Standardization of a Method for Characterizing Low-Concentration Biogels: Elastic Properties of Low-Concentration Agarose Gels

1999 ◽  
Vol 121 (2) ◽  
pp. 184-187 ◽  
Author(s):  
M. Benkherourou ◽  
C. Rochas ◽  
P. Tracqui ◽  
L. Tranqui ◽  
P. Y. Gume´ry
Keyword(s):  
Extracellular Matrix ◽  
Soft Tissues ◽  
Compression Tests ◽  
Low Concentration ◽  
Mechanical Responses ◽  
Young’S Moduli ◽  
Biological Phenomena ◽  
Low Concentrations ◽  
Gel Technique

Low-concentration biogels, which provide an extracellular matrix for cells in vitro, are involved in a number of important cell biological phenomena, such as cell motility and cell differentiation. In order to characterize soft tissues, which collapse under their own weight, we developed and standardized a new experimental device that enabled us to analyze the mechanical properties of floating biogels with low concentrations, i.e., with values ranging from 2 g/L to 5 g/L. In order to validate this approach, the mechanical responses of free floating agarose gel samples submitted to compression as well as stretching tests were quantified. The values of the Young’s moduli, measured in the range of 1000 to 10,000 Pa, are compared to the values obtained from other experimental techniques. Our results showed indeed that the values we obtained with our device closely match those obtained independently by performing compression tests on an Instron device. Thus, the floating gel technique is a useful tool first to characterize and then to model soft tissues that are used in biological science to study the interaction between cell and extracellular matrix.

scholarly journals Development of a novel hybrid bioactive hydrogel for future clinical applications

2018 ◽  
Vol 33 (3) ◽  
pp. 447-465 ◽  
Author(s):  
Lydia Francis ◽  
Karin V Greco ◽  
Aldo R Boccaccini ◽  
Judith J Roether ◽  
Nicholas R English ◽  
...  
Keyword(s):  
Clinical Application ◽  
Cellular Proliferation ◽  
Soft Tissues ◽  
Structural Integrity ◽  
Mechanical Stability ◽  
Cartilage Tissue ◽  
Compression Tests ◽  
Control Tissue ◽  
Acellular Tissue

Three-dimensional hydrogels are ideal for tissue engineering applications due to their structural integrity and similarity to native soft tissues; however, they can lack mechanical stability. Our objective was to develop a bioactive and mechanically stable hydrogel for clinical application. Auricular cartilage was decellularised using a combination of hypertonic and hypotonic solutions with and without enzymes to produce acellular tissue. Methacryloyl groups were crosslinked with alginate and PVA main chains via 2-aminoethylmathacrylate and the entire macromonomer further crosslinked with the acellular tissue. The resultant hydrogels were characterised for its physicochemical properties (using NMR), in vitro degradation (via GPC analysis), mechanical stability (compression tests) and in vitro biocompatibility (co-culture with bone marrow-derived mesenchymal stem cells). Following decellularisation, the cartilage tissue showed to be acellular at a significant level (DNA content 25.33 ng/mg vs. 351.46 ng/mg control tissue), with good structural and molecular integrity of the retained extra cellular matrix (s-GAG= 0.19 μg/mg vs. 0.65 μg/mg ±0.001 control tissue). Proteomic analysis showed that collagen subtypes and proteoglycans were retained, and SEM and TEM showed preserved matrix ultra-structure. The hybrid hydrogel was successfully cross-linked with biological and polymer components, and it was stable for 30 days in simulated body fluid (poly dispersal index for alginate with tissue was stable at 1.08 and for PVA with tissue was stable at 1.16). It was also mechanically stable (Young’s modulus of 0.46 ± 0.31 KPa) and biocompatible, as it was able to support the development of a multi-cellular feature with active cellular proliferation in vitro. We have shown that it is possible to successfully combine biological tissue with both a synthetic and natural polymer and create a hybrid bioactive hydrogel for clinical application.

