scholarly journals Src and ROCK Kinases Differentially Regulate Mineralization of Human Osteosarcoma Saos-2 Cells

2019 ◽  
Vol 20 (12) ◽  
pp. 2872 ◽  
Author(s):  
Agnieszka Strzelecka-Kiliszek ◽  
Marta Romiszewska ◽  
Lukasz Bozycki ◽  
Saida Mebarek ◽  
Joanna Bandorowicz-Pikula ◽  
...  
Keyword(s):  
Bone Development ◽  
Bone Mineralization ◽  
Src Kinase ◽  
Coiled Coil ◽  
Matrix Vesicles ◽  
Apatite Formation ◽  
Mineralization Process ◽  
Alizarin Red ◽  
Specific Alkaline Phosphatase ◽  
Human Osteosarcoma

Osteoblasts initiate bone mineralization by releasing matrix vesicles (MVs) into the extracellular matrix (ECM). MVs promote the nucleation process of apatite formation from Ca2+ and Pi in their lumen and bud from the microvilli of osteoblasts during bone development. Tissue non-specific alkaline phosphatase (TNAP) as well as annexins (among them, AnxA6) are abundant proteins in MVs that are engaged in mineralization. In addition, sarcoma proto-oncogene tyrosine-protein (Src) kinase and Rho-associated coiled-coil (ROCK) kinases, which are involved in vesicular transport, may also regulate the mineralization process. Upon stimulation in osteogenic medium containing 50 μg/mL of ascorbic acid (AA) and 7.5 mM of β-glycerophosphate (β-GP), human osteosarcoma Saos-2 cells initiated mineralization, as evidenced by Alizarin Red-S (AR-S) staining, TNAP activity, and the partial translocation of AnxA6 from cytoplasm to the plasma membrane. The addition of 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d] pyrimidine (PP2), which is an inhibitor of Src kinase, significantly inhibited the mineralization process when evaluated by the above criteria. In contrast, the addition of (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide hydrochloride (Y-27632), which is an inhibitor of ROCK kinase, did not affect significantly the mineralization induced in stimulated Saos-2 cells as denoted by AR-S and TNAP activity. In conclusion, mineralization by human osteosarcoma Saos-2 cells seems to be differently regulated by Src and ROCK kinases.

scholarly journals BMP3 Affects Cortical and Trabecular Long Bone Development in Mice

2022 ◽  
Vol 23 (2) ◽  
pp. 785
Author(s):  
Ivan Banovac ◽  
Lovorka Grgurevic ◽  
Viktorija Rumenovic ◽  
Slobodan Vukicevic ◽  
Igor Erjavec
Keyword(s):  
Trabecular Bone ◽  
Bone Tissue ◽  
Bone Development ◽  
Bone Mineralization ◽  
Antagonistic Effect ◽  
Hair Follicles ◽  
Long Bone ◽  
Long Bones ◽  
Epiphyseal Growth Plate ◽  
Alizarin Red

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3−/− mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3−/− mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3−/− mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3−/− mice.

scholarly journals Matrix Vesicles: Role in Bone Mineralization and Potential Use as Therapeutics

2021 ◽  
Vol 14 (4) ◽  
pp. 289
Author(s):  
Sana Ansari ◽  
Bregje W. M. de de Wildt ◽  
Michelle A. M. Vis ◽  
Carolina E. de de Korte ◽  
Keita Ito ◽  
...  
Keyword(s):  
Bone Formation ◽  
Bone Regeneration ◽  
Bone Cells ◽  
Bone Mineralization ◽  
Recipient Cell ◽  
Organic Matrix ◽  
Matrix Vesicles ◽  
Matrix Mineralization

