scholarly journals Effect of BMP- 2, -4, and -7 on Proliferation and Osteogenic Differentiation in Cultured Human PDLSCs

2021 ◽  
Author(s):  
Juedan Li ◽  
Min Wang ◽  
Min Cui ◽  
Cheng Chen ◽  
Zheng Cheng
Keyword(s):  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Collagen Type ◽  
Time Dependent ◽  
Control Group ◽  
Type I ◽  
Dependent Manner ◽  
Periodontal Ligament Stem Cells ◽  
Phosphatase Activities ◽  
Related Proteins

Abstract Aim To investigate the effects of bone morphogenetic proteins (BMPs) 2, 4, and 7 on proliferation and osteogenic differentiation in human periodontal ligament stem cells (PDLSCs). Methods PDLSCs were isolated by an immunomagnetic method. Expression of cell surface antigens CD146, CD44, and CD34, and pluripotency (osteogenic and adipogenic) were measured. Cultured PDLSCs were treated, in dose- and time-dependent experiments, with single BMPs, with 1:1 combinations, and with a mix of all three BMPs (1:3 each). For dose-dependent experiments, PDLSCs were incubated for 12 d with media containing BMPs at 0, 10, 25, 50, and 100 ng/ml. For time-dependent experiments, PDLSCs were treated with media containing 50 ng/ml BMPs for 0, 3, 6, 12, and 24 d. Cell growth and alkaline phosphatase activities were measured by MTT and an enzyme kit. Immunohistochemistry and western blotting were used to detect osteogenic differentiation-related proteins, i.e., osteocalcin, bone sialoprotein, collagen type I, and collagen type III. Results PDLSCs displayed CD146 (93%) and CD44 (91.2%) positive expression; CD34 (1.8%) showed negative expression. All cells exhibited osteogenic and adipogenic potential. The proliferation and alkaline phosphatase activities of PDLSCs treated with the aforesaid single and combined BMPs increased in a dose- and time-dependent manner; proliferation and alkaline phosphatase activity were greater with the BMP combinations. Compared with the control group, the levels of osteogenic differentiation-related proteins increased markedly in PDLSCs treated with 50 ng/ml BMPs for 12 d, whereas no significant differences were observed between the different BMP treatments. Conclusion BMP-2, -4, and -7, singly and in combination, promoted development and osteogenic differentiation of PDLSCs, and both cellular outcomes were more pronounced with BMP combinations.

Acute Myeloid Leukemia (AML) Cells Alter the Bone Marrow Microenvironment By Inducing Osteogenic and Suppressing Adipogenic Differentiation of MSCs through BMP-RUNX2-CTGF Mediated Mechanisms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2403-2403
Author(s):  
Venkata Lokesh Battula ◽  
Phuong M Le ◽  
Jeffrey Sun ◽  
Hong Mu ◽  
Teresa Mc.Queen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Cell Growth ◽  
Osteogenic Differentiation ◽  
Research Funding ◽  
Adipogenic Differentiation ◽  
Time Dependent ◽  
Dependent Manner ◽  
Osteoprogenitor Cells