EFFECTS OF LOW CONCENTRATIONS OF THYROID STIMULATING HORMONE: PHARMACOLOGICAL EXPERIMENTS IN VITRO

1970 ◽  
Vol 64 (1) ◽  
pp. 133-149 ◽  
Author(s):  
M.-L. Desbarats-Schönbaum ◽  
E. A. Sellers ◽  
Malle Laansoo ◽  
Eva Koves ◽  
E. Schönbaum
Keyword(s):  
Guinea Pig ◽  
Thyroid Tissue ◽  
Phosphodiesterase Inhibitor ◽  
Thyroid Stimulating Hormone ◽  
Low Concentration ◽  
Low Concentrations ◽  
Stimulating Hormone

ABSTRACT In guinea pig thyroid tissue incubated at 25°C for forty hours the binding (organification) of iodide appears to be the step most sensitive to TSH. Binding is independent of uptake in this system and physiological doses of TSH can stimulate binding while uptake is partially inhibited. At a suitably low concentration of PTU, the addition of TSH can counteract the effect of this drug. At less than maximal concentrations, the effects of TSH and theophylline (a phosphodiesterase inhibitor) are additive, which supports the hypothesis that TSH acts by stimulating the production of cyclic 3′,5′-AMP. The effect of TSH appears to be exerted equally on any iodide present in the thyroid at the beginning of, or taken up during, incubation in vitro. In this system deiodination has been excluded as a major complicating factor.

A comparison of the acylation of 1-acyl-sn-glycero-3-phosphoinositol and 1-acyl-sn-glycero-3-phosphocholine in neuronal nuclei in vitro using radioactive arachidonate and oleate

1986 ◽  
Vol 64 (1) ◽  
pp. 1-7 ◽  
Author(s):  
R. Roy Baker ◽  
Huu-Yi Chang
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Nuclear Fraction ◽  
Maximal Rate ◽  
Low Concentration ◽  
Neuronal Nuclei ◽  
Low Concentrations ◽  
High Concentrations

A neuronal nuclear fraction (N1) was isolated from cerebral cortices of 15-day-old rabbits. Samples of N1 were incubated with a radioactive fatty acid ([3H]arachidonate or [14C]oleate), acylation cofactors, and 1-acyl-sn-glycero-3-phosphoinositol (1-acyl-GPI) or 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC). In competition studies, both radioactive fatty acids were incubated with one lysophospholipid or the two lysophospholipids were incubated with one radioactive fatty acid. Using [3H]arachidonate and one lysophosphoglyceride, a maximal rate of incorporation into phosphatidylinositol (PI) was found at a relatively low concentration of 1-acyl-GPI (10 μM), while increasing rates of incorporation into phosphatidylcholine (PC) were seen with increasing concentrations of 1-acyl-GPC (to 65 μM). At low concentrations of lysophosphoglyceride (≤ 25 μM) the rate of arachidonate incorporation into PI greatly exceeded rates of arachidonate incorporation into PC. This higher rate of arachidonate incorporation into PI was also seen in incubations where both lysophospholipids were present. For oleate, greater rates of incorporation into PC were found in comparison with rates of labelling of PI in assays using relatively high concentrations of one or both lysophospholipids. When comparing arachidonate and oleate, in assays with one or both fatty acids, the polyunsaturate showed at least threefold higher rates of incorporation into PI. For PC labelling higher rates of arachidonate incorporation were evident at the higher concentrations of 1-acyl-GPC and the superiority over oleate was not as marked as that seen in PI labelling.

Fabricated HyalS Micropatterns and Surface Guidance of NCTC 2544 Continuous Cell Line: An in Vitro Study

2002 ◽  
Vol 25 (9) ◽  
pp. 892-898 ◽  
Author(s):  
M. Mattioli-Belmonte ◽  
F. Gabbanelli ◽  
T. Casoli ◽  
A. Delfino ◽  
F. Giantomassi ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Soft Tissues ◽  
In Vitro Study ◽  
Repair Process ◽  
Molecular Volume ◽  
Low Concentrations ◽  
Micrometer Size ◽  
Cytoskeletal Elements

