nucleotide long aptamer and chitosan 95/50 (degree of deacetylation 92.6–97.5%; molecular weight 80–220 kDa) were used. First, the dynamics of the FN in aqueous solution, in the presence or absence of anti‐FN‐specific DNA aptamers, were described. Thus, the same study was carried out on 2% (w/w) chitosan‐based films, functionalized with FN alone or through aptamers as selective spacers.ResultsAmide hydrogen/deuterium exchange mass spectrometry (HDX‐MS) analysis identified the SYRIGDTWSKKDNRGNL and YRVGDTYERPKDSMI, YVVGETWEKPYQGWMM and WERTYLGNAL fragments as presumably involved in the interaction of FN with aptamers. MS findings indicated the improved functionality of FN on chitosan when aptamers were used as selective spacer, and this may explain why cells preferentially attached to aptamer‐bound FN rather than on chitosan‐FN films.ConclusionsAptamers did not affect the amount of adsorbed FN, but influenced its conformation enhancing its biological activity toward adhesion and growth of osteoblasts. HDX‐MS data allowed the identification of the FN/aptamer interaction regions. Differences in FN flexibility on the chitosan film in the presence or absence of the aptamer were established providing insights at the molecular level to better understand the protein biological functionality on cell proliferation.

Functionalization of SF/HAP Scaffold with GO-PEI-miRNA inhibitor Complexes to Enhance Bone Regeneration through Activating Transcription Factor 4.

Evidence indicates that microRNAs (miRNAs) play vital roles in regulating osteogenic differentiation and bone formation.Methods: Here, we show that a polyethyleneimine (PEI)-functionalized graphene oxide (GO) complex efficiently loaded with the miR-214 inhibitor is assembled into silk fibroin/hydroxyapatite (SF/HAP) scaffolds that spatially control the release of the miR-214 inhibitor.Results: SF/HAP/GO scaffolds with nanosized GO show high mechanical strength, and their hierarchical microporous structures promote cell adhesion and growth. The SF/HAP/GO-PEI scaffolds loaded with mir-214 inhibitor (SF/HAP/GPM) were tested for their ability to enhance osteogenic differentiation by inhibiting the expression of miR-214 while inversely increasing the expression of activating transcription factor 4 (ATF4) and activating the Akt and ERK1/2 signaling pathways in mouse osteoblastic cells (MC3T3-E1) in vitro. Similarly, the scaffolds activated the osteoblastic activity of endogenous osteoblast cells to repair critical-sized bone defects in rats without the need for loading osteoblast cells.Conclusion: This technology is used to increase osteogenic differentiation and mineralized bone formation in bone defects, which helps to achieve cell-free scaffold-based miRNA-inhibitor therapy for bone tissue engineering.

The facile synthesis of copper oxide quantum dots on chitosan with assistance of phyto-angelica for enhancing the human osteoblast activity to the application of osteoporosis

Osteoblasts are an important key factor for the pathogenesis of several bone-related diseases, notably in osteoporosis. Osteoporosis is a disorder categorized based on the bone mineral density (BMD) and an alteration in the bone micro-architecture had been considered as the major determinant for increasing the fracture risk. The available medicine for curing the osteoporosis shows a minimal or no activity against the genesis or function of osteoblasts. The present study was conducted to determine the influence of phyto Angelica species (Ang.) mediated synthesized copper quantum dots decorated chitosan on human osteoblast cells for application of osteoporosis. The phyto compound of Angelica sp. was extracted by ethanol as solvent and it has been characterized through spectral analyses. An Angelica sp. mediated synthesized copper oxide quantum dots (CuO QDs) and the presence of CuO QDs on chitosan have been analyzed and exhibited by important spectral investigations. The morphological observation of CuO QDs on the chitosan (CS) was visualized by the microscopic analyses. The MTT assay results showed that cell growth of CuO QDs/CS-Ang. by the concentration dependent. The highest cell growth (87%) was noted at 5 μg/mL followed by 80 and 77% at 15 and 25 μg/mL respectively. The functional groups and potential compounds of Angelica sp. with CuO QDs/CS has been improved the osteoblast cell activity as prophylactic potentials against osteoporosis.

