Nutraceuticals Synergistically Promote Osteogenesis in Cultured 7F2 Osteoblasts and Mitigate Inhibition of Differentiation and Maturation in Simulated Microgravity

Microgravity is known to impact bone health, similar to mechanical unloading on Earth. In the absence of countermeasures, bone formation and mineral deposition are strongly inhibited in Space. There is an unmet need to identify nutritional countermeasures. Curcumin and carnosic acid are phytonutrients with anticancer, anti-inflammatory, and antioxidative effects and may exhibit osteogenic properties. Zinc is a trace element essential for bone formation. We hypothesized that these nutraceuticals could counteract the microgravity-induced inhibition of osteogenic differentiation and function. To test this hypothesis, we cultured 7F2 murine osteoblasts in simulated microgravity (SMG) in a Random Positioning Machine in the presence and absence of curcumin, carnosic acid, and zinc and evaluated cell proliferation, function, and differentiation. SMG enhanced cell proliferation in osteogenic medium. The nutraceuticals partially reversed the inhibitory effects of SMG on alkaline phosphatase (ALP) activity and did not alter the SMG-induced reduction in the expression of osteogenic marker genes in osteogenic medium, while they promoted osteoblast proliferation and ALP activity in the absence of traditional osteogenic media. We further observed a synergistic effect of the intermix of the phytonutrients on ALP activity. Intermixes of phytonutrients may serve as convenient and effective nutritional countermeasures against bone loss in space.

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The Rosmarinus Bioactive Compound Carnosic Acid Is a Novel PPAR Antagonist That Inhibits the Browning of White Adipocytes

Cells ◽

10.3390/cells9112433 ◽

2020 ◽

Vol 9 (11) ◽

pp. 2433

Author(s):

Cécilia Colson ◽

Pierre-Louis Batrow ◽

Nadine Gautier ◽

Nathalie Rochet ◽

Gérard Ailhaud ◽

...

Keyword(s):

Bioactive Compound ◽

Antagonistic Effect ◽

Carnosic Acid ◽

Brown Adipocyte ◽

Marker Genes ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

White Adipocyte ◽

White Adipocytes ◽

And Function

Thermogenic brown and brite adipocytes convert chemical energy from nutrients into heat. Therapeutics that regulate brown adipocyte recruitment and activity represent interesting strategies to control fat mass such as in obesity or cachexia. The peroxisome proliferator-activated receptor (PPAR) family plays key roles in the maintenance of adipose tissue and in the regulation of thermogenic activity. Activation of these receptors induce browning of white adipocyte. The purpose of this work was to characterize the role of carnosic acid (CA), a compound used in traditional medicine, in the control of brown/brite adipocyte formation and function. We used human multipotent adipose-derived stem (hMADS) cells differentiated into white or brite adipocytes. The expression of key marker genes was determined using RT-qPCR and western blotting. We show here that CA inhibits the browning of white adipocytes and favors decreased gene expression of thermogenic markers. CA treatment does not affect β-adrenergic response. Importantly, the effects of CA are fully reversible. We used transactivation assays to show that CA has a PPARα/γ antagonistic action. Our data pinpoint CA as a drug able to control PPAR activity through an antagonistic effect. These observations shed some light on the development of natural PPAR antagonists and their potential effects on thermogenic response.

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In vitro osteogenic activities of sulfated derivative of polysaccharide extracted from Tamarindus indica L.

Biological Chemistry ◽

10.1515/hsz-2021-0200 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Minh Thi Hong Nguyen ◽

Chien Van Tran ◽

Phuong Hong Nguyen ◽

Quang De Tran ◽

Min-Sung Kim ◽

...