Bone is a complex organ maintained by three main cell types: osteoblasts, osteoclasts, and osteocytes. During bone formation, osteoblasts deposit a mineralized organic matrix. Evidence shows that bone cells release extracellular vesicles (EVs): nano-sized bilayer vesicles, which are involved in intercellular communication by delivering their cargoes through protein–ligand interactions or fusion to the plasma membrane of the recipient cell. Osteoblasts shed a subset of EVs known as matrix vesicles (MtVs), which contain phosphatases, calcium, and inorganic phosphate. These vesicles are believed to have a major role in matrix mineralization, and they feature bone-targeting and osteo-inductive properties. Understanding their contribution in bone formation and mineralization could help to target bone pathologies or bone regeneration using novel approaches such as stimulating MtV secretion in vivo, or the administration of in vitro or biomimetically produced MtVs. This review attempts to discuss the role of MtVs in biomineralization and their potential application for bone pathologies and bone regeneration.

scholarly journals Apoptosis in the Extraosseous Calcification Process

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 131
Author(s):  
Federica Boraldi ◽  
Francesco Demetrio Lofaro ◽  
Daniela Quaglino
Keyword(s):  
Genetic Basis ◽  
Membrane Vesicles ◽  
Matrix Vesicles ◽  
Apoptotic Bodies ◽  
Connective Tissues ◽  
Mineralization Process ◽  
Mitochondrial Alterations ◽  
Age Related ◽  
And Oxidative Stress ◽  
And Cartilage

Extraosseous calcification is a pathologic mineralization process occurring in soft connective tissues (e.g., skin, vessels, tendons, and cartilage). It can take place on a genetic basis or as a consequence of acquired chronic diseases. In this last case, the etiology is multifactorial, including both extra- and intracellular mechanisms, such as the formation of membrane vesicles (e.g., matrix vesicles and apoptotic bodies), mitochondrial alterations, and oxidative stress. This review is an overview of extraosseous calcification mechanisms focusing on the relationships between apoptosis and mineralization in cartilage and vascular tissues, as these are the two tissues mostly affected by a number of age-related diseases having a progressively increased impact in Western Countries.

Tailoring the in vitro characteristics of poly(vinyl alcohol)-nanohydroxyapatite composite scaffolds for bone tissue engineering

2016 ◽  
Vol 36 (8) ◽  
pp. 771-784 ◽  
Author(s):  
Tejinder Kaur ◽  
Arunachalam Thirugnanam ◽  
Krishna Pramanik
Keyword(s):  
Mechanical Strength ◽  
Vinyl Alcohol ◽  
Contact Angle Measurement ◽  
Angle Measurement ◽  
Poly Vinyl Alcohol ◽  
Apatite Formation ◽  
Composite Scaffolds ◽  
Alizarin Red ◽  
Casting Technique

Abstract Poly(vinyl alcohol) reinforced with nanohydroxyapatite (PVA-nHA) composite scaffolds were developed by varying the nHA (1%, 2%, 3%, 4%, and 5%, w/v) composition in the PVA matrix by solvent casting technique. The developed composite scaffolds were characterized using scanning electron microscopy (SEM), X-ray powder diffraction (XRD), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, and contact angle measurement. The stability of the composite scaffolds in physiological environment was evaluated by swelling and degradation studies. Further, these composite scaffolds were tested for in vitro bioactivity, hemolysis, biocompatibility, and mechanical strength. SEM micrographs showed a homogenous distribution of nHA (3%, w/v) in the PVA matrix. XRD and ATR-FTIR analysis confirmed no phase contamination and the existence of the chemical bond between PVA-nHA at approximately 2474 cm-1. PVA-nHA composite scaffolds with 3% (w/v) concentration of nHA showed nominal swelling and degradation behavior with good mechanical strength. The mechanical strength and degradation properties of the scaffold above 3% (w/v) of nHA was found to deteriorate, which is due to the agglomeration of nHA. The in vitro bioactivity and hemolysis studies showed improved apatite formation and hemocompatibility of the developed scaffolds. In vitro cell adhesion, proliferation, alkaline phosphatase activity, and Alizarin red S staining confirmed the biocompatibility of the composite scaffolds.