Abstract Osteoprogenitor cells are a critical component of the bone marrow microenvironment (BME), support AML cell growth (Raaijmakers et al., Nature, 2010); however the mechanism has not been fully elucidated. Here, we hypothesize that AML cells induce osteogenic differentiation in mesenchymal stem/stromal cells to gain growth advantage. We have isolated age matched (between 40-70 years) bone marrow derived MSCs from AML patients (AML-MSC, n=20) and normal (N-MSC, n=10) individuals and analyzed their cell growth, cell surface phenotype and multi-lineage differentiation. AML-MSCs are phenotypically different with larger cell surface area and lower cell growth compared to N-MSCs. The average doubling time of AML-MSCs is 52±8hrs compared to 34±6hours for N-MSCs (p<0.01). Phenotypic and enzyme activity studies revealed that tissue non-specific alkaline phosphatase (TNAP), a protein that is highly expressed in naïve MSCs and osteoprogenitor cells and osteoblasts, is expressed 10-14 fold higher in AML-compared to N-MSCs (p<0.01). As TNAP is associated with osteogenic lineage, we next assessed the expression of osteogenic markers including RUNX-2, osteopontin, TNAP and osterix. Interestingly, these genes were up-regulated by 5-10-fold in AML-MSCs compared to N-MSCs. To validate these observations, N-MSCs were cultured with OCI-AML3 cell derived conditioned medium (CM) for 3-5 days and then induced to osteogenic or adipogenic differentiation. As expected, alkaline phosphatase enzyme activity and Alizarine Red S staining was twice as high in MSCs cultured with AML-CM compared to MSCs cultured in regular cell culture medium. In addition, we found expression of osteogenic genes including RUNX-2, osteopontin, TNAP and osterix 3-4 fold upregulated in MSCs cultured with AML-CM compared to control MSCs. These data indicate that AML cells prime MSCs to undergo osteogenic differentiation. Adipocyte differentiation was assessed by Oil-Red O staining for lipid droplets and revealed a >95% reduction (p<0.001) in the number of mature adipocytes in AML-MSCs compared to N-MSCs. Gene expression analysis by qRT-PCR revealed that adipogenic markers including aP2, lipoprotein lipase and PPARγ were down-regulated 10-20 fold in AML-MSCs compared to N-MSCs in a time-dependent manner suggesting that AML-MSCs lack the ability to differentiate into adipocytes. To evaluate these findings in-vivo, we developed a Human Bone Marrow Implant (HBMI) mouse model by subcutaneous implantation of human femur derived bone pieces into NSG mice. In this model, mice bearing HBMI with leukemia expressed higher osteogenic related proteins compared to mice without leukemia. Importantly, multispectral image analysis revealed 5-10 fold higher osteogenic activity in AML patient BM-biopsies compared to normal bones. Bone morphogenic proteins (BMP) are the most essential factors during osteogenic differentiation and new bone formation in humans. We therefore treated MSCs with AML cell-derived conditioned medium, and observed a 6-8 fold increase in pSmad1/5 levels in N-MSCs in a time dependent manner. In addition, AML induced pSmad1/5 up-regulation was inhibited when MSCs were treated with BMP-type1 receptor specific inhibitor LDN-212854, in a dose dependent manner. In addition, inhibition of BMP signaling also inhibited AML-induced osteogenic differentiation in MSCs. Mechanistic studies revealed that bone morphogenetic proteins (BMPs) derived from AML cells induce connective tissue growth factor (CTGF) expression in MSCs. When transplanted in Col1a2-CTGF transgenic mice, AML cells engrafted faster compared to wild type mice suggesting that CTGF enhances AML cell growth. Our data suggest that AML cells induce osteogenic differentiation in MSCs and receive growth advantage through BMP-CTGF signaling. Inhibition of these pathways may reduce leukemia propagation and improve AML patient survival. Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

Biocompatibility study of modified injectable hyaluronic acid hydrogel with mannitol/BSA to alveolar bone cells

2020 ◽  
pp. 088532822097174
Author(s):  
Kwanhatai Areevijit ◽  
Nirada Dhanesuan ◽  
Jittima Amie Luckanagul ◽  
Sorasun Rungsiyanont
Keyword(s):  
Alkaline Phosphatase ◽  
Hyaluronic Acid ◽  
Collagen Type ◽  
Alveolar Bone ◽  
Bone Cells ◽  
Collagen Type I ◽  
Control Group ◽  
Type I ◽  
Hyaluronic Acid Hydrogel ◽  
Alveolar Bone Cells

The quality and quantity of bone are crucial to the success of dental implant treatment. Recently, bone grafting materials have reached some limitations. This study aimed to evaluate the biocompatibility of novel drug delivery material, injectable methacrylated hyaluronic acid hydrogel incorporated with different ratios of mannitol and BSA (Man/BSA MeHA), to human alveolar bone cells. The three-dimensionally encapsulated cell culture was evaluated with the resazurin cell viability test, alkaline phosphatase activity assay, immunohistochemistry test for collagen type-I synthesis, and cell morphology. The results showed that the encapsulated cells were viable in all four ratios of Man/BSA MeHA hydrogel and the average metabolic rate was not less than the control group. The morphology test showed round shape cells at the upper portion of the hydrogel and fibroblast-like or polygonal shape at the lower portion of hydrogel next to the culture plate. All four groups could express enzyme alkaline phosphatase and collagen type-I. In conclusion, four ratios of Man/BSA MeHA hydrogel were biocompatible with primary human alveolar bone cells.

scholarly journals Comparison of in vitro biocompatibility and antibacterial activity of two calcium silicate-based materials

2021 ◽  
Vol 32 (5) ◽  
Author(s):  
Mingxiang Liu ◽  
Lu He ◽  
Hongyuan Wang ◽  
Wenpei Su ◽  
Hong Li
Keyword(s):  
Antibacterial Activity ◽  
Osteogenic Differentiation ◽  
Mineral Trioxide Aggregate ◽  
Antibacterial Activities ◽  
Control Group ◽  
Type I ◽  
Differentiation Markers ◽  
Periodontal Ligament Stem Cells ◽  
Significant Difference

