Beneficial Effects of Chondroitinase ABC Release From Lipid Microtubes Encapsulated in Chondrocyte-Seeded Hydrogel Constructs

2011 ◽  
Author(s):  
Grace D. O’Connell ◽  
Clare Gollnick ◽  
Gerard A. Ateshian ◽  
Ravi V. Bellamkonda ◽  
Clark T. Hung
Keyword(s):  
Enzyme Release ◽  
Compressive Properties ◽  
Hydrogel Scaffold ◽  
Beneficial Effects ◽  
Agarose Hydrogel ◽  
Vitro Development ◽  
Engineered Cartilage ◽  
Over Time

Tissue-engineered cartilage using a hydrogel scaffold is capable of achieving native compressive properties and glycosaminglycan (GAG) content [1]; however, promoting collagen growth towards native values has been challenging. As the cells in the cartilage constructs deposit matrix over time in culture, transport of nutrients to the construct center becomes increasingly hindered [2]. Digestion of mature tissue engineered constructs with chondroitinase (chABC) temporarily suppresses the GAG content, allowing an increase in the collagen content and eventually improving the mechanical properties after GAG content recovers [1]. However, adding chABC into the feeding media limits its effectiveness to the construct’s periphery, reflecting enzyme diffusion gradients. Additionally, long-term use of chABC, without re-application, is limited since its enzymatic activity degrades within 5 days at 37°C [3]. Lee and co-workers have developed a method for delivering thermostabilized chABC using sugar trehalose and hydrogel-microtubes for applications desiring extended enzyme release [4]. Lipid microtubes loaded with thermostabilized chABC may be incorporated into an agarose hydrogel scaffold to provide long-term release of the enzyme uniformly throughout the construct [3]. The objective of this study was to test the hypothesis that chABC-filled microtubes will enhance in vitro development of engineered cartilage.

Lipid Mictrotubes as a Nutrient Reservoir or Enzyme Delivery Vehicle in Engineered Cartilage

2012 ◽  
Author(s):  
Grace D. O’Connell ◽  
Clare Gollnick ◽  
Gerard A. Ateshian ◽  
Ravi V. Bellamkonda ◽  
Clark T. Hung
Keyword(s):  
Compressive Properties ◽  
Delivery Vehicle ◽  
Hydrogel Scaffold ◽  
Collagen Production ◽  
Injury Repair ◽  
Agarose Hydrogel ◽  
Spinal Chord ◽  
Nutrient Diffusion ◽  
Enzyme Delivery ◽  
Engineered Cartilage

Tissue-engineered cartilage using a hydrogel scaffold is capable of achieving native compressive properties and glycosaminglycan (GAG) content [1]. However, these tissues are limited in their collagen production and closer inspection of the localized mechanical properties demonstrates that mature constructs consist of a stiffer periphery region surrounding a softer core [1, 2]. Nutrient diffusion becomes increasingly more challenging as the cells in the construct periphery deposit extracellular matrix. Altering the scaffold porosity by adding microscopic porogens can improve the nutrient diffusion into the center of the construct [3]. Furthermore, chondroitinase ABC (chABC) has been shown to improve collagen production of mature engineered cartilage (i.e. tissue cultured for 2–4 weeks before chABC digestion). Lipid microtubes, designed to slowly release chABC for spinal chord injury repair can be incorporated into our agarose hydrogel scaffold in a chABC-loaded or unloaded form. The objective of this study was to explore the use of lipid microtubes in our scaffold as a tubular porogen and as a vehicle to deliver chABC throughout the scaffold to improve nutrient diffusion and collagen production into our engineered constructs.

50 Long-term Motility of Semen Following Breeding Soundness Exams for Yearling Beef Bulls

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 152-152
Author(s):  
Garrett R Seltzer ◽  
Ashley R Hartman ◽  
Sharon K Tucker ◽  
David M Grieger
Keyword(s):  
Gradual Decrease ◽  
Future Research ◽  
Motile Sperm ◽  
Biological Factors ◽  
Ph Values ◽  
Scrotal Circumference ◽  
Breeding Soundness ◽  
Over Time