Preparation and In Vitro Characterization of Polycaprolactone and Demineralized Bone Matrix Scaffolds

2012 ◽  
Vol 1417 ◽  
Author(s):  
Titilayo Moloye ◽  
Christopher Batich
Keyword(s):  
Demineralized Bone Matrix ◽  
Bone Matrix ◽  
Apatite Formation ◽  
Alizarin Red ◽  
Salt Leaching ◽  
Synthetic Materials ◽  
Calcium And Phosphorus ◽  
Demineralized Bone

ABSTRACTCylindrical porous polycaprolactone (PCL) scaffolds containing 25, 35, and 50 wt% demineralized bone matrix (DBM) were fabricated using a salt-leaching method for application in bone engineering. In the present work, PCL-DBM scaffolds were monitored for calcium and phosphorus deposition in both deionized (DI) water and simulated body fluid (SBF) for time periods of 5, 10, 15, and 20 days at 37°C under constant rotation. An in vitro assessment of the bioactivity of synthetic materials using SBF under physiological conditions can be used as a barometer of scaffold behavior in vivo. DBM, an osteoinductive material, was used to gauge if there was a correlation between the concentration of DBM within a scaffold and the apatite formation on its surface. Biochemical assays, alizarin red S staining, and scanning electron microscopy (SEM) with elemental analysis of calcium and phosphorus were consistent in that they confirmed that PCL scaffolds containing 35 wt% DBM in SBF at 14 days post-immersion showed signs of early apatite formation.

scholarly journals Effect of magnesium ions/Type I collagen promote the biological behavior of osteoblasts and its mechanism

2019 ◽  
Author(s):  
Xiaojing Nie ◽  
Xirao Sun ◽  
Chengyue Wang ◽  
Jingxin Yang
Keyword(s):  
Type I Collagen ◽  
Bone Development ◽  
Critical Role ◽  
Biological Behavior ◽  
Type I ◽  
Alizarin Red ◽  
Downstream Signaling ◽  
Proliferation And Differentiation ◽  
Main Component ◽  
Rhodamine B Isothiocyanate

Abstract Type I collagen (Col I) is a main component of extracellular matrix (ECM). Its safety, biocompatibility, hydrophilicity and pyrogen immunogenicity make it suitable for tissues engineering applications. Mg2+ also control a myriad of cellular processes, including the bone development by enhancing the attachment and differentiation of osteoblasts and accelerating mineralization to enhance bone healing. In our studies, Mg2+ bind collagen to promote the proliferation and differentiation of osteoblasts through the expression of integrins and downstream signaling pathways. In order to clarify the biological behavior effect of 10 mM Mg2+/Col I coating, we performed 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP), 4′6-diamidino-2-phenylindole (DAPI), Alizarin red staining and Rhodamine B-isothiocyanate (RITC)-labeled phalloidin experiments and found that 10 mM Mg2+ group, Col I-coating group, 10 mM Mg2+/Col I-coating group, respectively, promoted the proliferation and differentiation of osteoblasts, especially 10 mM Mg2+/Col I-coating group. We detected the mRNA expression of osteogenic-related genes (Runx2, ALP and OCN, OPN and BMP-2) and the protein expression of signaling pathway (integrin α2, integrin β1, FAK and ERK1/2), these results indicated that 10 mM Mg2+/Col I coating play an critical role in up-regulating the MC3T3-E1 cells activity. The potential mechanisms of this specific performance may be through activating via integrin α2β1-FAK-ERK1/2 protein-coupled receptor pathway.

Effects of Metal Ions on Apatite Formation and Bone Mineralization

1991 ◽  
Vol 252 ◽  
Author(s):  
N. C. Blumenthal ◽  
V. Cosma
Keyword(s):  
Bone Mineralization ◽  
Chemical Properties ◽  
Causative Factor ◽  
Apatite Formation ◽  
Mineral Phase ◽  
Considerable Evidence ◽  
Cellular Effects ◽  
Physical Chemical ◽  
Joint Prostheses ◽  
Defective Bone

Certain metals (such as Al) are known to cause bone pathologies in humans and animals. While little is known about the mechanism of action of metals on either the formation of bone or on the physical-chemical properties of the mineral phase, there is considerable evidence that (1) Al and Cd directly affect the formation and properties of HA and thus are a causative factor in metal-related defective bone formation—in addition to any cellular effects which they may induce; (2) that other transition metals (such as Cr, V, Ti, Ni, Co), which are used in joint prostheses, significantly affect HA formation; and (3) gallium, the most effective antihypercalcemic agent, affects apatite formation and solubility.