Keyword(s):

Bone Formation ◽

Bone Mineralization ◽

Marker Genes ◽

Elemental Analysis Data ◽

Research Approach ◽

Tamarindus Indica ◽

Treatment Of Osteoporosis ◽

Alp Activity ◽

Tamarind Seed

Abstract Osteoporosis, one of the most serious public health concerns caused by an imbalance between bone resorption and bone formation, has a major impact on the population. Therefore, finding the effective osteogenic compounds for the treatment of osteoporosis is a promising research approach. In our study, tamarind (Tamarindus indica L.) seed polysaccharide (TSP) extracted from tamarind seed was subjected to synthesize its sulfate derivatives. The 1H NMR, FT-IR, SEM, monosaccharide compositions and elemental analysis data revealed that tamarind seed polysaccharide sulfate (TSPS) was successfully prepared. As the result, TSPS showed potent effects on inducing osteoblast differentiation via increasing alkaline phosphatase (ALP) activity up to 20% after 10 days and bone mineralization approximately 58% after four weeks at concentration of 20 μg/mL, whereas no statistically increase for both ALP activity and bone mineralization was observed in TSP treatment. Furthermore, TSPS enhanced expression of several marker genes in bone formation. Overall, the obtained data provided novelty on osteogenic compounds originated from TSP of T. indica, as well as scientific fundamentals on drug development and bone tissue engineering for the treatment of osteoporosis and other bone-related diseases.

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miR-452-3p inhibited osteoblast differentiation by targeting Smad4

PeerJ ◽

10.7717/peerj.12228 ◽

2021 ◽

Vol 9 ◽

pp. e12228

Author(s):

Ming Wu ◽

Hongyan Wang ◽

Dece Kong ◽

Jin Shao ◽

Chao Song ◽

...

Keyword(s):

Bone Formation ◽

Osteoblast Differentiation ◽

Cell Model ◽

Pcr Analysis ◽

Marker Genes ◽

Alizarin Red ◽

Alp Activity ◽

Complex Process ◽

Collagen Type 1

Osteoblast differentiation is a complex process that is essential for normal bone formation. A growing number of studies have shown that microRNAs (miRNAs) are key regulators in a variety of physiological and pathological processes, including osteogenesis. In this study, BMP2 was used to induce MC3T3-E1 cells to construct osteoblast differentiation cell model. Then, we investigated the effect of miR-452-3p on osteoblast differentiation and the related molecular mechanism by RT-PCR analysis, Western blot analysis, ALP activity, and Alizarin Red Staining. We found that miR-452-3p was significantly downregulated in osteoblast differentiation. Overexpression miR-452-3p (miR-452-3p mimic) significantly inhibited the expression of osteoblast marker genes RUNX2, osteopontin (OPN), and collagen type 1 a1 chain (Col1A1), and decreased the number of calcium nodules and ALP activity. In contrast, knockdown miR-452-3p (miR-452-3p inhibitor) produced the opposite effect. In terms of mechanism, we found that Smad4 may be the target of miR-452-3p, and knockdown Smad4 (si-Smad4) partially inhibited the osteoblast differentiation enhanced by miR-452-3p. Our results suggested that miR-452-3p plays an important role in osteoblast differentiation by targeting Smad4. Therefore, miR-452-3p is expected to be used in the treatment of bone formation and regeneration.

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Ishige sinicola extract stimulates osteoblast proliferation and differentiation via the bone morphogenetic protein 2/runt-related gene 2 signalling pathway

Zeitschrift für Naturforschung C ◽

10.1515/znc-2018-0044 ◽

2019 ◽

Vol 74 (7-8) ◽

pp. 167-174

Author(s):

Mihyang Kim ◽

Jeong Hyeon Kang ◽

Geun Hye Oh ◽

Mi Hwa Park

Keyword(s):

Cell Proliferation ◽

Bone Formation ◽

Bone Morphogenetic Protein ◽

Bone Diseases ◽

Signalling Pathway ◽

Bone Morphogenetic Protein 2 ◽

Dependent Manner ◽

Alizarin Red ◽

Alp Activity ◽

Morphogenetic Protein

Abstract Osteoporosis is one of the most common bone diseases, occurring due to an imbalance between bone formation and bone resorption. The aim of this study was to investigate the effects of Ishige sinicola, a brown alga, on osteoblast differentiation through the activation of the bone morphogenetic protein 2 (BMP-2)/runt-related transcription factor 2 (Runx2) signalling pathway in MC3T3-E1 cells. A cell proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining, and expression analysis of osteoblastic genes were carried out to assess MC3T3-E1 cell proliferation and osteoblastic differentiation. We found that I. sinicola extract (ISE) increased cell proliferation in a dose-dependent manner. Ishige sinicola extract markedly promoted ALP activity and mineralization. Alizarin red S staining demonstrated that ISE treatment tended to increase extracellular matrix calcium accumulation. Moreover, ISE up-regulated the osteoprotegerin/receptor activator of nuclear factor κB ligand ratio. Ishige sinicola extract also increased the protein expression levels of type 1 collagen, ALP, osteocalcin, osterix, BMP-2, and Runx2. Therefore, ISE showed potential in stimulating osteoblastic bone formation, and it might be useful for the prevention and treatment of osteoporosis.