Abstract To find an in vitro predictor of in vivoM/em> semen motility prompted this study. Our objective was to evaluate semen motility for an 8-hour period immediately following a breeding soundness exam. Ejaculates from 52 Angus and 56 Charolais bulls were evaluated. Motility, morphology, scrotal circumference and pH of ejaculate were evaluated at the time of collection. Ejaculates were then extended using a one to one ratio and incubated in a water bath held at 37 degrees Celsius and evaluated hourly. Motility was evaluated hourly for 8 hours, or until motility of the sample reached zero. Data were analyzed for breed and hourly effects using the GLIMMIX procedure of SAS. There was statistical evidence for difference (P < 0.0001) between breeds for motility over time. Angus ejaculates had higher pH values than Charolais ejaculates showing an association between breed and pH (6.82 vs 6.76, respectively). Primary spermatozoa abnormalities were greater (P < 0.0001) for Angus bulls compared to Charolais bulls (13.33% vs. 10.91%, respectively). Scrotal circumference between breeds tended to be different (P < 0.07), with Charolais bulls having a larger scrotal circumference compared to Angus bulls (38.29 vs. 38.03 centimeters, respectively). There was no difference (P > 0.05) between breeds for secondary abnormalities. There was a significant interaction (P < 0.01) between breed and time of motility measurement. Angus bull’s motility decreased drastically until hour 4, it then had a more gradual decrease until hour 8. Charolais bulls had a more gradual decrease in the percentage of motile sperm over time. In conclusion, there was evidence for difference between breeds for pH, primary spermatozoa abnormalities, and long-term motility, and a scrotal tendency. Understanding the effects of breed and individual biological factors may help producers adjust BSE expectations and lead to future research in long term semen motility.

scholarly journals Bone Regeneration by Novel Bioactive Glasses Containing Strontium and/or Magnesium: A Preliminary In-Vivo Study

Materials ◽  
2018 ◽  
Vol 11 (11) ◽  
pp. 2223 ◽  
Author(s):  
Devis Bellucci ◽  
Valeria Cannillo ◽  
Alexandre Anesi ◽  
Roberta Salvatori ◽  
Luigi Chiarini ◽  
...  
Keyword(s):  
Statistical Significance ◽  
Formation Processes ◽  
Complete Dissolution ◽  
In Vitro Bioactivity ◽  
Bioactive Glasses ◽  
Beneficial Effects ◽  
Glass Dissolution

In this work, a set of novel bioactive glasses have been tested in vivo in an animal model. The new compositions, characterized by an exceptional thermal stability and high in vitro bioactivity, contain strontium and/or magnesium, whose biological benefits are well documented in the literature. To simulate a long-term implant and to study the effect of the complete dissolution of glasses, samples were implanted in the mid-shaft of rabbits’ femur and analyzed 60 days after the surgery; such samples were in undersized powder form. The statistical significance with respect to the type of bioactive glass was analyzed by Kruskal–Wallis test. The results show high levels of bone remodeling, several new bone formations containing granules of calcium phosphate (sometimes with amounts of strontium and/or magnesium), and the absence of adverse effects on bone processes due to the almost complete glass dissolution. In vivo results confirming the cell culture outcomes of a previous study highlighted that these novel bioglasses had osteostimulative effect without adverse skeletal reaction, thus indicating possible beneficial effects on bone formation processes. The presence of strontium in the glasses seems to be particularly interesting.

scholarly journals Functional properties of bone marrow-derived MSC-based engineered cartilage are unstable with very long-term in vitro culture

2014 ◽  
Vol 47 (9) ◽  
pp. 2173-2182 ◽  
Author(s):  
Megan J. Farrell ◽  
Matthew B. Fisher ◽  
Alice H. Huang ◽  
John I. Shin ◽  
Kimberly M. Farrell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Vitro Culture ◽  
Functional Properties ◽  
Engineered Cartilage

scholarly journals Delineation of precursors in murine spleen that develop in contact with splenic endothelium to give novel dendritic-like cells

Blood ◽  
2010 ◽  
Vol 115 (18) ◽  
pp. 3678-3685 ◽  
Author(s):  
Jonathan K. H. Tan ◽  
Pravin Periasamy ◽  
Helen C. O'Neill
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Culture System ◽  
In Vitro Development ◽  
Cell Lineages ◽  
Mhc Ii ◽  
Murine Spleen ◽  
Vitro Development

