Effect of a Simvastatin-Impregnated Chitosan Scaffold on Cell Growth and Osteoblastic Differentiation

This study aims to evaluate the effect of chitosan (CS) scaffold, alone, and the potential synergistic effect when impregnated with simvastatin (SIM), on immortalized human bone-marrow mesenchymal stem cells (hbMMSCs) compared to CollaCote (CL). CS scaffolds were fabricated and seeded with immortalized hBMMSCs. Samples were divided into control groups (negative with no added material and positive with CL added) and four experimental groups: CS alone, CS/SIM 0.01, 0.03, and 0.05 mg, respectively. Cell viability, osteoblastic differentiation and calcium deposition were investigated via AlamarBlue, alkaline phosphate activity assays and alizarin red S staining at 1 and 14 days, respectively. At day one, no significant difference was noted between the groups regarding cell viability. However, all CS/SIM groups showed significant cutback at day 14 in cell proliferation compared to CS alone and CL groups (p < 0.001). All groups supported osteoblastic differentiation with no significant difference. Alkaline phosphate activity increased in both time periods in the CS/SIM 0.05 mg group compared to the other SIM groups, with no significant difference among the experimental groups. Chitosan scaffold is a bioactive compatible material capable of regenerative potential of hBMMSCs and a promising material to be used for perforation repair.

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Osteogenic Effects of Naringenin Are Mediated Through the Activation of AMPK Pathway

Current Developments in Nutrition ◽

10.1093/cdn/nzaa040_012 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 12-12

Author(s):

Savion Carswell ◽

Tanzim Mridha ◽

Cynthia E Francis ◽

Shashidharamurthy Taval ◽

Srujana Rayalam

Keyword(s):

Bone Marrow ◽

Adenosine Monophosphate ◽

Calcium Deposition ◽

Alizarin Red ◽

Ampk Pathway ◽

Funding Sources ◽

Osteogenic Markers ◽

Age Related ◽

Marrow Mesenchymal Stem Cells ◽

Post Hoc

Abstract Objectives Bone marrow mesenchymal stem cells are the precursor cells for adipocytes and osteoblasts. The accumulation of adipocytes in bone marrow at the expense of osteoblastogenesis is a major factor contributing to age-related bone loss. Grapefruit flavonoid naringenin (NAR) was shown to promote osteogenesis but the mechanism behind these effects is not clear. In this study, we propose to demonstrate that the osteogenic effects of NAR are mediated through the activation of adenosine monophosphate-activated kinase (AMPK) pathway. Methods MC3T3-E1 pre-osteoblast cells were cultured using alpha MEM medium (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Osteogenic differentiation was induced by using differentiation medium purchased from Stem Cell Technologies. Western blotting, ELISA and alizarin red assays were conducted to test our hypothesis. Results NAR increased calcium deposition in MC3T3 cells compared to DMSO control after 21-day differentiation period. Additionally, in the presence of AMPK inhibitor Ara-A, NAR-induced calcium deposition was suppressed compared to NAR alone. Furthermore, while NAR increased phosphorylation of AMPK, the combination of NAR and Ara-A significantly decreased AMPK phosphorylation. Finally, Ara-A suppressed NAR-induced expression of osteogenic markers including Runx2, osteoprotegerin and osteocalcin suggesting that NAR-induced osteogenesis is partly mediated through AMPK pathway. All data were expressed as the mean ± SEM, minimum n = 3. Comparisons were made by using one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. Conclusions The suppression of osteogenic markers and calcium deposition in the presence of NAR plus Ara-A, an AMPK inhibitor, compared to NAR alone provide evidence that the observed osteogenic effects of NAR are in part mediated through the activation of AMPK pathway. These data combined with our previous reports of anti-adipogenic effects of NAR suggest that NAR may have potential therapeutic applications for osteoporosis by not only promoting osteogenesis but also inhibiting adipogenesis in the bone marrow. Funding Sources Division of Research, PCOM and NIH-1R03 AI128254–01A1.

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Evaluation of the Osteogenic Potential of Different Scaffolds Embedded with Human Stem Cells Originated from Schneiderian Membrane: An In Vitro Study

BioMed Research International ◽

10.1155/2019/2868673 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Rita Bou Assaf ◽

Mohammad Fayyad-Kazan ◽

Fatima Al-Nemer ◽

Rawan Makki ◽

Hussein Fayyad-Kazan ◽

...

