In vitro effects of alendronate on fibroblasts of the human rotator cuff tendon

Abstract Background Bone mineral density of the humeral head is an independent determining factor for postoperative rotator cuff tendon healing. Bisphosphonates, which are commonly used to treat osteoporosis, have raised concerns regarding their relationships to osteonecrosis of the jaw and to atypical fracture of the femur. In view of the prevalence of rotator cuff tear in osteoporotic elderly people, it is important to determine whether bisphosphonates affect rotator cuff tendon healing. However, no studies have investigated bisphosphonates’ cytotoxicity to human rotator cuff tendon fibroblasts (HRFs) or bisphosphonates’ effects on rotator cuff tendon healing. The purpose of this study was to evaluate the cytotoxicity of alendronate (Ald), a bisphosphonate, and its effects on HRF wound healing. Methods HRFs were obtained from human supraspinatus tendons, using primary cell cultures. The experimental groups were control, 0.1 μM Ald, 1 μM Ald, 10 μM Ald, and 100 μM Ald. Alendronate exposure was for 48 h, except during a cell viability analysis with durations from 1 day to 6 days. The experimental groups were evaluated for cell viability, cell cycle and cell proliferation, type of cell death, caspase activity, and wound-healing ability. Results The following findings regarding the 100 μM Ald group contrasted with those for all the other experimental groups: a significantly lower rate of live cells (p < 0.01), a higher rate of subG1 population, a lower rate of Ki-67 positive cells, higher rates of apoptosis and necrosis, a higher number of cells with DNA fragmentation, higher caspase-3/7 activity (p < 0.001), and a higher number of caspase-3 positive staining cells. In scratch-wound healing analyses of all the experimental groups, all the wounds healed within 48 h, except in the 100 μM Ald group (p < 0.001). Conclusions Low concentrations of alendronate appear to have little effect on HRF viability, proliferation, migration, and wound healing. However, high concentrations are significantly cytotoxic, impairing cellular proliferation, cellular migration, and wound healing in vitro.

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BMP-2 and BMP-7 affect human rotator cuff tendon cells in vitro

Journal of Shoulder and Elbow Surgery ◽

10.1016/j.jse.2011.01.015 ◽

2012 ◽

Vol 21 (4) ◽

pp. 464-473 ◽

Cited By ~ 38

Author(s):

Stephan Pauly ◽

Franka Klatte ◽

Catrin Strobel ◽

Gerhard Schmidmaier ◽

Stefan Greiner ◽

...

Keyword(s):

Rotator Cuff ◽

Rotator Cuff Tendon ◽

Tendon Cells ◽

Human Rotator Cuff

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Direct Effect of Septic Plasma in Human Cell Lines Viability

Blood Purification ◽

10.1159/000494597 ◽

2018 ◽

Vol 47 (1-3) ◽

pp. 270-276

Author(s):

Grazia Maria Virzì ◽

Chiara Borga ◽

Chiara Pasqualin ◽

Silvia Pastori ◽

Alessandra Brocca ◽

...

Keyword(s):

Cytochrome C ◽

Cell Viability ◽

Cell Lines ◽

Caspase 3 ◽

Test Group ◽

Organ Injury ◽

Enzyme Linked Immunosorbent Assay ◽

Caspase 9 ◽

Renal Tubular Cells

Background: Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients’ plasma. Methods: We enrolled 26 septic patients (“test” group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit. Results: We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937). Conclusion: Humoral mediators in septic patients’ plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages.

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Effects of a New Bioceramic Material on Human Apical Papilla Cells

Journal of Functional Biomaterials ◽

10.3390/jfb9040074 ◽

2018 ◽

Vol 9 (4) ◽

pp. 74 ◽

Cited By ~ 16

Author(s):

Diana Sequeira ◽

Catarina Seabra ◽

Paulo Palma ◽

Ana Cardoso ◽

João Peça ◽

...