189 The Effect of Vitamin K on the Differentiation of Adipose-Derived Stem Cells into Osteoblasts

2018 ◽  
Vol 30 (1) ◽  
pp. 234
Author(s):  
T. A. Bane ◽  
J. C. Bertels ◽  
K. M. Polkoff ◽  
M. Rubessa ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Vitamin K ◽  
Bone Mineralization ◽  
Negative Control ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Mineral Density ◽  
Alizarin Red ◽  
Significant Difference

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. This involves using scaffolds of various sizes and shapes that contain stem cells and other osteoinductive molecules. The aim of this project was to evaluate the effects of vitamin K in osteogenic medium and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Vitamin K has been shown to increase bone mineral density by acting as a coenzyme in the γ-carboxylation of osteocalcin, a protein involved in bone mineralization (Weber 2001 Nutrition 11–12, 1024). Our hypothesis was that the presence of vitamin K in the osteogenic medium would positively influence the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided into 7 different treatments: 5 concentrations of vitamin K in the osteogenic medium (10, 50 100, 500, 1000 nM) plus 2 control treatments (osteogenic medium without vitamin K and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 6 times. At the end of the culture period, cells were stained with Alizarin Red S and Von Kossa. In each well, we counted the nodules and then divided them in 2 categories: formed and forming nodules. Data were analysed by analysis of variance using the generalized linear model (GLM) procedure of SPSS (IBM/SPSS, Armonk, NY, USA); the least significant difference (l.s.d.) post hoc test was used to perform statistical multiple comparison, and the α-level was set at 0.05. The results showed (in Table 1) that there was no positive effect on nodule formation when vitamin K was added to the medium; however, when 1000 nM vitamin K was added, nodule formation decreased. More experiments need to be conducted to determine if vitamin K can act synergistically with other vitamins to produce a significant role in ASC differentiation into osteoblasts. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with vitamin K incorporated into their matrix. Table 1.The average number of formed and forming osteoblast nodules compared between treatment groups (SD in parentheses)

scholarly journals Preventive Effects of Evodiamine on Dexamethasone-Induced Osteoporosis in Zebrafish

2019 ◽  
Vol 2019 ◽  
pp. 1-6 ◽  
Author(s):  
Heng Yin ◽  
Jianwei Wang ◽  
Mao Wu ◽  
Yong Ma ◽  
Shanfu Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Mapk Pathway ◽  
Bone Mineralization ◽  
Tartrate Resistant Acid Phosphatase ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Calcium Phosphorus ◽  
Commercial Kits ◽  
Preventive Effects

The aim of this study was to investigate the effect of evodiamine (EV) on dexamethasone-induced osteoporosis in zebrafish. Zebrafish larvae were exposed to different concentrations of dexamethasone to obtain the osteoporosis in zebrafish. Calcium, phosphorus, and alizarin red staining determination were performed to evaluate the effects of EV on bone mineralization. Alkaline phosphatase (ALP), hydroxyproline (HP), and tartrate resistant acid phosphatase (TRAP) were also measured by commercial kits. The expression of MMP3-OPN-MAPK pathway in zebrafish was measured by Western blot. RT-PCR was used to determine mRNA levels of MMP3, OPN, and MAPK. EV could significantly increase the content of calcium and phosphorus. The results of alizarin red staining showed that EV could significantly increase the calcium sink of horse fish, increasing the area of bone formation. EV could increase the content of hydroxyproline in zebrafish. EV also increased ALP and TRAP in zebrafish. Western blot and RT-PCR results showed that EV restored the MMP3-OPN-MAPK pathway in zebrafish. In conclusion, we found that EV can alleviate dexamethasone-induced osteoporosis in zebrafish. The mechanism is related to activating MMP3-OPN-MAPK pathway and then activating bone remodeling.

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