Surface topography is important in establishing tissue organisation adjacent to implants, smooth surfaces generally being associated with fibrous encapsulation. By virtue of its large hydrated molecular volume and its capacity to form molecular matrix, hyaluronic acid can expand the interfibrillar collagen spaces to allow the movement of cells, although it can also hamper their locomotion. Low molecular-weight hyaluronan can also stimulate cell proliferation, especially at low concentrations. The aim of the present work was to evaluate in vitro the growth and migratory behaviour of NCTC 2544 keratinocytes cultured on different materials microstructured with hyaluronic acid or sulfated hyaluronic acid to assess the possibility of using these devices in the repair process of soft tissues. Ultrastructural morphological analyses, morphometric evaluations and detection of cytoskeletal elements were performed. Our observations provide evidence that micrometer-size parallel grooves of hyaluronic acid can influence cell growth behaviour since cells seeded onto the microstructured substrate arranged themselves according to a shape and an orientation that clearly reflected the chemotropism exerted on them by the two forms of acid. These data also highlight the importance of accurate microtexture fabrication. We intend to follow up these in vitro studies with in vivo experimental applications using PET and gelatin substrates structured with HyalS to evaluate wound healing responses, and to extend our investigations of the cytoskeletal modifications induced by different microstructures.

scholarly journals Dose- and time-dependent effects of hyaluronidase on structural cells and the extracellular matrix of the skin

2020 ◽  
Author(s):  
Bettina Alexandra Buhren ◽  
Holger Schrumpf ◽  
Katharina Gorges ◽  
Oliver Reiners ◽  
Edwin Boelke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Matrix ◽  
Wound Healing ◽  
Ex Vivo ◽  
Time Dependent ◽  
Low Concentrations ◽  
High Concentrations ◽  
Dose Dependent ◽  
Skin Samples

Abstract BACKGROUNDHyaluronic acid (hyaluronan; HA) is an essential component of the extracellular matrix (ECM) of the skin. The HA-degrading enzyme hyaluronidase (HYAL) is critically involved in the HA-metabolism. Yet, only little information is available regarding the skin´s HA-HYAL interactions on the molecular and cellular levels.To analyze the dose- and time-dependent molecular and cellular effects of HYAL on structural cells and the HA-metabolism in the skin.METHODSChip-based, genome-wide expression analyses (Affymetrix® GeneChip PrimeView™ Human Gene Expression Array), quantitative real-time PCR analyses, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (DAB), and in vitro wound healing assays were performed to assess dose-dependent and time-kinetic effects of HA and HYAL (bovine hyaluronidase, Hylase Dessau) on normal human dermal fibroblasts (NHDF), primary human keratinocytes in vitro and human skin samples ex vivo.RESULTSGenome-wide expression analyses revealed an upregulation of HA synthases (HAS) up to 1.8-fold change in HA- and HYAL-treated NHDF. HA and HYAL significantly accelerated wound closure in an in vitro model for cutaneous wound healing. HYAL induced HAS1 and HAS2 mRNA gene expression in NHDF. Interestingly, low concentrations of HYAL (0.015 U/ml) resulted in a significantly higher induction of HAS compared to moderate (0.15 and 1.5 U/ml) and high concentrations (15 U/ml) of HYAL. This observation corresponded to increased concentrations of HA measured by ELISA in conditioned supernatants of HYAL-treated NHDF with the highest concentrations observed for 0.015 U/ml of HYAL. Finally, immunohistochemical analysis of human skin samples incubated with HYAL for up to 48 hrs ex vivo demonstrated that low concentrations of HYAL (0.015 U/ml) led to a pronounced accumulation of HA, whereas high concentrations of HYAL (15 U/ml) reduced dermal HA-levels.CONCLUSIONSHYAL is a bioactive enzyme that exerts multiple effects on the HA-metabolism as well as on the structural cells of the skin. Our results indicate that HYAL promotes wound healing and exerts a dose-dependent induction of HA-synthesis in structural cells of the skin. Herein, interestingly the most significant induction of HAS and HA were observed for the lowest concentration of HYAL.

scholarly journals In-vitro and in-vivo comparisons of high versus low concentrations of inhaled epoprostenol to adult intubated patients

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jie Li ◽  
Ashley E. Augustynovich ◽  
Payal K. Gurnani ◽  
James B. Fink
Keyword(s):  
Pulmonary Hypertension ◽  
Clinical Study ◽  
In Vitro Study ◽  
Vitro Study ◽  
Invasive Ventilation ◽  
High Concentration ◽  
Low Concentration ◽  
Low Concentrations ◽  
Clinical Scenarios