AbstractThis study is aimed at comparing and evaluating the biocompatibility and antibacterial activities of mineral trioxide aggregate (MTA) and iRoot BP Plus as novel retro-filling materials. Discs of both materials were prepared and incubated for 72 h to obtain material extracts in medium. Flow cytometry and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were used to assess the rate of apoptosis and proliferation of human periodontal ligament stem cells (hPDLSCs) when exposed to eluates of both materials. The expression levels of alkaline phosphatase, collagen type I, osteocalcin, Runt-related transcription factor-2, and Osterix were tested for evaluating the osteogenic differentiation of hPDLSCs. The antibacterial activities of both materials were compared by the direct contact test. The hPDLSCs stimulated by MTA or iRoot BP Plus eluates showed significantly higher cell viability than that of the control group with no eluates. No significant differences were observed among the percentages of necrotic and apoptotic cells stimulated by MTA and iRoot BP Plus eluates and the control group. The expression of all osteogenic differentiation markers of hPDLSCs in both experimental groups were significantly higher than those of the control group, while the increment values in MTA group were significantly higher than those of the iRoot BP Plus group. The antibacterial activity against Enterococcus faecalis showed no significant difference between MTA and iRoot BP Plus. Therefore, both materials may be suitable for retro-filling applications.

scholarly journals CLEC-2 suppresses calcification in cultured osteoblasts

2019 ◽  
Author(s):  
Takenori Kanai ◽  
Yoshihiko Sawa ◽  
Kenyo Takara ◽  
Koichiro Kajiwara ◽  
Takahiro Fujita ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Collagen Type ◽  
Collagen Type I ◽  
Medullary Cavity ◽  
Type I ◽  
Alizarin Red ◽  
Phosphatase Activities

AbstractPodoplanin is the only counter-receptor of platelet CLEC-2 and is expressing on mature osteoblast, but there is no report on the role of podoplanin and CLEC-2 in calcification. This study aimed to investigate the role of podoplanin binding to CLEC-2 in the calcification of osteoblasts carrying homozygously deleted Pdpn alleles (PdpnΔ/Δ) by heterozygously expressing collagen type I alpha 1 promoter (Col1a)-driven Cre recombinase. There were no macroscopic abnormalities in the bone and dentin of Col1a11-Cre;PdpnΔ/Δ mice but the coccygeal bone medullary cavity was very narrow. In the quantitative analysis for alizarin red-stained products and alkaline phosphatase activities on the cultured calvarial osteoblasts, the amounts of calcified products and alkaline phosphatase activity of calvarial osteoblasts of both Pdpnfl/fl and Col1a11-Cre;PdpnΔ/Δ mice were significantly higher in the calcification medium than in the α-mem. Both the amounts of calcified products and alkaline phosphatase activity of calvarial osteoblasts from Pdpnfl/fl mice were significantly lower in the calcification medium with CLEC-2 than without CLEC-2 while there were no significant differences in the amounts of calcified products and alkaline phosphatase activities of calvarial osteoblasts from Col1a11-Cre;PdpnΔ/Δ mice with CLEC-2. Platelet CLEC-2 may play a role in regulating the calcification via binding to podoplanin on mature osteoblasts expressing podoplanin in the medullary cavity of a part of the bone.

scholarly journals NF-κB and FosB mediate inflammation and oxidative stress in the blast lung injury of rats exposed to shock waves

2021 ◽  
Author(s):  
Hong Wang ◽  
Wenjuan Zhang ◽  
Jinren Liu ◽  
Junhong Gao ◽  
Le Fang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lung Injury ◽  
Shock Waves ◽  
Time Dependent ◽  
Inflammatory Factors ◽  
Control Group ◽  
Dependent Manner ◽  
Total Antioxidant ◽  
Blast Lung Injury ◽  
And Oxidative Stress

