Demonstration of Osseous Tissue in Quail Embryo by Alizarin Red Technique

Цель исследования - оценка токсического действия сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов на предварительно поврежденную интиму аорты крыс. Методика. Токсическое действие сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов на поврежденную интиму брюшной аорты крыс линии Wistar (n = 10 на группу) оценивали путем их однократного внутривенного введения после баллонной ангиопластики с эксплантацией поврежденного участка аорты через 5 нед. Биоптаты анализировали: 1) классическими гистологическими методами (окрашивание гематоксилин-эозином, ализариновым красным, по Вейгерту-ван Гизону и по Расселлу-Мовату); 2) иммунофлюоресцентным окрашиванием криосрезов (сочетанное окрашивание на CD31 и CD34, на CD31 и α-гладкомышечный актин (α-ГМА), на виментин и α-ГМА, на коллаген IV типа и α-ГМА). Для оценки влияния системного воспаления на КФБ-индуцированную эндотелиотоксичность определяли содержание моноцитарного хемоаттрактантного белка (МСР-1/CCL2) и церулоплазмина в сыворотке крови прооперированных крыс посредством иммуноферментного анализа. Результаты. Сферические кальций-фосфатные бионы и игольчатые кальций-фосфатные бионы вызывали выраженную гипертрофию интимы брюшной аорты в 90% (9 из 10 крыс) и 80% случаев (8 из 10 крыс) соответственно, в то время как частота гипертрофии в группе контрольных крыс составила лишь 10% (1 из 10 крыс). Неоинтима при экспозиции интимы брюшной аорты обоим типам бионов характеризовалась переходом фенотипа клеток мезенхимального ряда с контрактильного (α-ГМА-положительные и виментин-отрицательные гладкомышечные клетки) и неактивного (α-ГМА-отрицательные и виментин-положительные фибробласты) на активный синтетический (α-ГМА- и виментин-положительные клетки), что приводило к формированию значительных количеств экстрацеллюлярного матрикса. Внутривенное введение сферических кальций-фосфатных бионов и игольчатых кальций-фосфатных бионов не приводило к изменению уровней МСР-1/CCL2 и церулоплазмина в сыворотке крови, что свидетельствовало об отсутствии их возможного влияния на развитие системного воспалительного ответа. Заключение. Внутривенное введение кальций-фосфатных бионов после повреждения интимы брюшной аорты крыс путем баллонной ангиопластики вызывает развитие гипертрофии интимы, частота и выраженность которой не зависит от формы кальций-фосфатных бионов и которая характеризуется переходом фенотипа клеток мезенхимального ряда из контрактильного/неактивного на активный синтетический. При этом эндотелиотоксическое действие кальций-фосфатных бионов обусловлено их непосредственным воздействием на эндотелий, а не развитием системного воспаления. Purpose. To compare toxicity of spherical calcium phosphate bions (SCPB) and needle-shaped calcium phosphate bions (NCPB) to injured intima of rat aortas. Methods. Toxicity of SCPB and NCPB to injured abdominal aortas of Wistar rats (n = 10 per group) was evaluated using intravenous administration of the bions after balloon angioplasty. Rats were sacrificed five weeks postoperation, and an injured aortic segment was excised. Tissue preparations were stained with hematoxylin and eosin, alizarin red S, Weigert-van Gieson, and Movat’s pentachrome stains. Selected tissue samples were then examined using combined immunofluorescence staining (CD31/CD34, CD31/α-smooth muscle actin (α-SMA), α-SMA/vimentin, and α-SMA/collagen IV). Possible influence of systemic inflammation on CPB-induced endothelial toxicity was assessed by measuring monocyte chemoattractant protein-1 and ceruloplasmin in rat serum using the enzyme-linked immunosorbent assay. Results. Intravenous administration of SCPB or NCPB provoked intimal hyperplasia in 90% (9 of 10) and 80% (8 of 10) of rats vs. 10% (1 of 10) in the control group. The neointima was characterized by a phenotypic switch of mesenchymal cells, i.e. transition of a contractile (α-SMA-positive, vimentin-negative vascular smooth muscle cells) and quiescent (α-SMA-negative vimentin-positive fibroblasts) to an active synthetic phenotype (double-positive cells), which resulted in deposition of the extracellular matrix. Neither SCPB nor NCPB changed serum levels of pro-inflammatory molecules, МСР-1/CCL2, and ceruloplasmin. Conclusions. Intravenous administration of CPB upon balloon-induced vascular injury caused intimal hyperplasia regardless of the CPB shape. Hyperplasia foci were characterized by a switch of mesenchymal cells from a contractile/quiescent to an active synthetic phenotype. Endothelial toxicity of CPBs was defined by their direct cytotoxic action rather than induction of systemic inflammation.