Keyword(s):

Wound Healing ◽

Mineral Trioxide Aggregate ◽

Cellular Adhesion ◽

Cellular Migration ◽

Cellular Viability ◽

Migration Rates ◽

Apical Papilla ◽

And Migration ◽

Proliferation And Migration

Background: The development of materials with bioregenerative properties is critically important for vital pulp therapies and regenerative endodontic procedures. The aim of this study was to evaluate the cytocompatibility and cytotoxicity of a new endodontic biomaterial, PulpGuard, in comparison with two other biomaterials widely used in endodontic procedures, ProRoot Mineral Trioxide Aggregate (MTA) and Biodentine. Methods: Apical papilla cells (APCs) were isolated from third molars with incomplete rhizogenesis from patients with orthodontic indication for dental extraction. Cultured APCs were incubated for 24, 48, or 72 h with different dilutions of eluates prepared from the three materials. Cellular viability, mobility, and proliferation were assessed in vitro using the Alamar Blue assay and a wound-healing test. The cells were also cultured in direct contact with the surface of each material. These were then analyzed via Scanning Electron Microscopy (SEM), and the surface chemical composition was determined by Energy-Dispersive Spectroscopy (EDS). Results: Cells incubated in the presence of eluates extracted from ProRoot MTA and PulpGuard presented rates of viability comparable to those of control cells; in contrast, undiluted Biodentine eluates induced a significant reduction of cellular viability. The wound-healing assay revealed that eluates from ProRoot MTA and PulpGuard allowed for unhindered cellular migration and proliferation. Cellular adhesion was observed on the surface of all materials tested. Consistent with their disclosed composition, EDS analysis found high relative abundance of calcium in Biodentine and ProRoot MTA and high abundance of silicon in PulpGuard. Significant amounts of zinc and calcium were also present in PulpGuard discs. Concerning solubility, Biodentine and ProRoot MTA presented mild weight loss after eluate extraction, while PulpGuard discs showed significant water uptake. Conclusions: PulpGuard displayed a good in vitro cytocompatibility profile and did not significantly affect the proliferation and migration rates of APCs. Cells cultured in the presence of PulpGuard eluates displayed a similar profile to those cultured with eluates from the widely used endodontic cement ProRoot MTA.

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Not So Fast: Platelet-Rich Fibrin Matrix May Potentially Have a Negative Effect on Rotator Cuff Tendon Healing

JBJS Orthopaedic Highlights Sports Medicine ◽

10.2106/jbjs.sm.l.00283 ◽

2012 ◽

Vol 2 (8) ◽

pp. e4

Author(s):

Stephen F. Brockmeier

Keyword(s):

Rotator Cuff ◽

Tendon Healing ◽

Platelet Rich Fibrin ◽

Rotator Cuff Tendon ◽

Fibrin Matrix ◽

Negative Effect

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Wound Healing Activity of the Essential Oil of Bursera morelensis, in Mice

Molecules ◽

10.3390/molecules25081795 ◽

2020 ◽

Vol 25 (8) ◽

pp. 1795

Author(s):

Judith Salas-Oropeza ◽

Manuel Jimenez-Estrada ◽

Armando Perez-Torres ◽

Andres Eliu Castell-Rodriguez ◽

Rodolfo Becerril-Millan ◽

...

Keyword(s):

Wound Healing ◽

Essential Oil ◽

Cell Viability ◽

Murine Model ◽

Biological Activities ◽

Healing Process ◽

In Vitro Tests ◽

Wound Healing Process ◽

Fibroblast Migration

Bursera morelensis is used in Mexican folk medicine to treat wounds on the skin. It is an endemic tree known as “aceitillo”, and the antibacterial and antifungal activity of its essential oil has been verified; it also acts as an anti-inflammatory. All of these reported biological activities make the essential oil of B. morelensis a candidate to accelerate the wound-healing process. The objective was to determine the wound-healing properties of B. morelensis’ essential oil on a murine model. The essential oil was obtained by hydro-distillation, and the chemical analysis was performed by gas chromatography-mass spectrometry (GC-MS). In the murine model, wound-healing efficacy (WHE) and wound contraction (WC) were evaluated. Cytotoxic activity was evaluated in vitro using peritoneal macrophages from BALB/c mice. The results showed that 18 terpenoid-type compounds were identified in the essential oil. The essential oil had remarkable WHE regardless of the dose and accelerated WC and was not cytotoxic. In vitro tests with fibroblasts showed that cell viability was dose-dependent; by adding 1 mg/mL of essential oil (EO) to the culture medium, cell viability decreased below 80%, while, at doses of 0.1 and 0.01 mg/mL, it remained around 90%; thus, EO did not intervene in fibroblast proliferation, but it did influence fibroblast migration when wound-like was done in monolayer cultures. The results of this study demonstrated that the essential oil was a pro-wound-healing agent because it had good healing effectiveness with scars with good tensile strength and accelerated repair. The probable mechanism of action of the EO of B. morelensis, during the healing process, is the promotion of the migration of fibroblasts to the site of the wound, making them active in the production of collagen and promoting the remodeling of this collagen.