Keyword(s):

Stem Cells ◽

Cell Viability ◽

Osteogenic Differentiation ◽

Bone Defects ◽

Osteogenic Potential ◽

Calcium Deposition ◽

Pcr Analysis ◽

Human Stem Cells ◽

Alizarin Red ◽

Alp Activity

Background. Novel treatments for bone defects, particularly in patients with poor regenerative capacity, are based on bone tissue engineering strategies which include mesenchymal stem cells (MSCs), bioactive factors, and convenient scaffold supports. Objective. In this study, we aimed at comparing the potential for different scaffolds to induce osteogenic differentiation of human maxillary Schneiderian sinus membrane- (hMSSM-) derived cells. Methods. hMSSM-derived cells were seeded on gelatin, collagen, or Hydroxyapatite β-Tricalcium phosphate-Fibrin (Haβ-TCP-Fibrin) scaffolds. Cell viability was determined using an MTT assay. Alizarin red staining method, Alkaline phosphatase (ALP) activity assay, and quantitative real-time PCR analysis were performed to assess hMSSM-derived cells osteogenic differentiation. Results. Cell viability, calcium deposition, ALP activity, and osteoblastic markers transcription levels were most striking in gelatin scaffold-embedded hMSSM-derived cells. Conclusion. Our findings suggest a promising potential for gelatin-hMSSM-derived cell construct for treating bone defects.

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Quaternary ammonium silane (k21) based intracanal medicament triggers biofilm destruction

BMC Oral Health ◽

10.1186/s12903-021-01470-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Esther Sook Kuan Kok ◽

Xian Jin Lim ◽

Soo Xiong Chew ◽

Shu Fen Ong ◽

Lok Yin See ◽

...

Keyword(s):

Laser Scanning ◽

Collagen Matrix ◽

Docking Studies ◽

Laser Scanning Microscopy ◽

Calcium Deposition ◽

Confocal Laser ◽

Alizarin Red ◽

Significant Difference ◽

Biofilm Destruction ◽

Root Canal Dentin

Abstract Background Compare antimicrobial efficacy of a quarternary ammonium silane (QAS)/k21 as an intracanal medicament against E. faecalis and C. albicans biofilms formed on root dentin. Methodology Dentin blocks were sterilized and E. faecalis and C. albicans microbial colonies were counted for colony-forming-units against 2%k21, 2%CHX and Ca(OH)2 medicaments. Biofilm colonies after 7 days on dentin were analysed using confocal laser scanning microscopy with live/dead bacterial viability staining. TEM was done to study dentin collagen matrix. Dentin discs from 3rd day and 7th day well plate was used for Raman spectra and observed under fluorescent-microscope. Docking studies were carried out on MMP-2 S1 binding-domain with k21. Results There was reduction of E. faecalis/C. albicans when k21, chlorhexidine and calcium hydroxide were used with highest percentage in 2%k21 treated specimens. 2%k21 showed dense and regular collagen network with intact cross-banding and decreased Raman intensity for 2%k21 on 3rd day. NaOCl + k21 showed least adherence, whereas saline groups showed highest adherence of E. faecalis and C. albicans to root-canal dentin. Alizarin red staining of hDPSCs revealed calcium deposition in all groups with significant difference seen amongst 2%k21 groups. MMP-2 ligand binding was seen accurately indicating possible target sites for k21 intervention. Conclusion 2%k21 can be considered as alternative intracanal medicament.

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Therapeutic potential of Liuwei Dihuang pill against KDM7A and Wnt/β-catenin signaling pathway in diabetic nephropathy-related osteoporosis

Bioscience Reports ◽

10.1042/bsr20201778 ◽

2020 ◽

Vol 40 (9) ◽

Cited By ~ 1

Author(s):

