Polarized Light Microscopy for Analyzing Tissue Mechanics During In Vitro Acupuncture

2008 ◽  
Author(s):  
Lowell Taylor Edgar ◽  
Margaret Julias ◽  
David I. Shreiber ◽  
Helen M. Buettner
Keyword(s):  
Connective Tissue ◽  
Light Microscopy ◽  
Type I Collagen ◽  
Polarized Light ◽  
Tissue Mechanics ◽  
Polarized Light Microscopy ◽  
Tissue Response ◽  
Type I ◽  
Collagen Gels

Acupuncture is a traditional therapy originating in China almost 2000 years ago. Acupuncture has slowly been growing in popularity in the West, and clinical evidence has shown the potential for acupuncture as a low-cost ‘alternative’ therapy for an assortment of ailments [1]. The practice of acupuncture involves inserting fine needles into the skin followed by needle manipulation, usually by rotation. Recent studies by Langevin et al demonstrate that this rotation causes the subcutaneous connective tissue to couple to and wind around the needle [2–4], which suggests that mechanotransduction in the connective tissue might play a role in the therapeutic mechanisms that underlay acupuncture [2, 3]. To begin to decompose and quantify this complex mechanism at the tissue level in a controlled setting, we have simulated acupuncture in type I collagen gels in vitro, and have developed algorithms to quantify the tissue response following imaging with polarized light microscopy (PLM).

Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Martin Liu ◽  
Angelos Karagiannis ◽  
Matthew Sis ◽  
Srivatsan Kidambi ◽  
Yiannis Chatzizisis
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Muscle Cells ◽  
Type I ◽  
Collagen Gels ◽  
Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

An in vitro Evaluation of Artificial Caries-like Lesions on Restored Overdenture Abutments

1988 ◽  
Vol 67 (3) ◽  
pp. 582-584 ◽  
Author(s):  
P.P. Kambhu ◽  
R.L. Ettinger ◽  
J.S. Wefel
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
The Other ◽  
Type Ii ◽  
Restorative Materials ◽  
Abutment Teeth ◽  
The Mean ◽  
Materials Used

An acidified dialyzed gelatin gel system was used to determine the caries resistance of a variety of restorative materials used to obturate the canal orifice of overdenture abutment teeth. The restorative materials used were Tytin, Tytin + Copalite, P30 + Scotchbond, Fuji Ionomer-Type II, and Miracle Mix. Polarized light microscopy and microradiography were used to examine the caries-like lesions adjacent to the restorations. The lesions formed in the Fuji Ionomer-Type II and Miracle Mix groups appeared arrested at the wall adjacent to the restoration, and did not penetrate apically down the wall as did those associated with the other restorative materials. The mean depths of lesions adjacent to Fuji Ionomer-Type II and Miracle Mix restorations were significantly less than those of Tytin, Tytin + Copalite, or P30 + Scotchbond.

scholarly journals Active Phagocytosis and Diachronic Processing of Calcium Oxalate Monohydrate Crystals in an in vitro Macrophage Model

2019 ◽  
Vol 44 (5) ◽  
pp. 1014-1025
Author(s):  
Atsushi Okada ◽  
Hiromasa Aoki ◽  
Daichi Onozato ◽  
Taiki Kato ◽  
Tadahiro Hashita ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Calcium Oxalate ◽  
Polarized Light ◽  
Calcium Oxalate Monohydrate ◽  
In Vitro Models ◽  
Polarized Light Microscopy ◽  
Transmission Electron ◽  
Imaging Cytometry ◽  
Acidic Organelles