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Anti-KRAS siRNA nanoparticles for targeted colorectal cancer therapy.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.4_suppl.636 ◽

2017 ◽

Vol 35 (4_suppl) ◽

pp. 636-636 ◽

Cited By ~ 1

Author(s):

Bradley Krasnick ◽

Matthew S. Strand ◽

Ye Bi ◽

Peter S. Goedegebuure ◽

Timothy Fleming ◽

...

Keyword(s):

Colorectal Cancer ◽

Cell Viability ◽

Caspase 3 ◽

Fluorescent Microscopy ◽

Mutant Type ◽

Viability Assay ◽

Mutational Status ◽

Promising Target ◽

Folfox Chemotherapy

636 Background: Standard treatment for metastatic colorectal cancer (mCRC) is systemic chemotherapy with anti-EGFR treatment, depending on KRAS mutational status. However, tumors harboring a KRAS mutation do not respond to existing targeted therapy. Moreover, targeting mutant KRAS has, to date, not been possible. Herein, we explore using a KRAS inhibitory nanoparticle (NP), to directly knock down mutant KRAS. Methods: Utilizing fluorescent-labeled small interfering RNA (siRNA) NPs, uptake was assessed via fluorescent microscopy. KRAS mutant CT26 and wild-type MC38 CRC cell lines were incubated with either scramble (Sc) sequence siRNA NP, KRAS siRNA NP, or FOLFOX (fluorouracil + oxaliplatin) chemotherapy ± KRAS siRNA NP. Cell viability was assessed via a luminescent viability assay. KRAS and cleaved caspase 3 protein expression were assessed using western blotting. Results: Fluorescent NP uptake was demonstrated in CT26 cells as early as 260 minutes post treatment, with increased uptake through 780 minutes. Decreased cellular viability was seen with KRAS siRNA NP treated CT26 cells, as compared to both Sc siRNA NP and non-treated CT26 cells (both p < 0.0001). Cell viability was significantly diminished with FOLFOX combined with KRAS siRNA NP as compared to FOLFOX alone for CT26 cells ( p = 0.0003), but not MC38 cells (p = 0.2259). Western blot demonstrated decreased KRAS and increased cleaved caspase 3 expression in CT26 cells treated with KRAS siRNA NP. Conclusions: A KRAS siRNA tagged NP was internalized by the CRC cells in vitro, and induced cellular death via apoptosis in mutant type KRAS CRC. In addition, KRAS siRNA NP acted synergistically with FOLFOX chemotherapy to enhance cell death. We believe KRAS inhibition based NP treatment is a promising target for mutant type KRAS CRC. [Table: see text]

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Lipid emulsion attenuates apoptosis induced by a toxic dose of bupivacaine in H9c2 rat cardiomyoblast cells

Human & Experimental Toxicology ◽

10.1177/0960327115608930 ◽

2016 ◽

Vol 35 (9) ◽

pp. 929-937 ◽

Cited By ~ 8

Author(s):

S-H Ok ◽

J Yu ◽

Y Lee ◽

H Cho ◽

I-W Shin ◽

...

Keyword(s):

Cell Viability ◽

Lipid Emulsion ◽

Caspase 3 ◽

In Vitro Study ◽

Terminal Deoxynucleotidyl Transferase ◽

Caspase 8 ◽

Lipid Solubility ◽

H9c2 Cells ◽

Toxic Dose

The goal of this in vitro study was to investigate the effect of lipid emulsion on apoptosis induced by a toxic dose of bupivacaine (BPV) in H9c2 rat cardiomyoblast cell lines. The effect of lipid emulsion on the decreased cell viability and count induced by BPV or mepivacaine (MPV) in the H9c2 cells was assessed using an 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay or a cell count assay. The effect of BPV or lipid emulsion combined with BPV on cleaved caspase 3, caspase 8, and Bax in H9c2 cells was investigated using Western blotting. A terminal deoxynucleotidyl transferase dUTP2′-deoxyuridine 5′-triphosphate nick end-labeling (TUNEL) assay was performed to detect apoptosis of H9c2 cells treated with BPV alone or lipid emulsion combined with BPV. The magnitude of lipid emulsion-mediated attenuation of decreased cell viability induced by BPV was higher than that of lipid emulsion-mediated attenuation of decreased cell viability induced by MPV. Lipid emulsion attenuated the increases in cleaved caspase 3, caspase 8 and Bax induced by BPV. Lipid emulsion attenuated the increases in TUNEL-positive cells induced by BPV. These results suggest that lipid emulsion attenuates a toxic dose of BPV-induced apoptosis via inhibition of the extrinsic and intrinsic apoptotic pathways. The protective effect of lipid emulsion may be partially associated with the relatively high lipid solubility of BPV.