Lycopene influences osteoblast functional activity and prevents femur bone loss in female rats submitted to an experimental model of osteoporosis

Antioxidant properties of several nutrients may influence bone metabolism, affording protection against damaging effects caused by oxidative stress. Thus, we hypothesized that lycopene may benefit bone tissue metabolism and functional activity of osteoblastic cells from bone marrow of osteoporotic female rats. Wistar rats were ovariectomized and paired with sham animals. In vitro evaluations were performed after 60days of surgery, when cells were cultured in osteogenic medium and divided in control (C), ovariectomized (OVX) and ovariectomized + 1μmol/L lycopene (OVXL) groups. Besides, in vivo studies were carried out to evaluate femur bone remodeling by histological and histomorphometric analyses after daily intake of 10mg/kg of lycopene for 30 and 60days after ovariectomy. Cell proliferation was significantly higher in OVX and OVXL groups after 10days of culture. Alkaline phosphatase activity (ALP) was higher in OVXL group in later periods of cell culture, whereas its in situ detection was higher for this group in all experimental periods; nevertheless, mineralization did not show significant differences among the groups. There was a significant upregulation of genes Sp7, Runx2 and Bsp after 3days and genes Runx2 and Bglap after 10days from OVXL when compared to OVX. In vivo results demonstrated that daily intake of 10mg/kg of lycopene for 60days decreased bone loss in femur epiphysis in ovariectomized rats by maintaining trabecular bone similar to controls. Data obtained suggest that lycopene might benefit the functional activity of osteoblastic cells from ovariectomized rats, as well as avoid further bone resorption.

Contribution of osteocalcin-mimetic peptide enhances osteogenic activity and extracellular matrix mineralization of human osteoblast-like cells.

A natural peptide motif in the first helix of osteocalcin (OCN) is used to promote nucleation and crystallization of hydroxyapatite (HA) in hard tissue. The capability of osteocalcin mimetic peptides to induce osteogenic activity of osteoblast cells leading to in-vitro mineralization is demonstrated. An osteocalcin-derived peptide consisting of thirteen amino acids is synthesized in both acidic (OSC) and amidic (OSN) forms and added into the human osteoblast-like cells (MG63) culture. The viability, proliferation, alkaline phosphatase activity, HA deposition and osteogenic gene expression by osteoblast cells are evaluated. It is revealed that the addition of 100 μg/ml of peptides enhances the proliferation rate and total protein content of osteoblast cells. Alkaline phosphatase activity is significantly higher in the presence of peptides which in turn stimulated RNA expression of collagen type I and osteopontin in a phosphate-dependent manner. Alizarin red staining and calcium content measurement show that mineral deposition is considerably increased. Ultrastructural characterization of MG63 cultures confirms the crystalline nature and chemical composition of HA mineral formation in the presence of peptides. It is confirmed that the osteocalcin-derived peptide, particularly in amidic form (OSN), is able to act as a bioactive inducer of mineralization process and hence accelerating bone tissue regeneration.

Development of nanocomposite scaffolds based on biomineralization of N,O-carboxymethyl chitosan/fucoidan conjugates for bone tissue engineering

Bone tissue engineering holds great promise and clinical efficacy for the regeneration of bone defects. In this study, an amphoteric N,O-carboxymethyl chitosan (NOCC) and fucoidan (FD) were covalently cross-linked via an amidation reaction to synthesize NOCC/FD composite hydrogels. The hydrogels were lyophilized and then three-dimensional scaffolds with interconnected macropores were obtained. To enhance the mechanical properties and osteogenic activity, the NOCC/FD scaffolds were biomineralized for the growth of hydroxyapatite crystals. A comparative assessment of the structures, morphologies, and physical properties of the original and mineralized scaffolds were performed by SEM, EDS, X-ray diffraction and FT-IR analysis. FD regulated the growth of hydroxyapatite nanocrystallites (n-HAp) and thus the NOCC/FD scaffolds showed better mineralization efficiency than NOCC scaffolds. The compressive strength of the scaffolds was greatly enhanced after mineralization with n-HAp. The n-HAp/NOCC/FD scaffolds enhanced the proliferation, ALP activity, and mineralization of osteoblast cells more strongly than the original and mineralized NOCC scaffolds. Hence, the n-HAp-mineralized NOCC/FD scaffolds may prove to be an excellent and versatile scaffold for bone tissue engineering.