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MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis

Current Pharmaceutical Design ◽

10.2174/1381612825666190930094019 ◽

2020 ◽

Vol 25 (45) ◽

pp. 4806-4812 ◽

Cited By ~ 3

Author(s):

Zhibo Sun ◽

Fei Wu ◽

Yue Yang ◽

Feng Liu ◽

Fengbo Mo ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Nuclear Translocation ◽

Bone Diseases ◽

Luciferase Reporter ◽

Reporter Assay ◽

Alizarin Red ◽

Over Expression ◽

Negative Effect ◽

Proliferation Ability

Background: MicroRNAs have recently been recognized to be engaged in the development of bone diseases. Objective: This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. Method: The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. Results: ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased β-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. Conclusion: MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

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Polyhedral Oligomeric Silsesquioxane /Platelets Rich Plasma/Gelrite-Based Hydrogel Scaffold for Bone Tissue Engineering

Current Pharmaceutical Design ◽

10.2174/1381612826666200311124732 ◽

2020 ◽

Vol 26 (26) ◽

pp. 3147-3160

Author(s):

Saeedeh Ahmadipour ◽

Jaleh Varshosaz ◽

Batool Hashemibeni ◽

Leila Safaeian ◽

Maziar Manshaei

Keyword(s):

Bone Fractures ◽

Compressive Strain ◽

Gelation Time ◽

Polyhedral Oligomeric Silsesquioxane ◽

Local Drug Delivery ◽

Gelling Agent ◽

Control Group ◽

Hydrogel Scaffold ◽

Alizarin Red ◽

Oligomeric Silsesquioxane

Background: Polyhedral oligomeric silsesquioxane (POSS) is a monomer with silicon structure and an internal nanometric cage. Objective: The purpose of this study was to provide an injectable hydrogel that could be easily located in open or closed bone fractures and injuries, and also to reduce the possible risks of infections caused by bone graft either as an allograft or an autograft. Methods: Various formulations of temperature sensitive hydrogels containing hydroxyapatite, Gelrite, POSS and platelets rich plasma (PRP), such as the co-gelling agent and cell growth enhancer, were prepared. The hydrogels were characterized for their injectability, gelation time, phase transition temperature and viscosity. Other physical properties of the optimized formulation including compressive stress, compressive strain and Young’s modulus as mechanical properties, as well as storage and loss modulus, swelling ratio, biodegradation behavior and cell toxicity as rheometrical parameters were studied on human osteoblast MG-63 cells. Alizarin red tests were conducted to study the qualitative and quantitative osteogenic capability of the designed scaffold, and the cell adhesion to the scaffold was visualized by scanning electron microscopy. Results: The results demonstrated that the hydrogel scaffold mechanical force and injectability were 3.34±0.44 Mpa and 12.57 N, respectively. Moreover, the scaffold showed higher calcium granules production in alizarin red staining compared to the control group. The proliferation of the cells in G4.5H1P0.03PRP10 formulation was significantly higher than in other formulations (p<0.05). Conclusion: The optimized Gelrite/Hydroxyapatite/POSS/PRP hydrogel scaffold has useful impacts on osteoblasts activity, and may be beneficial for local drug delivery in complications including a break or bone loss.