Abstract Background Inhaled epoprostenol (iEPO) has been shown to reduce pulmonary artery pressure and improve oxygenation. iEPO is mainly delivered via a syringe pump with feed tubing connected to a vibrating mesh nebulizer with high or low formulation concentration delivery. Methods An in vitro study and a two-period retrospective case–control study were implemented. The in vitro study compared iEPO delivery via invasive ventilation at low concentrations of 7.5, and 15 mcg/mL and high concentration at 30 mcg/mL, to deliver the ordered dose of 30 and 50 ng/kg/min for three clinical scenarios with predicted body weight of 50, 70 and 90 kg. While in the clinical study, adult patients receiving iEPO via invasive ventilation to treat refractory hypoxemia, pulmonary hypertension, or right ventricular failure were included. 80 patients received low concentration iEPO at multiple concentrations (2.5, 7.5, and 15 mcg/mL, depending on the ordered dose) from 2015 to 2017, while 84 patients received high concentration iEPO at 30 mcg/mL from 2018 to 2019. Results In the in vitro study, there were no significant differences in aerosol deposition between high vs low concentrations of iEPO at a dose of 50 ng/kg/min. In the clinical study, age, gender, ethnicity, and indications for iEPO were similar between high and low concentration groups. After 30–120 min of iEPO administration, both delivery strategies significantly improved oxygenation in hypoxemic patients and reduced mean pulmonary arterial pressure (mPAP) for patients with pulmonary hypertension. However, no significant differences of the incremental changes were found between two delivery groups. Compared to low concentration, high concentration delivery group had better adherence to the iEPO weaning protocol (96% vs 71%, p < 0.001), fewer iEPO syringes utilized per patient (5 [3, 10] vs 12 [6, 22], p = 0.001), and shorter duration of invasive ventilation (6 [3, 12] vs 9 [5, 18] days, p = 0.028). Intensive care unit length of stay and mortality were similar between two groups. Conclusion Compared to low concentration delivery of iEPO, high concentration iEPO via a vibrating mesh nebulizer maintained clinical benefits and increased clinician compliance with an iEPO weaning protocol, required less medication preparation time, and shortened duration of invasive ventilation.

Neutrophil migration through in vitro interstitial matrix

1992 ◽  
Vol 50 (1) ◽  
pp. 894-895
Author(s):  
J. Roemer ◽  
S.R. Simon
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cells ◽  
Inflammatory Cell ◽  
Neutrophil Migration ◽  
Culture Conditions ◽  
Aortic Smooth Muscle ◽  
Specific Culture

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.

Biological and biomedical applications of collagenous biomaterials

1990 ◽  
Vol 48 (3) ◽  
pp. 856-857
Author(s):  
Yasushi P. Kato ◽  
Michael G. Dunn ◽  
Frederick H. Silver ◽  
Arthur J. Wasserman
Keyword(s):  
Biomedical Applications ◽  
Soft Tissues ◽  
Collagen Fibers ◽  
Bone Collagen ◽  
Cover Slip ◽  
Fibroblast Migration ◽  
Cellular Expression ◽  
Defect Repair

Collagenous biomaterials have been used for growing cells in vitro as well as for augmentation and replacement of hard and soft tissues. The substratum used for culturing cells is implicated in the modulation of phenotypic cellular expression, cellular orientation and adhesion. Collagen may have a strong influence on these cellular parameters when used as a substrate in vitro. Clinically, collagen has many applications to wound healing including, skin and bone substitution, tendon, ligament, and nerve replacement. In this report we demonstrate two uses of collagen. First as a fiber to support fibroblast growth in vitro, and second as a demineralized bone/collagen sponge for radial bone defect repair in vivo.For the in vitro study, collagen fibers were prepared as described previously. Primary rat tendon fibroblasts (1° RTF) were isolated and cultured for 5 days on 1 X 15 mm sterile cover slips. Six to seven collagen fibers, were glued parallel to each other onto a circular cover slip (D=18mm) and the 1 X 15mm cover slip populated with 1° RTF was placed at the center perpendicular to the collagen fibers. Fibroblast migration from the 1 x 15mm cover slip onto and along the collagen fibers was measured daily using a phase contrast microscope (Olympus CK-2) with a calibrated eyepiece. Migratory rates for fibroblasts were determined from 36 fibers over 4 days.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

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