Tolerance to sustained activation of the cAMP/Creb pathway activity in osteoblastic cells is enabled by loss of p53

The loss of p53 function is a central event in the genesis of osteosarcoma (OS). How mutation of p53 enables OS development from osteoblastic lineage cells is poorly understood. We and others have reported a key role for elevated and persistent activation of the cAMP/PKA/Creb1 pathway in maintenance of OS. In view of the osteoblast lineage being the cell of origin of OS, we sought to determine how these pathways interact within the context of the normal osteoblast. Normal osteoblasts (p53 WT) rapidly underwent apoptosis in response to acute elevation of cAMP levels or activity, whereas p53-deficient osteoblasts tolerated this aberrant cAMP/Creb level and activity. Using the p53 activating small-molecule Nutlin-3a and cAMP/Creb1 activator forskolin, we addressed the question of how p53 responds to the activation of cAMP. We observed that p53 acts dominantly to protect cells from excessive cAMP accumulation. We identify a Creb1-Cbp complex that functions together with and interacts with p53. Finally, translating these results we find that a selective small-molecule inhibitor of the Creb1-Cbp interaction demonstrates selective toxicity to OS cells where this pathway is constitutively active. This highlights the cAMP/Creb axis as a potentially actionable therapeutic vulnerability in p53-deficient tumors such as OS. These results define a mechanism through which p53 protects normal osteoblasts from excessive or abnormal cAMP accumulation, which becomes fundamentally compromised in OS.

Hypoxia regulates angeogenic-osteogenic coupling process via up-regulating IL-6 and IL-8 in human osteoblastic cells through hypoxia-inducible factor-1α pathway

Inappropriate angiogenesis and osteogenesis are considered as the crucial factors of osteoporotic fracture. Hypoxia is a primary driving force for regulating the angiogenic-osteogenic coupling process. Our recent results indicated that hypoxia could improve angiogenesis as well as differentiation and activity of osteoblastic cells via up-regulating VEGF through HIF-1α pathway. Here we demonstrated that in human osteoblastic MG-63, U2-OS and Saos-2 cells, besides VEGF, the other two pro-angiogenic factors IL-6 and IL-8 were also up-regulated by hypoxia and CoCl2 (a mimic of hypoxia). Mechanism studies indicated overexpression of HIF-1α (generated from transfection with a plasmid encoding sense HIF-1α) markedly increased the levels of IL-6 and IL-8 in osteoblastic cells. Furthermore, a luciferase reporter assay was performed using the reporter vector containing the IL-6 or IL-8 promoter sequence to illustrate observably increased activity of hypoxia-induced IL-6 and IL-8 promoter caused by overexpression of HIF-1α. Additionally, chromatin immune-precipitation analysis showed hypoxia increased the DNA binding ability of HIF-1α to IL-6 or IL-8 promoter. Analysis in vitro by MTT test and Boyden chamber assay showed exogenous IL-6 and IL-8 (a relatively short period of treatment with recombinant IL-6 or IL-8 equivalent to the autocrine levels) could significantly promote the proliferation of human osteoblastic, endothelial and monocytic cells, as well as the migration of human endothelial cells. Taken together, these results indicate that IL-6 and IL-8 in osteoblastic cells may also contribute to the angiogenic-osteogenic coupling process via HIF-1α pathway. Besides VEGF, IL-6- or IL-8-targeted adjunctive therapy maybe a new strategy to improve the treatment of osteoporosis.

Shaping the craniofacial skeleton of Mexican cavefish (Astyanax mexicanus); Role of Osteoblast and Osteoclast

The vertebrate bony skeleton is remodelled continuously by bone deposition and reabsorption, which is essential for normal development, growth, repair and for the mineral homeostasis. Remodelling of the skeleton usually involves the removal of the bone by osteoclasts and the subsequent formation of new bone by osteoblasts. These remodelling mechanisms can be found in the teleost fish skeleton with special adaptation to the aquatic life. Most of the fish species have teeth in their craniofacial region and replace their dentitions though out the life which lead to continuous bone remodelling activity in the tooth‐bearing bones. The Mexican tetra (Astyanax mexicanus), is a small teleost fish that exists as two morphs; sighted and blind cave form. The degeneration of the eyes, result in the characteristics craniofacial phenotype in cavefish. However, little is known about the cellular mechanisms underlying the remodelling activity of the skeleton of this species. This study was designed to identify the cellular interactions during the development of craniofacial skeleton of Mexican cavefish. Histological and whole mount analysis was performed to compare the development of the skeleton in these two morphs. The cavefish showed delayed eruption of teeth in tooth‐bearing bones but has large jaw bones compared to the surface larval fish. Sites of bone resorption in the bony skeleton will be investigated by RNA in situ hybridization and ultrastructural analysis. Live bone staining techniques will be used to identify the pattern of bone deposition. This study will provide insights into the evolution of osteoblastic and osteoclastic cell activity and will highlight the use of this species as a model in mammalian skeletal disorders.Support or Funding InformationThis research was funded by the University of Manitoba.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.