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Biomimetic reduced graphene oxide coated collagen scaffold for in situ bone regeneration

Scientific Reports ◽

10.1038/s41598-021-96271-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sajad Bahrami ◽

Nafiseh Baheiraei ◽

Mostafa Shahrezaee

Keyword(s):

Graphene Oxide ◽

Reduced Graphene Oxide ◽

Cranial Bone ◽

Drying Methods ◽

Porous Scaffolds ◽

Alizarin Red ◽

Reduced Graphene ◽

Coated Collagen

AbstractA variety of bone-related diseases and injures and limitations of traditional regeneration methods require new tissue substitutes. Tissue engineering and regeneration combined with nanomedicine can provide different natural or synthetic and combined scaffolds with bone mimicking properties for implantation in the injured area. In this study, we synthesized collagen (Col) and reduced graphene oxide coated collagen (Col-rGO) scaffolds, and we evaluated their in vitro and in vivo effects on bone tissue repair. Col and Col-rGO scaffolds were synthesized by chemical crosslinking and freeze-drying methods. The surface topography, and the mechanical and chemical properties of scaffolds were characterized, showing three-dimensional (3D) porous scaffolds and successful coating of rGO on Col. The rGO coating enhanced the mechanical strength of Col-rGO scaffolds to a greater extent than Col scaffolds by 2.8 times. Furthermore, Col-rGO scaffolds confirmed that graphene addition induced no cytotoxic effects and enhanced the viability and proliferation of human bone marrow-derived mesenchymal stem cells (hBMSCs) with 3D adherence and expansion. Finally, scaffold implantation into rabbit cranial bone defects for 12 weeks showed increased bone formation, confirmed by Hematoxylin–Eosin (H&E) and alizarin red staining. Overall, the study showed that rGO coating improves Col scaffold properties and could be a promising implant for bone injuries.

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Globular adiponectin inhibits osteoblastic differentiation of vascular smooth muscle cells through the PI3K/AKT and Wnt/β-catenin pathway

Journal of Molecular Histology ◽

10.1007/s10735-021-10012-2 ◽

2021 ◽

Author(s):

Yun Zhou ◽

Li-Long Wei ◽

Rui-Ping Zhang ◽

Cheng-Wu Han ◽

Yongtong Cao

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Osteoblastic Differentiation ◽

Alizarin Red S ◽

Regulation Mechanism ◽

Alizarin Red ◽

Globular Adiponectin

AbstractLipid metabolism is closely related to the improvement of vascular calcification (VC) in chronic kidney disease (CKD). Globular adiponectin (gAd) has been reported to be involved in the development of VC in CKD, but the detailed regulatory role remains unclear. The present study is aimed to investigate the biological function and the underlying regulation mechanism of gAd in the process of VC during CKD. Vascular smooth muscle cells (VSMCs) calcification was determined by Alizarin Red S staining. Protein signaling related with VC was tested by western blotting. The expression and intracellular localization of runt-related transcription factor 2 (Runx2) was detected by immunofluorescence and uraemic rat with VC was established by a two-step nephrectomy. Combined with the results of Alizarin Red S staining, we discovered that β-glycerophosphate (β-Gp)-induced the osteoblastic differentiation of VSMCs was significantly reversed by gAd treatment. Along with the VSMCs calcification and the increase of Runx2 in β-Gp-exposed VSMCs, the activities of protein kinase B (AKT) and Wnt/β-catenin pathway were enhanced, but that were counteracted by the exposure of gAd in rat and human VSMCs. After administration with agonists of the Wnt (SKL2001) and AKT (SC79), there appeared more osteoblastic differentiation and higher expression of Runx2 in gAd-treated VSMCs, but showing lower impact in the presence of SC79 than that in the presence of SKL2001. In the in vivo experiments, intravenous injection of gAd also significantly inhibited VC and Runx2 level in uraemic rat in a dose-dependent manner, possibly through regulating Wnt/β-catenin pathway. This study demonstrates that gAd ameliorates osteoblastic differentiation of VSMCs possibly by blocking PI3K/AKT and Wnt/β-catenin signaling transduction. The findings provide an important foundation for gAd in treating VC in kidney diseases.