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Biochanin A Promotes Osteogenic but Inhibits Adipogenic Differentiation: Evidence with Primary Adipose-Derived Stem Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/846039 ◽

2013 ◽

Vol 2013 ◽

pp. 1-12 ◽

Cited By ~ 15

Author(s):

Shu-Jem Su ◽

Yao-Tsung Yeh ◽

Shu-Hui Su ◽

Kee-Lung Chang ◽

Huey-Wen Shyu ◽

...

Keyword(s):

Stem Cells ◽

Bone Formation ◽

Adipogenic Differentiation ◽

Biochanin A ◽

Marker Genes ◽

Dependent Manner ◽

Adipose Derived Stem Cells ◽

Alizarin Red ◽

Alp Activity ◽

Cytoplasmic Lipid Droplet

Biochanin A has promising effects on bone formationin vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1–1 μM) were assessedin vitrousing various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 μM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.

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Effect of Short-Time Povidone-Iodine Application on Osteoblast Proliferation and Differentiation

The Open Dentistry Journal ◽

10.2174/1874210600903010208 ◽

2009 ◽

Vol 3 (1) ◽

pp. 208-212 ◽

Cited By ~ 14

Author(s):

P.R Schmidlin ◽

T Imfeld ◽

P Sahrmann ◽

A Tchouboukov ◽

F.E Weber

Keyword(s):

Cell Proliferation ◽

Povidone Iodine ◽

Bone Morphogenetic Protein 2 ◽

Separate Experiment ◽

Short Exposure ◽

Alizarin Red ◽

Alp Activity ◽

And Function ◽

Short Time

Background and Objective: Povidone-iodine [polyvinylpyrrolidone-iodine complex (PVP-I)] is a broad-spectrum antimicrobial agent, frequently used in dentistry. In this study we investigated the short- and longterm effects on osteoblast number, viability, and function after short exposure to PVP-I with and without additional bone-morphogenetic protein-2 (BMP-2). Material and Methods: Confluent osteoblast-like cell line (MC3T3-E1, subclone 24) cultures were exposed to pure PVP-I solution (7.7 mg/ml) and dilutions of 1:10, 1:100 and 1:1000 for 10 seconds and washed with phosphate buffer solution. Cell proliferation and viability was determined by MTT and differentiation status by alkaline phosphatase (ALP) activity 6 days after initial plating. In a separate experiment, long-term cell proliferation, viability and function were assessed 4 weeks after PVP-I treatment by MTT and deposited calcium using an Alizarin-red staining test. Results: PVP-I decreased ALP activity substantially. Stimulation by BMP-2 recovered ALP activity to near control levels at 1:100 and 1:1000 dilutions of PVP-I. The MTT assay showed reduced proliferation of the preosteoblastic cells for all treatments, irrespective whether BMP-2 was used or not. Only at PVP-I dilutions of 1:1000 proliferation rate was back to normal levels (95.6±2.4 %). No adverse long-term effect of PVP-I on mineralization of the extracellular matrix (Alizarinred) for dilutions higher than 1:100 was observed. Interestingly, undiluted and 1:10 diluted PVP-I even showed a significant increase in mineral deposition, especially in the presence of BMP-2. Conclusion: Short-time application of PVP-I in concentrations of 1:10 and higher lead to decreased viability and impaired differentiation. However, surviving cells showed good recovery and mineralization potential.

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Tead1 Reciprocally Regulates Adult β-Cell Proliferation and Function to Maintain Glucose Homeostasis

SSRN Electronic Journal ◽

10.2139/ssrn.3704009 ◽

2020 ◽

Author(s):

Jeongkyung Lee ◽

Ruya Liu ◽

Byung S. Kim ◽

Yiqun Zhang ◽

Feng Li ◽

...