Background: We previously discovered that renal macrophages (Mφs) phagocytose renal calcium oxalate monohydrate (COM) crystals. This study investigated the processing of engulfed crystals using in vitro models. Methods: J774.1 mouse Mφs were exposed to COM crystals and observed for 24 h using polarized light microscopy with/without cytochalasin B (CB), an inhibitor of phagocytosis, to confirm active crystal phagocytosis. LysoTracker and immunohistochemical staining using transmission electron microscopy for lysosomal-associated membrane protein 1 were used to confirm engulfed COM crystal uptake into lysosomes. Diachronic tracking of specific Mφs was performed to capture the entire course of engulfed COM crystal processing using polarized light microscopy. Follow-up studies of fluorescent COM (f-COM) crystals using imaging cytometry were performed in the presence and absence of nigericin to dissipate the pH gradient in acidic organelles. Results: Phagocytosis rates increased with COM density and were significantly lower in cells treated with CB (p < 0.01). We observed that engulfed crystals colocalized within lysosomes of the Mφs; moreover, diachronic observation indicated that the engulfed COM crystals were subdivided during Mφ division and eliminated by the 7th day of culture. Additionally, imaging cytometry showed that the fluorescence level of f-COM crystals in the nigericin (–) group after 48 h was significantly lower than that in the nigericin (+) group. Conclusions: This study confirmed active phagocytosis and lysosomal processing of engulfed COM crystals by Mφs. This discovery is expected to contribute to the development of future drugs that enhance the COM crystal phagocytic ability of Mφs.

scholarly journals Identification and characterization of a fibroblast marker: FSP1.

1995 ◽  
Vol 130 (2) ◽  
pp. 393-405 ◽  
Author(s):  
F Strutz ◽  
H Okada ◽  
C W Lo ◽  
T Danoff ◽  
R L Carone ◽  
...  
Keyword(s):  
Cell Fate ◽  
Calcium Binding ◽  
Type I Collagen ◽  
De Novo ◽  
Mesangial Cells ◽  
In Vivo Studies ◽  
Type I ◽  
Tubular Epithelium ◽  
Collagen Gels

We performed subtractive and differential hybridization for transcript comparison between murine fibroblasts and isogenic epithelium, and observed only a few novel intracellular genes which were relatively specific for fibroblasts. One such gene encodes a filament-associated, calcium-binding protein, fibroblast-specific protein 1 (FSP1). The promoter/enhancer region driving this gene is active in fibroblasts but not in epithelium, mesangial cells or embryonic endoderm. During development, FSP1 is first detected by in situ hybridization after day 8.5 as a postgastrulation event, and is associated with cells of mesenchymal origin or of fibroblastic phenotype. Polyclonal antiserum raised to recombinant FSP1 protein stained the cytoplasm of fibroblasts, but not epithelium. Only occasional cells stain with specific anti-FSP1 antibodies in normal parenchymal tissue. However, in kidneys fibrosing from persistent inflammation, many fibroblasts could be identified in interstitial sites of collagen deposition and also in tubular epithelium adjacent to the inflammatory process. This pattern of anti-FSP1 staining during tissue fibrosis suggests, as a hypothesis, that fibroblasts in some cases arise, as needed, from the local conversion of epithelium. Consistent with this notion that FSP1 may be involved in the transition from epithelium to fibroblasts are experiments in which the in vitro overexpression of FSP1 cDNA in tubular epithelium is accompanied by conversion to a mesenchymal phenotype, as characterized by a more stellate and elongated fibroblast-like appearance, a reduction in cytokeratin, and new expression of vimentin. Similarly, tubular epithelium submerged in type I collagen gels exhibited the conversion to a fibroblast phenotype which includes de novo expression of FSP1 and vimentin. Use of the FSP1 marker, therefore, should further facilitate both the in vivo studies of fibrogenesis and the mapping of cell fate among fibroblasts.

Advances Toward Forming Synthetic Mimetic Tendon

MRS Advances ◽  
2016 ◽  
Vol 1 (18) ◽  
pp. 1283-1288
Author(s):  
Dilinazi Aishanjiang ◽  
Emily C. Green ◽  
Heng Li ◽  
Marilyn L. Minus
Keyword(s):  
Type I Collagen ◽  
Fibrillar Structure ◽  
Type I ◽  
Structural Development ◽  
Collagen Gels ◽  
Connective Tissues ◽  
Synthetic Fibers ◽  
Spinning Process ◽  
Bovine Type