Abstract Hematopoietic cell lineages are best described in terms of distinct progenitors with limited differentiative capacity. To distinguish cell lineages, it is necessary to define progenitors and induce their differentiation in vitro. We previously reported in vitro development of immature dendritic-like cells (DCs) in long-term cultures (LTCs) of murine spleen, and in cocultures of spleen or bone marrow (BM) over splenic endothelial cell lines derived from LTCs. Cells produced are phenotypically distinct CD11bhiCD11cloCD8−MHC-II− cells, tentatively named L-DCs. Here we delineate L-DC progenitors as different from known DC progenitors in BM and DC precursors in spleen. The progenitor is contained within the lineage-negative (Lin)−c-kit+ subset in neonatal and adult spleen. This subset has multipotential reconstituting ability in mice. In neonatal spleen, the progenitor is further enriched within the c-kitlo and CD34+ subsets of Lin−c-kit+ cells. These cells seed cocultures of splenic endothelial cells, differentiating to give L-DCs that can activate T cells. L-DC progenitors are distinguishable from described splenic CD11clo DC precursors and from Fms-like tyrosine kinase 3+ DC progenitors in BM. Overall, this study confirms that LTCs are a physiologically relevant culture system for in vitro development of a novel DC type from spleen progenitors.

scholarly journals Determination of two fluoroquinolones and their combinations with hyaluronan effect in in vitro canine cartilage explants

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6553
Author(s):  
Puntita Siengdee ◽  
Waranee Pradit ◽  
Siriwadee Chomdej ◽  
Korakot Nganvongpanit
Keyword(s):  
Adverse Effects ◽  
Interleukin 1 ◽  
Expression Patterns ◽  
Cartilage Explants ◽  
Beneficial Effects ◽  
Regulated Expression ◽  
Safranin O

Background Previous studies reported the effect of enrofloxacin (Enro) and marbofloxacin (Mar) on cell death and alteration of the key genes involved in catabolic and anabolic processes and demonstrated the beneficial effects of hyaluronan (HA) combined with fluoroquinolones (FQs) on primary canine chondrocytes. This study further determines the effects of these treatments on canine cartilage explants in both normal and interleukin-1 beta (IL-1β)-stimulated conditions. Methods We examined sulfate glycosaminoglycan (s-GAG) release, uronic acid (UA) content, and safranin-O staining, as well as the expression patterns of inflammatory, extracellular matrix (ECM) component and enzymes. Results Enro treatment alone effectively stimulated proteoglycan anabolism by increasing UA content and glycosaminoglycans (GAGs) in normal and pre-IL-1β-stimulated explant, whereas Mar showed opposite results. The combination of HA and FQs increased s-GAG release and UA content in normal explants in addition to effective down-regulated expression of MMP3. HA reduced the adverse effects of Mar by enhancing UA and GAG contents in both normal and pre-IL-1β-explants. Moreover, HA effectively induced HAS1and ACANup-regulation and reduced MMP9, TNF, PTGS2,and NFKB1 expression for a long term. Discussion Our results suggest the direct effects of Enro and Mar may selectively stimulate the conditioned explants to express MMP-codinggenes and promote gene expression involved in matrix production, pro-inflammatory cytokines, and cell degradation in different directions. HA successfully reduced the adverse effects of FQs by enhancing s-GAG and UA contents and down-regulated expression of MMPs.

67 A HOLLOW FIBER VITRIFICATION METHOD ENABLES CRYOBANKING OF IN VITRO-MATURATION/IN VITRO-FERTILIZATION-DERIVED TRANSGENIC PIG EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 181
Author(s):  
M. Maehara ◽  
H. Matsunari ◽  
K. Honda ◽  
K. Nakano ◽  
Y. Takeuchi ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Hollow Fiber ◽  
Production Efficiency ◽  
Sucrose Solution ◽  
Transgenic Pig ◽  
In Vitro Development ◽  
Vitro Development

