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Poultry Science Jul 2021As the largest single bone, avian sterna are very different from those of mammals in terms of morphology and functions. Moreover, years of artificial selection in...
As the largest single bone, avian sterna are very different from those of mammals in terms of morphology and functions. Moreover, years of artificial selection in poultry led to incomplete sternal ossification at slaughter age, which may cause diseases, sternal injury, and restriction to breast muscle growth. However, in living birds, studies have rarely described the ossification pattern and underlying mechanisms of the sterna. Here, we examined the pattern (timeline, ossification centers, ossification directions, weekly changes of different parts, quantified differences in ossification degree among sexes and parts) and developmental changes (histological structure, gene expression) of postnatal duck sternal ossification. Direct observation and alcian blue and alizarin red staining of whole sterna samples revealed that, duck sterna mainly ossified during 5 to 9 wk old with five ossification centers. These centers and their ossification directions were different from and more complex than the previously studied birds. The weekly changes of sterna and the quantitative analysis of ossification-related traits showed that ossifications in the three parts of duck sterna (sternum body, keel, posterolateral processes) were mutually independent in space and time, meanwhile, the male duck sterna were more late-maturing than the female. The results of hematoxylin-eosin, alcian blue, and toluidine blue stainings and the expression levels of COL2A1, COL10A1, COL1A2, and CTSK together supported that, duck sternal ossification was highly similar to typical endochondral ossification. Furthermore, continuously high expression of MMP13 and SPARC and their significant (P < 0.05) co-expression with COL2A1, COL10A1, COL1A2, and CTSK suggested the importance of MMP13 and SPARC in duck sternal ossification. Taken together, our results may be helpful for the understanding of avian sternal ossification and the improvement of the performance and welfare of poultry from a new perspective.
Topics: Animals; Chickens; Ducks; Female; Gene Expression; Male; Osteogenesis; Sternum
PubMed: 34116350
DOI: 10.1016/j.psj.2021.101112 -
Bioscience Reports Jan 2019Various miRNAs have been reported to regulate the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs); however, whether miR-134 plays a role in...
Various miRNAs have been reported to regulate the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs); however, whether miR-134 plays a role in this biological process remains undetermined. In the present study, we first evaluated the chondrogenic differentiation of BMSCs by Alcian blue staining, and examined the miR-134 expression by quantitative real-time PCR (qRT-PCR) during this process. And miR-134 inhibitor was used to investigate the functions of miR-134 in chondrogenic differentiation of BMSCs by Alcian blue staining, qRT-PCR, and Western blot. Subsequently, the correlation between miR-134 and SMAD6 was assessed via bioinformatics analysis and dual-luciferase reporter assay. Finally, the role of SMAD6 in chondrogenic differentiation of BMSCs was also determined through Alcian blue staining, qRT-PCR, and Western blot. As results showed that miR-134 expression was significantly down-regulated during chondrogenic differentiation, and inhibition of miR-134 obviously promoted chondrogenic differentiation. Dual-luciferase reporter assay indicated that miR-134 could directly target the 3'-UTRs of SMAD6, inhibit miR-134 expression in BMSCs, and up-regulate SMAD6 expression. Moreover, we found that overexpression of SMAD6 significantly promoted chondrogenic differentiation, and that SMAD6-induced promotion of chondrogenic differentiation could be reversed by miR-134 mimics. In conclusion, our findings suggest that miR-134 may act as a negative regulator during chondrogenic differentiation of BMSCs by interacting with SMAD6.
Topics: 3' Untranslated Regions; Animals; Bone Marrow Cells; Cell Differentiation; Mesenchymal Stem Cells; MicroRNAs; Rats, Sprague-Dawley; Smad6 Protein
PubMed: 30135141
DOI: 10.1042/BSR20180921 -
Stem Cells International 2016Objective. To find a convenient and efficient way to isolate MSCs from human menstrual blood and to investigate their biological characteristics, proliferative capacity,...