Keyword(s):

Cell Proliferation ◽

Glucose Homeostasis ◽

Β Cell ◽

And Function

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P1237TISSUE-NONSPECIFIC ALKALINE PHOSPHATASE, A CULPRIT DURING ARTERIAL MEDIA CALCIFICATION BUT INDISPENSABLE DURING PHYSIOLOGICAL BONE FORMATION/-MINERALIZATION IN RATS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p1237 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Britt Opdebeeck ◽

José Millan Luis ◽

Anthony Pinkerton ◽

Anja Verhulst ◽

Patrick D'Haese ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Bone Formation ◽

Vascular Calcification ◽

Rat Model ◽

Bone Mineralization ◽

Calcium Content ◽

Marker Genes ◽

Peripheral Arteries ◽

Calcium Phosphorus ◽

Chondrogenic Marker

Abstract Background and Aims Vascular media calcification is frequently seen in elderly and patients with chronic kidney disease (CKD), diabetes and osteoporosis. Pyrophosphate is a well-known calcification inhibitor that binds to nascent hydroxyapatite crystals and prevents further incorporation of inorganic phosphate into these crystals. However, the enzyme tissue-nonspecific alkaline phosphatase (TNAP), which is highly expressed in calcified arteries, degrades extracellular pyrophosphate into phosphate ions, by which pyrophosphate loses its ability to block vascular calcification. Here, we aimed to evaluate whether a TNAP inhibitor is able to prevent the development of arterial calcification in a rat model of warfarin-induced vascular calcification. Method To induce vascular calcification, rats received a diet containing 0.30% warfarin and 0.15% vitamin K1 throughout the entire study and were subjected to the following daily treatments: (i) vehicle (n=10) or (ii) 10 mg/kg/day TNAP-inhibitor (n=10) administered via an intraperitoneal catheter from start of the study until sacrifice at week 7. Calcium, phosphorus and parathyroid hormone (PTH) levels were determined in serum samples as these are important determinants of vascular calcification. As TNAP is also expressed in the liver, serum alanine aminotransferase (ALT) and aspartate (AST) levels were analyzed. At sacrifice, vascular calcification was evaluated by measurement of the total calcium content in the arteries and quantification of the area % calcification on Von Kossa stained sections of the aorta. The mRNA expression of osteo/chondrogenic marker genes (runx2, TNAP, SOX9, collagen 1 and collagen 2) was analyzed in the aorta by qPCR to verify whether vascular smooth muscle cells underwent reprogramming towards bone-like cells. Bone histomorphometry was performed on the left tibia to measure static and dynamic bone parameters as TNAP also regulates physiological bone mineralization. Results No differences in serum calcium, phosphorus and PTH levels was observed between both study groups. Warfarin exposure resulted in distinct calcification in the aorta and peripheral arteries. Daily dosing with the TNAP inhibitor (10 mg/kg/day) for 7 weeks significantly reduced vascular calcification as indicated by a significant decrease in calcium content in the aorta (vehicle 3.84±0.64 mg calcium/g wet tissue vs TNAP inhibitor 0.70±0.23 mg calcium/g wet tissue) and peripheral arteries and a distinct reduction in area % calcification on Von Kossa stained aortic sections as compared to vehicle condition. The inhibitory effects of SBI-425 on vascular calcification were without altering serum liver markers ALT and AST levels. Furthermore, TNAP-inhibitor SBI-425 did not modulate the mRNA expression of osteo/chondrogenic marker genes runx2, TNAP, SOX9, collagen 1 and 2. Dosing with SBI-425 resulted in decreased bone formation rate and mineral apposition rate, and increased osteoid maturation time and this without significant changes in osteoclast- and eroded perimeter. Conclusion Dosing with TNAP inhibitor SBI-425 significantly reduced the calcification in the aorta and peripheral arteries of a rat model of warfarin-induced vascular calcification and this without affecting liver function. However, suppression of TNAP activity should be limited in order to maintain adequate physiological bone mineralization.