We have recently developed a novel high-performance embryo cryopreservation method: the hollow fiber vitrification (HFV) method (Matsunari et al. 2012 J. Reprod. Dev., in press). In this study, we aimed to demonstrate the utility of the HFV method for the cryopreservation of transgenic pig embryos produced by in vitro oocyte maturation/fertilization (IVM/IVF). In vitro-matured oocytes were inseminated with cryopreserved epididymal sperm (Kikuchi et al. 1998 Theriogenology 50, 615–623) from a transgenic pig carrying the humanized Kusabira-Orange gene (Matsunari et al. 2008 Cloning Stem Cell 10, 313–323) and then cultured for 96 h. Morulae with normal morphology were divided into the vitrification and nonvitrification groups. The vitrification of embryos was performed by the HFV method using 20-mM HEPES-buffered TCM199 containing 20% calf serum as a base medium. Cellulose acetate hollow fibers (25 mm), each containing 10 to 20 embryos, were placed in an equilibration solution containing 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 5 to 7 min and were then placed for 1 min in the vitrification solution containing 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5 M sucrose. The embryos were then vitrified by immersion in liquid nitrogen and held there for 5 to 10 min. The embryos were warmed by immersing the hollow fiber in a 1-M sucrose solution at 38.5°C, followed by a stepwise dilution of the cryoprotectants using 0.5-M sucrose solution (3 min) and the base medium (10 min). Vitrified and nonvitrified embryos were cultured for 40 h, and their development into blastocysts was evaluated. The in vitro development of vitrified embryos to the blastocyst stage was compared with that of the nonvitrified controls on Day 6. In the embryo-transfer experiments, blastocysts at either Day 5 or Day 6 from both the vitrification and nonvitrification groups were transferred to 3 recipient gilts per group (25–32 blastocysts/gilt), and their development through farrowing was compared. To test long-term preservation, some of the vitrified morulae were kept in liquid nitrogen for 43 days, and their development to Day 30 fetuses was evaluated after transfer to an additional recipient. The differences in proportional data between the 2 groups were analyzed with the χ2-test. Of the 393 putative zygotes obtained by IVM/IVF, 169 (43.0%) developed into morulae. In vitro development of the vitrified morulae to blastocysts (66/85, 77.6%) was comparable with that of the nonvitrified morulae (67/84, 79.8%, not significant: NS). The embryo-transfer experiments resulted in pregnancy in all 6 of the recipients. The production efficiency of piglets (piglets/embryos transferred) was 17/88 (19.3%) for the vitrification group and 27/88 (27.7%, NS) for the nonvitrification group. Approximately 50% of the offspring in both groups were transgenic. Long-term cryopreservation using the HFV method resulted in similar piglet production efficiency (7 piglets produced out of 32 embryos transferred). This study demonstrated for the first time that the HFV method effectively cryopreserves IVM/IVF-derived transgenic pig embryos. Supported by the JST CREST program.

Bone Morphogenetic Proteins-2, -12, and -13 Modulate in Vitro Development of Engineered Cartilage

2002 ◽  
Vol 8 (4) ◽  
pp. 591-601 ◽  
Author(s):  
K.J. Gooch ◽  
T. Blunk ◽  
D.L. Courter ◽  
A.L. Sieminski ◽  
G. Vunjak-Novakovic ◽  
...  
Keyword(s):  
Bone Morphogenetic Proteins ◽  
In Vitro Development ◽  
Vitro Development ◽  
Engineered Cartilage

Evidence for paracrine/autocrine regulation of GLP-1-producing cells

2013 ◽  
Vol 305 (10) ◽  
pp. C1041-C1049 ◽  
Author(s):  
Camilla Kappe ◽  
Qimin Zhang ◽  
Jens J. Holst ◽  
Thomas Nyström ◽  
Åke Sjöholm
Keyword(s):  
Cell Viability ◽  
Insulin Signaling ◽  
Dipeptidyl Peptidase 4 ◽  
Beneficial Effects ◽  
Novel Drugs ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1

Glucagon-like peptide-1 (GLP-1), secreted from gut L cells upon nutrient intake, forms the basis for novel drugs against type 2 diabetes (T2D). Secretion of GLP-1 has been suggested to be impaired in T2D and in conditions associated with hyperlipidemia and insulin resistance. Further, recent studies support lipotoxicity of GLP-1-producing cells in vitro. However, little is known about the regulation of L-cell viability/function, the effects of insulin signaling, or the potential effects of stable GLP-1 analogs and dipeptidyl peptidase-4 (DPP-4) inhibitors. We determined effects of insulin as well as possible autocrine action of GLP-1 on viability/apoptosis of GLP-1-secreting cells in the presence/absence of palmitate, while also assessing direct effects on function. The studies were performed using the GLP-1-secreting cell line GLUTag, and palmitate was used to simulate hyperlipidemia. Our results show that palmitate induced production of reactive oxygen species and caspase-3 activity and reduced cell viability are significantly attenuated by preincubation with insulin/exendin-4. The indicated lipoprotective effect of insulin/exendin-4 was not detectable in the presence of the GLP-1 receptor (GLP-1R) antagonist exendin (9–39) and attenuated in response to pharmacological inhibition of exchange protein activated by cAMP (Epac) signaling, while protein kinase A inhibition had no significant effect. Insulin/exendin-4 also significantly stimulate acute and long-term GLP-1 secretion in the presence of glucose, suggesting novel beneficial effects of insulin signaling and GLP-1R activation on glycemia through enhanced mass of GLP-1-producing cells and enhanced GLP-1 secretion. In addition, the effects of insulin indicate that not only is GLP-1 important for insulin secretion but altered insulin signaling may contribute to an altered GLP-1 secretion.

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