Abstract Blast lung injury (BLI) is the major cause of death in explosion-derived shock waves; however, the mechanisms of BLI are not well understood. To identify the time-dependent manner of BLI, a model of lung injury of rats induced by shock waves was established by a fuel air explosive. The model was evaluated by hematoxylin and eosin staining and pathological score. The inflammation and oxidative stress of lung injury were also investigated. The pathological scores of rats’ lung injury at 2 h, 24 h, 3 days, and 7 days post-blast were 9.75±2.96, 13.00±1.85, 8.50±1.51, and 4.00±1.41, respectively, which were significantly increased compared with those in the control group (1.13±0.64; P&lt;0.05). The respiratory frequency and pause were increased significantly, while minute expiratory volume, inspiratory time, and inspiratory peak flow rate were decreased in a time-dependent manner at 2 and 24 h post-blast compared with those in the control group. In addition, the expressions of inflammatory factors such as interleukin (IL)-6, IL-8, FosB, and NF-κB were increased significantly at 2 h and peaked at 24 h, which gradually decreased after 3 days and returned to normal in 2 weeks. The levels of total antioxidant capacity, total superoxide dismutase, and glutathione peroxidase were significantly decreased 24 h after the shock wave blast. Conversely, the malondialdehyde level reached the peak at 24 h. These results indicated that inflammatory and oxidative stress induced by shock waves changed significantly in a time-dependent manner, which may be the important factors and novel therapeutic targets for the treatment of BLI.

scholarly journals The Selective Histone Deacetylase Inhibitor MI192 Enhances the Osteogenic Differentiation Efficacy of Human Dental Pulp Stromal Cells

2021 ◽  
Vol 22 (10) ◽  
pp. 5224
Author(s):  
Kenny Man ◽  
Liam Lawlor ◽  
Lin-Hua Jiang ◽  
Xuebin B. Yang
Keyword(s):  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Dental Pulp ◽  
Selective Inhibitor ◽  
Epigenetic Reprogramming ◽  
Type I ◽  
Human Dental Pulp ◽  
Pre Treatment ◽  
Dose Dependent

The use of human dental pulp stromal cells (hDPSCs) has gained increasing attention as an alternative stem cell source for bone tissue engineering. The modification of the cells’ epigenetics has been found to play an important role in regulating differentiation, with the inhibition of histone deacetylases 3 (HDAC3) being linked to increased osteogenic differentiation. This study aimed to induce epigenetic reprogramming using the HDAC2 and 3 selective inhibitor, MI192 to promote hDPSCs osteogenic capacity for bone regeneration. MI192 treatment caused a time–dose-dependent change in hDPSC morphology and reduction in viability. Additionally, MI192 successfully augmented hDPSC epigenetic functionality, which resulted in increased histone acetylation and cell cycle arrest at the G2/M phase. MI192 pre-treatment exhibited a dose-dependent effect on hDPSCs alkaline phosphatase activity. Quantitative PCR and In-Cell Western further demonstrated that MI192 pre-treatment significantly upregulated hDPSCs osteoblast-related gene and protein expression (alkaline phosphatase, bone morphogenic protein 2, type I collagen and osteocalcin) during osteogenic differentiation. Importantly, MI192 pre-treatment significantly increased hDPSCs extracellular matrix collagen production and mineralisation. As such, for the first time, our findings show that epigenetic reprogramming with the HDAC2 and 3 selective inhibitor MI192 accelerates the osteogenic differentiation of hDPSCs, demonstrating the considerable utility of this MSCs engineering approach for bone augmentation strategies.

scholarly journals SAT0298 IS INTERLEUKIN 6 A FACTOR OF FIBROGENESIS IN DERMAL FIBROBLASTS?

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1094.1-1094
Author(s):  
A. S. Siebuhr ◽  
P. Juhl ◽  
M. Karsdal ◽  
A. C. Bay-Jensen
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Collagen Type ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Full Time ◽  
Collagen Formation ◽  
Type Iii