Ming Ming Liu ◽

Rui Dong ◽

Zhen Hua ◽

Nan Ning Lv ◽

Yong Ma ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Cell Viability ◽

Signaling Pathway ◽

High Glucose ◽

Bending Strength ◽

Therapeutic Potential ◽

Osteoblastic Differentiation ◽

Sprague Dawley ◽

Mineral Density ◽

Alizarin Red

Abstract The effects of Liuwei Dihuang pill (LWDH) on diabetic nephropathy-related osteoporosis (DNOP) are unclear. The present study aimed to evaluate the effects of LWDH on KDM7A and Wnt/β-catenin signaling pathway in DNOP rats and the high glucose-induced MC3T3-E1 cells. A DNOP model was prepared by streptozotocin in 9-week-old male Sprague-Dawley (SD) rats to evaluate the effects of LWDH. The cell viability and differentiation capacity of high glucose-induced MC3T3-E1 cells were determined by CCK-8 assay, Alizarin Red staining, and alkaline phosphatase (ALP) staining, respectively. Furthermore, the expressions of KDM7A and Wnt1/β-catenin pathway-related proteins were determined by Western blot analysis. Treatment of DNOP rats with LWDH could significantly ameliorate the general state, degradation of renal function, and renal pathological changes. LWDH decreased the levels of TNF-α, IL-6, IL-8, IL-1β, ALP, and TRAP, and increased the calcium, phosphorus in serum, as well as decreased the level of the calcium and phosphorus in the urine. Besides, LWDH significantly improved bone mineral density (BMD), bone volume (BV), and the bone microstructure of DNOP rats. Moreover, LWDH increased the levels of the elastic modulus, ultimate load, and bending strength in the femurs. In MC3T3-E1 cells, serum-containing LWDH significantly increases in cell viability and osteoblastic differentiation capability. The expression of α-SMA, vimentin, KDM7A, Wnt1 and β-catenin were significantly down-regulated, and the E-cadherin, H3K9-Me2, H3K27-Me2, BMP-4, BMP-7, Runx2, osteocalcin, and Col1a1 were significantly up-regulated with LWDH treatment. The present study shows that LWDH has a therapeutic effect on DNOP, in part, through down-regulation of KDM7A and Wnt/β-catenin pathway.

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Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.672518 ◽

2021 ◽

Vol 8 ◽

Author(s):

Iñigo Gaitán-Salvatella ◽

Edgar Oliver López-Villegas ◽

Patricia González-Alva ◽

Fernando Susate-Olmos ◽

Marco Antonio Álvarez-Pérez

Keyword(s):

Magnetic Nanoparticles ◽

Cell Viability ◽

Cellular Response ◽

Magnetic Levitation ◽

Engineering Application ◽

Bone Defects ◽

Calcium Deposition ◽

Sem Images ◽

Alizarin Red ◽

Matrix Deposition

Skeletal reconstruction is necessary in cases of bone defects created by tumors, trauma, and abnormalities. Regeneration of bone defects remains a critical problem, and current approaches are based on biocompatible scaffolds. Spheroids represent a simple 3D system since no supporting material is required for cell growth. Different techniques are used to generate spheroids, such as hanging drop, low-attachment plates, and magnetic nanoparticles. The idea of using magnetic nanoparticles is to cross-link through cell membrane overnight to create complex 3D cellular spheroid by using magnets to guide the cellular response. Herein, the current study aimed to achieve 3D human fetal osteoblast (hFOB) spheroid under magnetic levitation. Formation of 3D spheroid culture under magnetic levitation was evaluated by cell viability at 3, 7, and 14 days. Morphology of the 3D hFOB spheroid was analyzed by SEM and fluorescence microscopy and the differentiation towards mineralized lineage by ALP assay, qPCR, and alizarin red staining. The cell viability indicated that the 3D hFOB spheroid still viable after 14 days of culture. ALP assay, qPCR analysis expression of Col1, ALP, and Itg-β1 molecules, and calcium deposition with alizarin red showed a high level of bioactivity of the 3D hFOB spheroid. SEM images allowed the morphological analysis of the 3D microtissue-like spheroid with the presence of matrix deposition. These results indicate that magnetic levitation culture enables 3D stable osteoblast spheroids and could be a promising strategy for engineering application in the 3D construct in surgery regeneration of mineralized tissue.

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MiR-486-3p promotes osteogenic differentiation of BMSCs by targeting CTNNBIP1 and activating Wnt/β-catenin pathway

10.21203/rs.3.rs-396210/v1 ◽

2021 ◽

Author(s):

Zheng Zhang ◽

Weiwei Jiang ◽

Miao Hu ◽

Yichen Meng ◽

Jun Ma ◽

...

Keyword(s):