Objective. To find a convenient and efficient way to isolate MSCs from human menstrual blood and to investigate their biological characteristics, proliferative capacity, and secretion levels. Methods. MSCs were isolated from menstrual blood of 3 healthy women using adherence. Cell immunological phenotype was examined by flow cytometry; the adipogenic, osteogenic, and chondrogenic differentiation of MSCs was examined by Oil-Red-O staining, ALP staining, and Alcian Blue staining, respectively; and the secretion of cytokines, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1), was detected using enzyme-linked immunosorbent assay. Results. MB-MSCs were successfully isolated from human menstrual blood using adherence. They were positive for CD73, CD105, CD29, and CD44, but negative for CD31 and CD45. The differentiated MB-MSCs were positive for ALP staining, Oil-Red-O staining, and Alcian Blue staining. In addition, they could secrete antiapoptotic cytokines, such as VEGF, IGF-1, and HGF. Conclusion. It is feasible to isolate MSCs from human menstrual blood, thus avoiding invasive procedures and ethical controversies. Adherence could be a promising alternative to the density gradient centrifugation for the isolation of MSCs from menstrual blood.
PubMed: 26681948
DOI: 10.1155/2016/3573846 -
Analytical Biochemistry Jun 1986Oligomeric and polymeric fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. A ladder-like series...
Oligomeric and polymeric fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. A ladder-like series of bands is observed, in which adjacent major bands correspond to species differing in chain length by one disaccharide unit. The component species are detected by a combined alcian blue and silver staining protocol. Detection limits are less than 50 ng per band, or approximately 2-5 micrograms total load for polydisperse samples. Densitometry of the stained gel may be used to determine molecular weight averages and distribution. The applicable molecular weight ranges are approximately 4000 to 100,000 for hyaluronate, or 1500 to 40,000 for chondroitin and dermatan sulfate samples of moderate charge density heterogeneity.
Topics: Alcian Blue; Electrophoresis, Polyacrylamide Gel; Glycosaminoglycans; Hyaluronic Acid; Indoles; Molecular Weight; Silver; Staining and Labeling; Viscosity
PubMed: 2425661
DOI: 10.1016/0003-2697(86)90437-9 -
The Saudi Dental Journal Nov 2021Mesenchymal stem cells (MSCs) are extensively used in tissue regenerative procedures. One source of MSCs is the periodontal ligament (PDL) of teeth. Isolation of MSCs...
UNLABELLED
Mesenchymal stem cells (MSCs) are extensively used in tissue regenerative procedures. One source of MSCs is the periodontal ligament (PDL) of teeth. Isolation of MSCs from extracted teeth is reasonably simple, being less invasive and presenting fewer ethical concerns than does the harvesting of MSC's from other sites. The objectives of this study were to isolate and characterize the PDL stem cells (PDLSC) from healthy adults' extracted teeth and then to characterize them by comparing them with bone-marrow derived MSCs (BMMSC).
METHODS
The PDL tissue was scraped from the roots of freshly extracted teeth to enzymatically digest using collagenase. The cells were sub-cultured. Flow-cytometric analysis for the MSC surface-markers CD105, CD73, CD166, CD90, CD34, CD45 and HLA-DR was performed. To confirm the phenotype, total RNA was extracted to synthesize cDNA and which was then subjected to RT-PCR. The gene-expression for Oct4A, Sox2, NANOG and GAPDH was determined by gel-electrophoresis. To assess their multilineage potential, cells were cultured with osteogenic, chondrogenic and adipogenic medium and then stained by Alizarin-red, Alcian-blue and Oil-Red-O respectively. MSCs from the bone-marrow were processed similarly to serve as controls.
RESULTS
The cells isolated from extracted teeth expanded successfully. On flow-cytometric analysis, the cells were positive for CD73, CD90, CD105, CD166 and negative for CD34, CD45 and HLA-DR. The PDLSCs expressed Oct4A, Sox2, and NANOG mRNA with GAPDH expression. Cells cultured in the osteogenic, chondrogenic and adipogenic media stained positive for Alizarin-red, Alcian-blue and Oil- Red-O respectively. The surface marker expression and the trilineage differentiation characteristics were comparable to those of the BMMSCs.
CONCLUSIONS
The periodontal ligament tissue of extracted teeth is a potential source of therapeutically useful MSCs. Harvesting them is not invasive and are a promising source of MSC as the PDLSCs showed characteristics similar to those of the highly regarded MSC's derived from bone-marrow.
PubMed: 34803321
DOI: 10.1016/j.sdentj.2020.04.007 -
Journal of Acupuncture and Meridian... Oct 2012By spraying and injecting Alcian blue into the lateral ventricle, we were able to visualize the network of the nerve primo vascular system above the pia mater of the...