ABSTRACTCollagen is the most abundant protein present in the human body and found in connective tissues, bone, and tendon. It is also known as a natural resource for healing damaged skin tissues [1]. In this study, under specific microenvironment conditions, mimetic collagen gels were successfully formed synthetically from reconstituted Bovine type I collagen monomers. This was achieved by controlling ionic strength, temperature and pH, allowing fibrils with native mimetic D periodic banding structure to assemble spontaneously within the gels. In addition, by providing appropriate aging temperatures and times, mature collagen fibril growth is also realized in the gels in vitro. Mimetic gels were subsequently formed into fibers through a wet-spinning process. These spun fibers were found to preserve the native mimetic D periodic banding and fibrillar structure formed in the initial gels. As a result, the synthetic fibers resemble native tendon. Here structural development within the gel samples and fibers as a function of processing was analyzed by scanning electron microscopy (SEM). Results in this study also show a potentially new route for the fabrication of synthetic collagen fibers mimicking tendon, which may find applications as engineered tissues or scaffolding materials.

Bradykinin augments fibroblast-mediated contraction of released collagen gels

2001 ◽  
Vol 281 (1) ◽  
pp. L164-L171 ◽  
Author(s):  
Tadashi Mio ◽  
Xiangde Liu ◽  
Myron L. Toews ◽  
Yuichi Adachi ◽  
Debra J. Romberger ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Type I Collagen ◽  
Inflammatory Diseases ◽  
Fetal Lung ◽  
Lung Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Collagen Gels ◽  
Protein Kinase C Inhibitors

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10−9 and 10−8 M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.

Microaligned collagen matrices by hydrodynamic focusing: controlling the pH-induced self-assembly

2005 ◽  
Vol 898 ◽  
Author(s):  
Sarah Köester ◽  
Jennie B Leach ◽  
Thomas Pfohl ◽  
Joyce Y Wong
Keyword(s):  
Self Assembly ◽  
Type I Collagen ◽  
Polarized Light ◽  
Type I ◽  
Hydrodynamic Focusing ◽  
Microfluidic Mixing ◽  
Ph Gradients ◽  
The Hierarchical Structure ◽  
Local Ph

AbstractThe hierarchical structure of type I collagen fibrils is a key contributor to the mechanical properties of the extracellular matrix (ECM). It is known that the process of in vitro fibrillogenesis strongly depends on the pH of the collagen solution. To date, there are few methods available for precisely controlling and investigating the dependence of collagen fibril assembly on the local pH. The objective of this work was to create highly defined pH gradients to systematically determine the effects of local pH on microscale collagen fibrillogenesis and alignment. We use a microfluidic mixing device to create a diffusion controlled pH gradient, which in turn initiates the self-assembly and concurrent flow-alignment of soluble collagen. Finite element method simulations of the hydrodynamic and diffusive phenomena are used to calculate the local concentrations of the components involved in the reaction. We develop a model to analytically calculate the local pH in the microfluidic device from these concentrations. A comparison with the experimental results from polarized light microscopy are in good agreement with the simulations.

351 POLARIZED LIGHT MICROSCOPY: DETECTION OF MICROTUBULES AND ITS EFFECTS ON THE VIABILITY OF IN VITRO-MATURED PORCINE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 332 ◽  
Author(s):  
I. Molina ◽  
M. Muñoz ◽  
C. Díez ◽  
E. Gómez ◽  
E. A. Martínez ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
Developmental Competence ◽  
Meiotic Spindle ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Oocyte Developmental Competence ◽  
Blastocyst Rate