Bone Marrow ◽

Osteogenic Differentiation ◽

Osteoblastic Differentiation ◽

Luciferase Reporter ◽

Negative Effects ◽

Alizarin Red ◽

Interacting Protein ◽

Marrow Mesenchymal Stem Cells ◽

Osteogenic Gene Expression ◽

Canonical Wnt

Abstract Background Dysfunction in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) leads to bone loss/osteoporosis. CTNNBIP1 (Catenin beta interacting protein 1) is an inhibitor of Wnt/β-catenin signaling, whose role in osteogenesis remains elusive. This study aims to reveal the effects of miR-486-3p/CTNNBIP1 in osteogenesis. Methods Bone marrow samples from control and osteoporosis patients were collected and ovariectomy was performed on mice and levels of CTNNBIP1 and miR-486-3p levels were assessed. Dual-luciferase reporter assay was used to confirm their interactions. MiR-486-3p mimics/inhibitor or CTNNBIP1 overexpression lentivirus were transfected in human BMSCs (hBMSCs) and then osteogenic assay was performed. Alizarin red S (ARS) and Alkaline phosphatase (ALP) intensity with osteogenic gene expression including Runx2, Alp, Bglap and OCN was measured. Key proteins in Wnt/β-catenin pathway including active β-catenin, Bcl-2 and Cyclin D1 were assessed. Results CTNNBIP1, an inhibitor of Wnt/β-catenin signaling was upregulated while miR-486-3p was downregulated in ovariectomized (OVX) mice. CTNNBIP1 was confirmed as a target of miR-486-3p. MiR-486-3p overexpression promoted but miR-486-3p knockdown suppressed osteogenic differentiation and Wnt/β-catenin pathway. Rescue experiments further elucidated that the negative effects of CTNNBIP1 overexpression on osteoblastic differentiation and canonical Wnt signaling could be reversed by miR-486-3p mimics. Conclusion This study demonstrated that miR-486-3p sponges CTNNBIP1 thus activating Wnt/β-catenin signaling pathway to promote osteogenesis of BMSCs.

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Osteogenic Properties of Novel Methylsulfonylmethane-Coated Hydroxyapatite Scaffold

International Journal of Molecular Sciences ◽

10.3390/ijms21228501 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8501

Author(s):

Jeong-Hyun Ryu ◽

Tae-Yun Kang ◽

Hyunjung Shin ◽

Kwang-Mahn Kim ◽

Min-Ho Hong ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Bone Regeneration ◽

Porous Scaffold ◽

Calcium Deposition ◽

Burst Release ◽

Alizarin Red ◽

Porous Hydroxyapatite ◽

Level Of Activity ◽

Significant Difference ◽

Hydroxyapatite Scaffold

Despite numerous advantages of using porous hydroxyapatite (HAp) scaffolds in bone regeneration, the material is limited in terms of osteoinduction. In this study, the porous scaffold made from nanosized HAp was coated with different concentrations of osteoinductive aqueous methylsulfonylmethane (MSM) solution (2.5, 5, 10, and 20%) and the corresponding MH scaffolds were referred to as MH2.5, MH5, MH10, and MH20, respectively. The results showed that all MH scaffolds resulted in burst release of MSM for up to 7 d. Cellular experiments were conducted using MC3T3-E1 preosteoblast cells, which showed no significant difference between the MH2.5 scaffold and the control with respect to the rate of cell proliferation (p > 0.05). There was no significant difference between each group at day 4 for alkaline phosphatase (ALP) activity, though the MH2.5 group showed higher level of activity than other groups at day 10. Calcium deposition, using alizarin red staining, showed that cell mineralization was significantly higher in the MH2.5 scaffold than that in the HAp scaffold (p < 0.0001). This study indicated that the MH2.5 scaffold has potential for both osteoinduction and osteoconduction in bone regeneration.

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INTS7–ABCD3 Interaction Stimulates the Proliferation and Osteoblastic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells by Suppressing Oxidative Stress

Frontiers in Physiology ◽

10.3389/fphys.2021.758607 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yubo Liu ◽

Xiao Yu ◽

Anquan Huang ◽

Xiangxin Zhang ◽

Yijun Wang ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Osteoblastic Differentiation ◽

Mouse Bone Marrow ◽

Osteoporosis Therapy ◽

Alizarin Red ◽

Marrow Mesenchymal Stem Cells

Increased adipocyte and decreased osteoblast differentiation, combined with the ectopic proliferation of bone marrow mesenchymal stem cells (BM-MSCs), represent the primary causes of osteoporosis. The dysregulation of numerous intracellular bioactive factors is responsible for the aberrant differentiation and growth of BM-MSCs. In this study, we focused on a new stimulative factor, integrator complex subunit 7 (INTS7), and its cooperative protein ATP-binding cassette subfamily D member 3 (ABCD3)/high-density lipoprotein-binding protein (HDLBP) in mouse BM-MSCs. We aimed to uncover the effects of the INTS7–ABCD3/HDLBP interaction on BM-MSC biological behaviors and the potential mechanism underlying these effects. Functional in vitro experiments showed that the suppression of the INTS7–ABCD3 interaction rather than HDLBP could impair BM-MSC proliferation and induce cell apoptosis. Moreover, Alizarin Red S and Oil Red O staining, respectively, revealed that INTS7 and ABCD3 knockdown but not HDLBP knockdown could decrease osteoblastic differentiation and accelerate the adipogenic differentiation of BM-MSCs. Mechanistically, reactive oxygen species (ROS) and histone γ-H2AX quantities significantly increased, whereas the levels of antioxidants declined due to INTS7 and ABCD3 inhibition in BM-MSCs. These findings indicated that the suppression of oxidative stress could be involved in the INTS7/ABCD3 co-regulatory mechanisms for BM-MSC proliferation and differentiation, identifying new potential candidates for osteoporosis therapy.