Background:Interleukin 6 (IL-6) is known to have both pro- and anti-inflammatory properties, depending on the receptor activation. The classical IL-6 signaling via the membrane bound receptor is mainly anti-inflammatory, whereas signaling through the soluble receptor (sIL-6R) is pro-inflammatory/pro-fibrotic. However, the direct fibrotic effect of IL-6 stimulation on dermal fibroblasts is unknown.Objectives:We investigated the fibrotic effect of IL-6 + sIL-6R in a dermal fibroblast model and assessed fibrosis by neo-epitope biomarkers of extracellular matrix proteins.Methods:Primary healthy human dermal fibroblasts were grown for up to 17 days in DMEM medium with 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid. IL-6 [1-90 nM]+sIL-6R [0.1-9 nM] alone or in combination with TGFβ [1 nM] were tested in three different donors. TGFβ [1 nM], PDGF-AB [3 nM] and non-stimulated cells (w/o) were used as controls. Tocilizumab (TCZ) with TGFβ + IL-6 + sIL-6R stimulation was tested in one donor. Collagen type I, III and VI formation (PRO-C1, PRO-C3 and PRO-C6) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Western blot analysis investigated signal cascades. Gene expression of selected ECM proteins was analyzed. Statistical analyses included One-way and 2-way ANOVA and area under the curve analysis.Results:formation by the end of the culture period. The fibronectin and collagen type VI signal were consistent between the three tested donors, whereas the formation of type III collagen was only increased in one donor, but in several trials. Type I collagen formation was unchanged by IL-6 + sIL-6R stimulation. The gene expression of type I collagen was induced by IL-6 + sIL-6R. Western blot analysis validated trans-signaling by the IL-6+sIL-6R stimulation as expected.IL-6 + sIL-6R stimulation in combination with TGFβ decreased fibronectin levels compared to TGFβ alone but did not reach the level of unstimulated fibroblasts. The formation of collagen type IV was generally unchanged with IL-6 + sIL-6R + TGFβ compared to TGFβ alone. Collagen type I and III formation was more scattered in the signals when IL-6 + sIL-6R was in combination with TGFβ, as the biomarker level could be either decreased or increased compared to TGFβ alone. In two studies the type I collagen level was synergistic increased by IL-6 + sIL-6R + TGFβ, whereas another study found the level to be decreased compared to TGFβ alone. The gene expression of fibronectin and type I collagen was increased with TGFβ +IL-6+sIL-6R compared to TGFβ alone.Inhibition of IL-6R by TCZ in combination with IL-6 + sIL-6R did only decrease the fibronectin level with the lowest TCZ concentration (p=0.03). TCZ alone decreased the fibronectin level in a dose-dependent manner (One-way ANOVA p=0.0002).Conclusion:We investigated the fibrotic response of dermal fibroblasts to IL-6 + sIL-6R stimulation. IL-6 modulated the fibronectin level and modulated the collagen type III formation level in a somewhat dose-dependent manner. In combination with TGFβ, IL-6 decreased collagen type I and IV formation and fibronectin. However, in this study inhibition of IL-6R by TCZ did not change the fibrotic response of the dermal fibroblasts. This study indicated that IL-6 did not induce collagen formation in dermal fibroblasts, except type III collagen formation with high IL-6 concentration.Figure:Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Pernille Juhl Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S.

Effects of Berberine on Expression of LOX-1 and SR-BI in Human Macrophage-Derived Foam Cells Induced by ox-LDL

2010 ◽  
Vol 38 (06) ◽  
pp. 1161-1169 ◽  
Author(s):  
Siming Guan ◽  
Bin Wang ◽  
Wei Li ◽  
Jinghuan Guan ◽  
Xin Fang
Keyword(s):  
Scavenger Receptor ◽  
Ldl Receptor ◽  
Foam Cells ◽  
Time Dependent ◽  
Human Macrophage ◽  
Type I ◽  
Dependent Manner ◽  
Atp Binding Cassette Transporter ◽  
Abca1 Expression ◽  
Class B

This study investigates the effects of beriberine on the expression of lectin-like ox-LDL receptor-1 (LOX-1), scavenger receptor A (SR-A), SR class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) in human macrophage-derived foam cells induced by ox-LDL. Different concentrations of Berberine were co-cultured with THP-1 derived foam cells. The mRNA and protein expressions of LOX-1, SR-A, SR-BI and ABCA1 were determined by RT-PCR and Western blot analysis, respectively. Ox-LDL significantly increased the expression of LOX-1 and inhibited the expression of SR-BI in a dose- and time-dependent manner. Berberine significantly inhibited the effects of ox-LDL in a dose- and time-dependent manner. Moreover, ox-LDL significantly promoted ABCA1 expression. However, berberine had no effect on SR-A or ABCA1 expression. Berberine can inhibit the expression of LOX-1 and promote the expression of SR-BI in macrophage-derived foam cells. Therefore, berberine could be used to treat atherosclerotic diseases.

scholarly journals Effects of Naringin on Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells In Vitro and In Vivo

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Lihua Yin ◽  
Wenxiao Cheng ◽  
Zishun Qin ◽  
Hongdou Yu ◽  
Zhanhai Yu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Trabecular Structure ◽  
Control Group ◽  
Periodontal Ligament Stem Cells ◽  
Expression Levels ◽  
Proliferation And Differentiation

This study is to explore the osteogenesis potential of the human periodontal ligament stem cells (hPDLSCs) induced by naringin in vitro and in vitro. The results confirmed that 1 μM naringin performs the best effect and a collection of bone-related genes (RUNX2,COL1A2, OPN, and OCN) had significantly higher expression levels compared to the control group. Furthermore, a typical trabecular structure was observed in vivo, surrounded by a large amount of osteoblasts. These results demonstrated that naringin, at a concentration of 1 μM, can efficiently promote the proliferation and differentiation of hPDLSCs both in vitro and in vivo.

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