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Carnosic Acid Induces Apoptosis and Inhibits Akt/mTOR Signaling in Human Gastric Cancer Cell Lines

Pharmaceuticals ◽

10.3390/ph14030230 ◽

2021 ◽

Vol 14 (3) ◽

pp. 230

Author(s):

Waseem El-Huneidi ◽

Khuloud Bajbouj ◽

Jibran Sualeh Muhammad ◽

Arya Vinod ◽

Jasmin Shafarin ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Proliferation ◽

Cancer Cell ◽

Gastric Cancer Cell ◽

Carnosic Acid ◽

Human Gastric Cancer ◽

Cancer Cell Proliferation ◽

Human Gastric Cancer Cell ◽

Gastric Cancer Cell Lines ◽

Gastric Cancer Cell Proliferation

Gastric cancer is among the most common malignancies worldwide. Due to limited availability of therapeutic options, there is a constant need to find new therapies that could target advanced, recurrent, and metastatic gastric cancer. Carnosic acid is a naturally occurring polyphenolic abietane diterpene derived from Rosmarinus officinalis and reported to have numerous pharmacological effects. In this study, the cytotoxicity assay, Annexin V-FITC/PI, caspases 3, 8, and 9, cell cycle analysis, and Western blotting were used to assess the effect of carnosic acid on the growth and survival of human gastric cancer cell lines (AGS and MKN-45). Our findings showed that carnosic acid inhibited human gastric cancer cell proliferation and survival in a dose-dependent manner. Additionally, carnosic acid is found to inhibit the phosphorylation/activation of Akt and mTOR. Moreover, carnosic acid enhanced the cleavage of PARP and downregulated survivin expression, both being known markers of apoptosis. In conclusion, carnosic acid exhibits antitumor activity against human gastric cancer cells via modulating the Akt-mTOR signaling pathway that plays a crucial role in gastric cancer cell proliferation and survival.

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A novel gelatin/chitooligosaccharide/demineralized bone matrix composite scaffold and periosteum-derived mesenchymal stem cells for bone tissue engineering

Biomaterials Research ◽

10.1186/s40824-021-00220-y ◽

2021 ◽

Vol 25 (1) ◽

Author(s):

Thakoon Thitiset ◽

Siriporn Damrongsakkul ◽

Supansa Yodmuang ◽

Wilairat Leeanansaksiri ◽

Jirun Apinun ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Demineralized Bone Matrix ◽

Bone Matrix ◽

Alp Activity

Abstract Background A novel biodegradable scaffold including gelatin (G), chitooligosaccharide (COS), and demineralized bone matrix (DBM) could play a significant part in bone tissue engineering. The present study aimed to investigate the biological characteristics of composite scaffolds in combination of G, COS, and DBM for in vitro cell culture and in vivo animal bioassays. Methods Three-dimensional scaffolds from the mixture of G, COS, and DBM were fabricated into 3 groups, namely, G, GC, and GCD using a lyophilization technique. The scaffolds were cultured with mesenchymal stem cells (MSCs) for 4 weeks to determine biological responses such as cell attachment and cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, cell morphology, and cell surface elemental composition. For the in vivo bioassay, G, GC, and GCD, acellular scaffolds were implanted subcutaneously in 8-week-old male Wistar rats for 4 weeks and 8 weeks. The explants were assessed for new bone formation using hematoxylin and eosin (H&E) staining and von Kossa staining. Results The MSCs could attach and proliferate on all three groups of scaffolds. Interestingly, the ALP activity of MSCs reached the greatest value on day 7 after cultured on the scaffolds, whereas the calcium assay displayed the highest level of calcium in MSCs on day 28. Furthermore, weight percentages of calcium and phosphorus on the surface of MSCs after cultivation on the GCD scaffolds increased when compared to those on other scaffolds. The scanning electron microscopy images showed that MSCs attached and proliferated on the scaffold surface thoroughly over the cultivation time. Mineral crystal aggregation was evident in GC and greatly in GCD scaffolds. H&E staining illustrated that G, GC, and GCD scaffolds displayed osteoid after 4 weeks of implantation and von Kossa staining confirmed the mineralization at 8 weeks in G, GC, and GCD scaffolds. Conclusion The MSCs cultured in GCD scaffolds revealed greater osteogenic differentiation than those cultured in G and GC scaffolds. Additionally, the G, GC, and GCD scaffolds could promote in vivo ectopic bone formation in rat model. The GCD scaffolds exhibited maximum osteoinductive capability compared with others and may be potentially used for bone regeneration.

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