By spraying and injecting Alcian blue into the lateral ventricle, we were able to visualize the network of the nerve primo vascular system above the pia mater of the brain and spine of rats. Staining these novel structures above the pia mater with 4',6-diamidino-2-phenylindole demonstrated that they coexisted in cellular and extracellular DNA forms. The cellular primo node consisted of many cells surrounded by rod-shaped nuclei while the extracellular primo node had a different morphology from that of a general cell in terms of DNA signals, showing granular DNA in a threadlike network of extracellular DNA. Also, differently from F-actin in general cells, the F-actin in the primo vessel was short and rod-shaped. Light and transmission electron microscopic images of the PN showed that the nerve primo vascular system above the pia mater of the brain and spine was a novel dynamic network, suggesting the coexistence of DNA and extracellular DNA. Based on these data, we suggest that a novel dynamic system with a certain function exists above the pia mater of the central nerve system. We also discuss the potential of this novel network system in the brain and spine as related to acupuncture meridians and neural regeneration.
Topics: Acupuncture Points; Alcian Blue; Animals; Blood Vessels; Brain; Brain Chemistry; Female; Meridians; Pia Mater; Rats; Rats, Wistar; Spine; Staining and Labeling
PubMed: 23040102
DOI: 10.1016/j.jams.2012.07.005 -
Pathology Oncology Research : POR Oct 2016Among the scientific communities, there is a convergence of results supporting a direct relationship between dysregulated sialylation and poor prognosis in many human...
Among the scientific communities, there is a convergence of results supporting a direct relationship between dysregulated sialylation and poor prognosis in many human cancers. For this reason, we have retrospectively investigated 169 cases of invasive ductal carcinoma of the breast, coming from female patients aged between 31 and 76 years old. The whole series was subdivided into two prognostic groups: the first group consisted of 138 patients, who showed a post-treatment survival time more than 5 years, while the second group was made up by 31 patients, died within 5 years despite of chemotherapy. All the surgical specimens were fixed in 10 % neutral buffered formalin, paraffin embedded and, then, submitted to routinely haematoxylin/eosin staining and to a further histochemical (Alcian Blue, DDD-Fast Blue B, Mercury Orange), immunohistochemical (ST3GAL5 sialyltransferase, Ki67, c-erbB2, ER, PR) and chemico-elemental characterization. In the 31 cases of breast cancer belonging to the second group, an overexpression of sialomucins and sialyltransferases has been detected. Our results lead us to support that in aggressive chemoresistant breast cancers, the altered expression of sialic acid, due to an uncontrolled sialylation, creates an excessive negative charge on cell membranes, which stimulates repulsion between neoplastic cells and their subsequent access into the blood stream. This event implies an early metastatization and a rapid disease progression with fatal outcome. The early application of Alcian Blue stain on diagnostic biopsies of breast cancer is able to cheaply reveal the sialomucin accumulations, providing for the disease course.
Topics: Adult; Aged; Breast Neoplasms; Cell Membrane; Disease Progression; Drug Resistance, Neoplasm; Female; Humans; Middle Aged; N-Acetylneuraminic Acid; Prognosis; Retrospective Studies; Sialomucins; Sialyltransferases
PubMed: 27037559
DOI: 10.1007/s12253-016-0057-6 -
The Journal of Cell Biology Jun 1965Histochemical reactions and radioautography were used to investigate the sequence of mast cell development in rat embryos. Mast cells arise ubiquitously in and are...
Histochemical reactions and radioautography were used to investigate the sequence of mast cell development in rat embryos. Mast cells arise ubiquitously in and are confined to the loose connective tissue in the embryo. The alcian blue-safranin reaction distinguishes between weakly sulfated and strongly sulfated mucopolysaccharides by a shift from alcian blue to safranin staining. Based on this reaction and morphologic characteristics, four stages were identified. Stage I mast cells are lymphocyte-like cells with cytoplasmic granules which invariably stain blue with the alcian blue-safranin reaction. In Stage II cells the majority of granules are alcian blue-positive, but some safranin-positive granules have appeared. Stage III mast cells are distinguished by a majority of safranin-positive cytoplasmic granules; some alcian blue-positive granules still remain. Stage IV cells contain only safranin-positive granules. Thymidine-H(3) uptake and identification of mitotic figures indicates that mast cells in Stages I and II comprise a mitotic pool while those in Stages III and IV are mitotically inactive. The pattern of S(35)O(4) incorporation and the sequence of appearance of histochemically identifiable mast cell constituents corroborates division of the proliferation and differentiation of embryonic mast cells into the four stages described above. The process of formation of mast cell granules is interpreted as reflecting the synthesis and accumulation of a heparin precursor in alcian blue positive granules followed by the synthesis and accumulation of highly N-sulfated heparin along with mast cell chymase and finally histamine in safranin-positive granules.