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The Effects of Andrographolide on the Enhancement of Chondrogenesis and Osteogenesis in Human Suprapatellar Fat Pad Derived Mesenchymal Stem Cells

Molecules ◽

10.3390/molecules26071831 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1831

Author(s):

Thitianan Kulsirirat ◽

Sittisak Honsawek ◽

Mariko Takeda-Morishita ◽

Nuttanan Sinchaipanid ◽

Wanvisa Udomsinprasert ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Adipogenic Differentiation ◽

Human Mesenchymal Stem Cells ◽

Fat Pad ◽

Alizarin Red ◽

Lineage Differentiation ◽

Expression Of Genes ◽

Dose Dependent ◽

Oil Red O Staining

Andrographolide is a labdane diterpenoid herb, which is isolated from the leaves of Andrographis paniculata, and widely used for its potential medical properties. However, there are no reports on the effects of andrographolide on the human suprapatellar fat pad of osteoarthritis patients. In the present study, our goal was to evaluate the innovative effects of andrographolide on viability and Tri-lineage differentiation of human mesenchymal stem cells from suprapatellar fat pad tissues. The results revealed that andrographolide had no cytotoxic effects when the concentration was less than 12.5 µM. Interestingly, andrographolide had significantly enhanced, dose dependent, osteogenesis and chondrogenesis as evidenced by a significantly intensified stain for Alizarin Red S, Toluidine Blue and Alcian Blue. Moreover, andrographolide can upregulate the expression of genes related to osteogenic and chondrogenic differentiation, including Runx2, OPN, Sox9, and Aggrecan in mesenchymal stem cells from human suprapatellar fat pad tissues. In contrast, andrographolide suppressed adipogenic differentiation as evidenced by significantly diminished Oil Red O staining and expression levels for adipogenic-specific genes for PPAR-γ2 and LPL. These findings confirm that andrographolide can specifically enhance osteogenesis and chondrogenesis of mesenchymal stem cells from human suprapatellar fat pad tissues. It has potential as a therapeutic agent derived from natural sources for regenerative medicine.

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An automated technique for double staining of bone and cartilage in fetal mouse skeletal specimens using alizarin red S and Alcian blue

Biotechnic & Histochemistry ◽

10.1080/10520295.2021.1933179 ◽

2021 ◽

pp. 1-6

Author(s):

Matthew Booth ◽

Nancy Powell ◽

Clare Corfield ◽

Julian M. French

Keyword(s):

Alcian Blue ◽

Alizarin Red S ◽

Double Staining ◽

Alizarin Red ◽

Fetal Mouse ◽

Automated Technique ◽

And Cartilage

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Basal Implant: A Remedy to Restore Resorbed Alveolar Ridges

Dental Journal of Advance Studies ◽

10.1055/s-0041-1731102 ◽

2021 ◽

Author(s):

Prakhar Thakur ◽

Tarun Kalra ◽

Manjit Kumar ◽

Ajay Bansal ◽

Shefali Malik

Keyword(s):

Cortical Bone ◽

Immediate Loading ◽

Osseous Tissue ◽

Alveolar Process ◽

Implant Stability ◽

Implant Placement ◽

Cortical Screw ◽

Functional Differences ◽

Basal Bone ◽

Cortical Implants

AbstractThe conventional crestal implants are used only when there is adequate jawbone height and width. Results of conventional implants are good in patients with healthy bone at the time of treatment, but prognosis gets deteriorated when surgical augmentation of bone is included with implant placement. These augmentation procedures have surgical risks and are costlier to the patients. Patients with atrophied jawbones are given no treatment, until crestal implants are seen as the last option. In this article, the indications for basal implants and functional differences between basal implants and crestal implants have been discussed.Patients with extreme jawbone atrophy do not benefit from crestal implants. The basal bone is the (cortical) osseous tissue of the mandible and maxilla, and lies below the alveolar process, which has a relatively strong and no resorbing framework.Basal osseointegrated and basal cortical screw (BCS) are two types of implants designed to take anchorage from the cortical bone of the jaw. BCS implants have long shafts and can be placed immediately in the socket after extraction and provided with immediate loading within 72 hours of implant placement. Basal implants are also called bicortical or cortical implants as they utilize the cortical portion of the jawbones for anchorage and implant stability. The basal bone has better quality and quantity of cortical bone for retention of these unique and highly advanced implants. The other names for these implants are lateral implants or disk implants.