The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) is used as a tool in human and, recently, in farm animals assisted reproductive technologies. PLM could be useful for a non-invasive evaluation of the meiotic spindle. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein within in vitro-matured porcine oocytes and to examine the effects of PLM on the oocyte developmental competence. Cumulus-oocyte complexes from slaughterhouse ovaries were matured in vitro for 42 h as described by Gil et al. (2004 Theriogenology 62, 544-552). In the first experiment, a total of 97 oocytes from 6 replicates were placed individually in 10-μL drops of TCM-199-Hepes-FCS in a glass Petri dish. PLM was used to detect the presence of polymerized protein which could be forming a meiotic spindle. The presence of polymerized protein and a meiotic spindle was confirmed in individual oocytes by inmunostaining and chromatin detection as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191-201). In the second experiment, a total of 160 oocytes from 4 replicates were exposed or not (controls) to PLM for 10 minutes. Thereafter, the oocytes were parthenogenetically activated and cultured in vitro. Cleavage rate, total blastocyst rate, expanded blastocyst rate on Day 7 and total cell numbers in expanded blastocysts were assessed. Data were analyzed by GLM procedure of SAS. There was a positive correlation (r = 1; P < 0.0001) between the signal obtained by PLM and the presence of microtubule-polymerized protein as confirmed by inmunostaining. A positive PLM signal was detected in 98.9% of the oocytes. A barrel-shape spindle was observed in 94.8% of the individual samples by inmunostaining and all of these oocytes were positive to PLM. Moreover, oocytes exposed to PLM did not differ significantly from controls on cleavage rate (83.7 ± 1.5 v. 84.4 ± 1.5), total blastocyst rate (36.9 ± 3.6 v. 41.2 ± 3.6) and expanded blastocyst rate on Day 7 (21.9 ± 1.7 v. 26.2 ± 1.7), respectively. There were also no differences in total cell numbers counted in expanded blastocysts (32.8 ± 2.6 v. 35.6 ± 2.5). These results indicate that polarized light microscopy did not exert detrimental effects on porcine oocyte developmental competence and it seems an efficient system to detect polymerized protein in in vitro-matured porcine oocytes. Grant support: INIA: RZ2007-00013-00-00. I. Molina, M. Muñoz, B. Trigal and D. Martín are sponsored by INIA, RYC08-03454, Cajastur and PTA2007-0268-I, respectively.

262 MEIOTIC SPINDLE CONFORMATION ASSESSMENT BY POLARIZED LIGHT MICROSCOPY IN SHEEP AND GOAT OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 279
Author(s):  
J. N. Caamaño ◽  
F. Cuadrado ◽  
C. Díez ◽  
M. Muñoz ◽  
D. Martín ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Linear Models ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
Meiotic Spindle ◽  
System Research ◽  
Grant Support ◽  
Meiotic Spindles ◽  
The Mean

Polarized light microscopy (PLM) allows detection of microtubule-polymerized protein in in vitro-matured sheep and goat oocytes (Caamaño et al. 2011 Reprod. Fertil. Dev. 23, 226–227). Spindle birefringence, measured as the mean retardance value, has been proposed as a marker of meiotic spindle conformation in humans and mice. Because the conformation of the meiotic spindle cannot be readily assessed by PLM, in this study we aimed to measure the mean retardance value of normal and abnormal meiotic spindles from in vitro-matured prepubertal sheep and goat oocytes. Oocytes were matured in vitro for 27 h and were then individually assessed by PLM (Oosight System, Research Instruments Ltd., Falmouth, Cornwall, UK), and the mean retardance value was analysed in meiotic spindles using specific software (Oosight). Meiotic spindle conformation was determined in individual oocytes by immunostaining and chromatin detection, as described by Morató et al. (2008 Mol. Reprod. Dev. 75, 191–201). A barrel-shaped spindle was considered a normal spindle configuration. Only oocytes with meiotic spindles identified by both PLM and immunostaining were used in this study. The experiment was replicated four times. Data were analysed by the general linear models procedure of SAS (SAS Institute Inc., Cary, NC, USA). A normal barrel-shaped spindle was observed in 80.8% of the sheep oocytes (n = 47) and in 83.9% of the goat oocytes (n = 62). In sheep, the mean retardance value in oocytes with a normal meiotic spindle conformation did not differ from that in oocytes with abnormal spindles (4.42 ± 0.26 nm v. 3.92 ± 0.54 nm). Similar results were obtained with goat oocytes with normal (2.94 ± 0.20 nm) v. abnormal spindle conformation (2.77 ± 0.08 nm). These results indicate that the mean retardance value does not distinguish between oocytes with normal and abnormal meiotic spindle conformation, as assessed by subsequent immunostaining. Grant support from INIA-RZ2007-00013-00-00. M. Muñoz was sponsored by RYC08-03454.

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