Topics: Animals; Autoradiography; Cell Differentiation; Cell Division; Glycosaminoglycans; Heparin; Histamine; Histocytochemistry; In Vitro Techniques; Mast Cells; Peptide Hydrolases; Periodic Acid; Rats; Sulfates; Thymidine
PubMed: 4284572
DOI: 10.1083/jcb.25.3.577 -
Nagoya Journal of Medical Science Nov 2022Primary laryngeal cryptococcosis is an extremely rare infection and presents with non-specific symptoms such as hoarseness or sore throat, resulting in delayed...
Primary laryngeal cryptococcosis is an extremely rare infection and presents with non-specific symptoms such as hoarseness or sore throat, resulting in delayed diagnosis. Here, we report the patient of a 56-year-old female patient with primary laryngeal cryptococcosis, who was being treated with oral and inhaled steroids for rheumatoid arthritis and bronchial asthma. The patient suffered from prolonged hoarseness and sore throat, and endoscopic biopsy was performed several times under local anesthesia, demonstrating only inflammatory cell infiltration. Considering the possibility of laryngeal malignancy, a third biopsy was performed by endoscopic laryngomicrosurgery under general anesthesia. Intraoperative frozen section revealed non-neoplastic laryngeal mucosa with erosion and severe inflammatory cell infiltration. However, we could not confirm the definite diagnosis of the lesion in the intraoperative consultation. Postoperative histopathological examination revealed a small number of yeast-type fungi and a definitive diagnosis was established by special stains including Alcian blue stain. Finally, the patient was diagnosed as primary laryngeal cryptococcosis. Daily oral administration of fluconazole (400 mg/day) was performed for 6 months according to the treatment protocol for pulmonary cryptococcosis. The symptoms gradually improved, and endoscopy revealed no recurrence 6 months post-treatment. Clinicians should consider the possibility of laryngeal cryptococcosis when severe inflammation is found in the larynx and discuss the disease history and pathological results with pathologists more closely.
Topics: Female; Humans; Middle Aged; Laryngeal Neoplasms; Hoarseness; Larynx; Cryptococcosis; Pharyngitis
PubMed: 36544607
DOI: 10.18999/nagjms.84.4.900 -
Tissue Engineering. Part C, Methods Apr 2016To date, the outcomes of cartilage repair have been inconsistent and have frequently yielded mechanically inferior fibrocartilage, thereby increasing the chances of...
To date, the outcomes of cartilage repair have been inconsistent and have frequently yielded mechanically inferior fibrocartilage, thereby increasing the chances of damage recurrence. Implantation of constructs with biochemical composition and mechanical properties comparable to natural cartilage could be advantageous for long-term repair. This study attempted to create such constructs, in vitro, using tissue engineering principles. Bovine synoviocytes were seeded on nonwoven polyethylene terephthalate fiber scaffolds and cultured in chondrogenic medium for 4 weeks, after which uniaxial compressive loading was applied using an in-house bioreactor for 1 h per day, at a frequency of 1 Hz, for a further 84 days. The initial loading conditions, determined from the mechanical properties of the immature constructs after 4 weeks in chondrogenic culture, were strains ranging between 13% and 23%. After 56 days (sustained at 84 days) of loading, the constructs were stained homogenously with Alcian blue and for type-II collagen. Dynamic compressive moduli were comparable to the high end values for native cartilage and proportional to Alcian blue staining intensity. We suggest that these high moduli values were attributable to the bioreactor setup, which caused the loading regime to change as the constructs developed, that is, the applied stress and strain increased with construct thickness and stiffness, providing continued sufficient cell stimulation as further matrix was deposited. Constructs containing cartilage-like matrix with response to load similar to that of native cartilage could produce long-term effective cartilage repair when implanted.
Topics: Animals; Cartilage; Cattle; Chondrogenesis; Collagen Type II; Polyethylene Terephthalates; Stress, Mechanical; Synovial Membrane; Tissue Engineering; Tissue Scaffolds
PubMed: 26850081
DOI: 10.1089/ten.TEC.2015.0351