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Targeting EZH2 Ameliorates the LPS-Inhibited PDLSC Osteogenesis via Wnt/β-Catenin Pathway

Cells Tissues Organs ◽

10.1159/000511702 ◽

2021 ◽

pp. 1-9

Author(s):

Mosha Cheng ◽

Qing Zhou

Keyword(s):

Bone Morphogenetic Protein 2 ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Protein Levels ◽

Inflammatory Conditions ◽

Morphogenetic Protein ◽

Related Transcription Factor ◽

Underlying Mechanisms ◽

Factor Α ◽

Osteoblast Maturation

As a histone methyltransferase, enhancer of zeste homolog 2 (EZH2), suppresses osteoblast maturation and is involved in inflammation. However, the role of EZH2 in human periodontal ligament stem cells (PDLSCs) under inflammation still needs to be further investigated. This study aimed to identify the underlying mechanisms and explore the function of EZH2 in PDLSC osteogenesis under inflammation. PDLSCs were treated with sh-EZH2, DZNep or DKK1 under inflammation. The alkaline phosphatase (ALP) activity, alizarin red staining, and osteogenesis-related protein levels were analyzed. Lipopolysaccharide (LPS)-induced inflammation restrained osteogenic differentiation. Under inflammation, the upregulation of EZH2 suppressed the expression of osteogenic markers, including osteocalcin, runt-related transcription factor 2, and bone morphogenetic protein-2, the activity of ALP, and the accumulation of mineralization through the Wnt/β-catenin pathway. EZH2 knockdown inhibited the levels of proinflammatory cytokines such as interleukin-6 and tumor necrosis factor-α. These results suggested that LPS-induced overexpression of EZH2 suppressed PDLSC osteogenesis under inflammatory conditions through the Wnt/β-catenin pathway. These findings give new insights into the physiological differentiation and pathological inflammation of PDLSC osteogenesis, and provide an underlying therapeutic target for periodontitis.

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Colocalization of Erythrocytes and Vascular Calcification in Human Atherosclerosis: A Systematic Histomorphometric Analysis

TH Open ◽

10.1055/s-0041-1725042 ◽

2021 ◽

Vol 05 (02) ◽

pp. e113-e124

Author(s):

Elsa Wilma Böhm ◽

Maria Pavlaki ◽

Georgios Chalikias ◽

Dimitrios Mikroulis ◽

George S. Georgiadis ◽

...

Keyword(s):

Calcium Phosphate ◽

Aortic Valve ◽

Carotid Artery ◽

Vascular Tissue ◽

Osteoblastic Differentiation ◽

Aortic Aneurysms ◽

Vascular Lesions ◽

Erythrocyte Membranes ◽

Intraplaque Hemorrhage ◽

Alizarin Red

Abstract Background Intimal calcification typically develops in advanced atherosclerosis, and microcalcification may promote plaque progression and instability. Conversely, intraplaque hemorrhage and erythrocyte extravasation may stimulate osteoblastic differentiation and intralesional calcium phosphate deposition. The presence of erythrocytes and their main cellular components (membranes, hemoglobin, and iron) and colocalization with calcification has never been systematically studied. Methods and Results We examined three types of diseased vascular tissue specimens, namely, degenerative aortic valve stenosis (n = 46), atherosclerotic carotid artery plaques (n = 9), and abdominal aortic aneurysms (n = 14). Biomaterial was obtained from symptomatic patients undergoing elective aortic valve replacement, carotid artery endatherectomy, or aortic aneurysm repair, respectively. Serial sections were stained using Masson–Goldner trichrome, Alizarin red S, and Perl's iron stain to visualize erythrocytes, extracelluar matrix and osteoid, calcium phosphate deposition, or the presence of iron and hemosiderin, respectively. Immunohistochemistry was employed to detect erythrocyte membranes (CD235a), hemoglobin or the hemoglobin scavenger receptor (CD163), endothelial cells (CD31), myofibroblasts (SMA), mesenchymal cells (osteopontin), or osteoblasts (periostin). Our analyses revealed a varying degree of intraplaque hemorrhage and that the majority of extravasated erythrocytes were lysed. Osteoid and calcifications also were frequently present, and erythrocyte membranes were significantly more prevalent in areas with calcification. Areas with extravasated erythrocytes frequently contained CD163-positive cells, although calcification also occurred in areas without CD163 immunosignals. Conclusion Our findings underline the presence of extravasated erythrocytes and their membranes in different types of vascular lesions, and their association with areas of calcification suggests an active role of erythrocytes in vascular disease processes.

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