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CBX4-dependent regulation of HDAC3 nuclear translocation reduces Bmp2-induced osteoblastic differentiation and calcification in adamantinomatous craniopharyngioma

Cell Communication and Signaling ◽

10.1186/s12964-021-00797-w ◽

2022 ◽

Vol 20 (1) ◽

Author(s):

Xiaorong Yan ◽

Dezhi Kang ◽

Yuanxiang Lin ◽

Songtao Qi ◽

Changzhen Jiang

Keyword(s):

Nuclear Localization ◽

Nuclear Translocation ◽

Tumor Resection ◽

Osteoblastic Differentiation ◽

Therapeutic Interventions ◽

Bone Morphogenetic Protein 2 ◽

Luciferase Reporter ◽

Calcium Deposition ◽

Alizarin Red ◽

Adamantinomatous Craniopharyngioma

Abstract Background Calcification of adamantinomatous craniopharyngioma (ACP) often causes problems with tumor resection, leading to a high incidence of deadly complications and tumor recurrence. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are 2 key enzymes that regulate histone acetylation and play important roles in tumor development. However, the roles of HAT and HDAC in the calcification and osteoblastic differentiation of ACP are not known. Methods In this study, primary cells were isolated from ACP tissues, and calcification was induced with bone morphogenetic protein 2 (Bmp2). HDAC3 expression was assessed in 12 tissue samples by Western blotting and immunohistochemistry. ACP calcification was assessed by Alizarin red staining. A luciferase reporter assay was performed to examine the interaction between miR-181b and the 3’-untranslated region of the polycomb chromobox 4 (CBX4) gene. Results Our results showed that the expression of HDAC3 was increased in the calcified ACP samples, but inhibition of HDAC3 promoted ACP cell calcification and osteoblastic differentiation. Mechanistically, HDAC3 nuclear translocation was suppressed by Bmp2, leading to Runx2 protein expression and Osterix, osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP) mRNA expression. In addition, this process was suppressed by CBX4, which stabilized the nuclear localization of HDAC3. miR-181b, the expression of which was increased in Bmp2-induced ACP cells, directly targeted and decreased CBX4 expression and inhibited the nuclear localization of HDAC3. Conclusions Our results demonstrate that Bmp2 increases miR-181b levels to directly target and inhibit CBX4 expression, leading to a reduction in the CBX4-dependent regulation of HDAC3 nuclear translocation, which results in Runx2 activation/osteoblastic differentiation and calcium deposition in ACP. Further studies targeting these cascades may contribute to therapeutic interventions used for recurrent ACP.

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Effects of Virgin Coconut Oil as Adjunct Therapy in the Treatment of Allergic Rhinitis

Journal of Clinical and Health Sciences ◽

10.24191/jchs.v1i1.5850 ◽

2016 ◽

Vol 1 (1) ◽

pp. 22

Author(s):

Nazli Zainuddin ◽

Nurul Azira Mohd Shah ◽

Rosdan Salim

Keyword(s):

Allergic Rhinitis ◽

Side Effects ◽

Coconut Oil ◽

Test Group ◽

Nasal Symptom ◽

Control Group ◽

Virgin Coconut Oil ◽

Control Groups ◽

Significant Difference ◽

And Control

Introduction: The role of virgin coconut oil in the treatment of allergic rhinitis is controversial. Thus, the aim of the present study is to determine the effects of virgin coconut oil ingestion, in addition to standard medications, on allergic rhinitis. We also studied the side effects of consumption of virgin coconut oil. Methods: Fifty two subjects were equally divided into test and control groups. All subjects received a daily dose of 10mg of loratadine for 28 days. The test group was given 10ml of virgin coconut oil three times a day in addition to loratadine. The symptoms of allergic rhinitis were scored at the beginning and end of the study. Results:, the symptom score were divided into nasal and non-nasal symptom scores. Sneezing score showed a significant difference, however the score was more in control group than test group, indicating that improvement in symptom was more in control group. The rest of the nasal symptom and non-nasal symptom score showed no significant difference between test and control groups. Approximately 58% of the test subjects developed side effects from consumption of virgin coconut oil, mainly gastrointestinal side effects. Conclusion: In the present study, ingestion of virgin coconut oil does not improve the overall and individual symptoms of allergic rhinitis, furthermore it has side effects.

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