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Antioxidant effects on hypoxia-induced oxidative stress and apoptosis in rat rotator cuff fibroblasts

10.22203/ecm.v041a44 ◽

2021 ◽

Vol 41 ◽

pp. 680-693

Author(s):

RJ Kim ◽

◽

SH An ◽

JY Gwark ◽

HB Park

Keyword(s):

Rotator Cuff ◽

Cell Viability ◽

Caspase 3 ◽

Hypoxia Inducible Factor ◽

Matrix Metalloproteinase 2 ◽

Heme Oxygenase 1 ◽

Ros Production ◽

Hypoxia Group ◽

Pre Treatment ◽

Parp 1

Most cells, highly sensitive to oxygen levels, undergo apoptosis under hypoxia. Therefore, the involvement of hypoxia in rotator cuff tendon degeneration has been proposed. While previous studies have reported that hypoxia induces apoptosis in rotator cuff fibroblasts (RCFs), little research has investigated whether antioxidants have cytoprotective effects against RCF apoptosis. The present study aimed at determining whether the antioxidant N-acetylcysteine (NAC) exerted cytoprotective effects against hypoxia-induced RCF apoptosis. Third-passage rat RCFs were divided into normoxia, NAC, hypoxia and NAC-hypoxia groups. The hypoxia inducer was 1,000 µmol/L cobalt chloride (CoCl2); the antioxidant was 20 mmol/L NAC. Expressions of hypoxia-inducible factor-1α (HIF-1α) and heme oxygenase-1 (HO-1), cell viability, intracellular reactive oxygen species (ROS) production, apoptosis rates as well as expressions of cleaved caspase-3, cleaved poly ADP-ribose polymerase-1 (PARP-1), vascular endothelial growth factors-β (VEGF-β) and matrix metalloproteinase-2 (MMP-2) were evaluated. Expression of HIF-1α and HO-1 was significantly higher in the hypoxia group than in the normoxia group (p < 0.001). Cell viability was significantly lower in the hypoxia group than in the normoxia group (p < 0.001). Intracellular ROS production, apoptosis rate and expressions of cleaved caspase-3, cleaved PARP-1, VEGF-β and MMP-2 were significantly higher in the hypoxia group than in the normoxia group (p < 0.001). All these responses were significantly attenuated by pre-treatment with NAC (p ≤ 0.001). ROS were involved in hypoxic RCF apoptosis induced by CoCl2; NAC, an ROS scavenger, inhibited hypoxia-induced RCF apoptosis by inhibiting ROS production.

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An image J plugin for the high throughput image analysis of in vitro scratch wound healing assays

10.1101/2020.04.20.050831 ◽

2020 ◽

Author(s):

Alejandra Suarez-Arnedo ◽

Felipe Torres Figueroa ◽

Camila Clavijo ◽

Pablo Arbeláez ◽

Juan C. Cruz ◽

...

Keyword(s):

Wound Healing ◽

Image Analysis ◽

Low Cost ◽

Cellular Migration ◽

Wound Healing Assay ◽

Wound Area ◽

Scratch Wound ◽

Scratch Wound Healing Assay ◽

Imagej Plugin

AbstractIn vitro scratch wound healing assay, a simple and low-cost technique that works along with other image analysis tools, is one of the most widely used 2D methods to determine the cellular migration and proliferation in processes such as regeneration and disease. There are open-source programs such as imageJ to analyze images of in vitro scratch wound healing assays, but these tools require manual tuning of various parameters, which is time-consuming and limits image throughput. For that reason, we developed an optimized plugin for imageJ to automatically recognize the wound healing size, correct the average wound width by considering its inclination, and quantify other important parameters such as: area, wound area fraction, average wound width, and width deviation of the wound images obtained from a scratch/ wound healing assay. Our plugin is easy to install and can be used with different operating systems. It can be adapted to analyze both individual images and stacks. Additionally, it allows the analysis of images obtained from bright field, phase contrast, and fluorescence microscopes. In conclusion, this new imageJ plugin is a robust tool to automatically standardize and facilitate quantification of different in vitro wound parameters with high accuracy compared with other tools and manual identification.

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