scholarly journals Low kindlin-3 levels in osteoclasts of kindlin-3 hypomorphic mice result in osteopetrosis due to leaky sealing zones

2021 ◽  
Author(s):  
Sarah Klapproth ◽  
Karsten Richter ◽  
Clara Türk ◽  
Theresa Bock ◽  
Thomas Bromberger ◽  
...  
Keyword(s):  
Electron Micrographs ◽  
Immunohistochemical Staining ◽  
Bone Matrix ◽  
Cell Spreading ◽  
Bone Surface ◽  
Actin Organization ◽  
Functional Assays

Osteoclasts form special integrin-mediated adhesion structures called sealing zones that enable them to adhere to and resorb bone. Sealing zones consist of densely packed podosomes tightly inter-connected by actin fibers. Their formation requires the presence of the hematopoietic integrin regulator kindlin-3. In this study, we investigated osteoclasts and their adhesion structures in kindlin-3 hypomorphic mice expressing only 5-10% of kindlin-3. Low kindlin-3 expression reduces integrin activity, results in impaired osteoclast adhesion and signaling, and delays cell spreading. Despite these defects, in vitro generated kindlin-3-hypomorphic osteoclast-like cells arrange their podosomes into adhesion patches and belts but their podosome and actin organization is abnormal. Remarkably, kindlin-3-hypomorphic osteoclasts form sealing zones when cultured on calcified matrix in vitro and on bone surface in vivo. However, functional assays, immunohistochemical staining and electron micrographs of bone sections showed that they fail to seal the resorption lacunae properly, which is required for secreted proteinases to digest bone matrix. This results in mild osteopetrosis. Our study reveals a new, hitherto understudied function of kindlin-3 as an essential organizer of integrin-mediated adhesion structures, such as sealing zones.

scholarly journals A Novel Human Ada2 Homologue Functions with Gcn5 or Brg1 To Coactivate Transcription

2003 ◽  
Vol 23 (19) ◽  
pp. 6944-6957 ◽  
Author(s):  
Nickolai A. Barlev ◽  
Alexander V. Emelyanov ◽  
Paola Castagnino ◽  
Philip Zegerman ◽  
Andrew J. Bannister ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Chromatin Remodeling ◽  
Adaptor Protein ◽  
Saga Complex ◽  
Yeast Two Hybrid ◽  
Functional Assays ◽  
Two Hybrid ◽  
Histone Acetyltransferase Activity

ABSTRACT In yeast, the transcriptional adaptor yeast Ada2 (yAda2) is a part of the multicomponent SAGA complex, which possesses histone acetyltransferase activity through action of the yGcn5 catalytic enzyme. yAda2, among several SAGA proteins, serves to recruit SAGA to genes via interactions with promoter-bound transcription factors. Here we report identification of a new human Ada2 homologue, hAda2β. Ada2β differs both biochemically and functionally from the previously characterized hAda2α, which is a stable component of the human PCAF (human Gcn5 homologue) acetylase complex. Ada2β, relative to Ada2α, interacted selectively, although not stably, with the Gcn5-containing histone acetylation complex TFTC/STAGA. In addition, Ada2β interacted with Baf57 (a component of the human Swi/Snf complex) in a yeast two-hybrid screen and associated with human Swi/Snf in vitro. In functional assays, hAda2β (but not Ada2α), working in concert with Gcn5 (but not PCAF) or Brg1 (the catalytic component of hSwi/Snf complex), increased transcription via the B-cell-specific transcription factor Pax5/BSAP. These findings support the view that Gcn5 and PCAF have distinct roles in vivo and suggest a new mechanism of coactivator function, in which a single adaptor protein (Ada2β) can coordinate targeting of both histone acetylation and chromatin remodeling activities.

Preparation and In Vitro Characterization of Polycaprolactone and Demineralized Bone Matrix Scaffolds

2012 ◽  
Vol 1417 ◽  
Author(s):  
Titilayo Moloye ◽  
Christopher Batich
Keyword(s):  
Demineralized Bone Matrix ◽  
Bone Matrix ◽  
Apatite Formation ◽  
Alizarin Red ◽  
Salt Leaching ◽  
Synthetic Materials ◽  
Calcium And Phosphorus ◽  
Demineralized Bone

ABSTRACTCylindrical porous polycaprolactone (PCL) scaffolds containing 25, 35, and 50 wt% demineralized bone matrix (DBM) were fabricated using a salt-leaching method for application in bone engineering. In the present work, PCL-DBM scaffolds were monitored for calcium and phosphorus deposition in both deionized (DI) water and simulated body fluid (SBF) for time periods of 5, 10, 15, and 20 days at 37°C under constant rotation. An in vitro assessment of the bioactivity of synthetic materials using SBF under physiological conditions can be used as a barometer of scaffold behavior in vivo. DBM, an osteoinductive material, was used to gauge if there was a correlation between the concentration of DBM within a scaffold and the apatite formation on its surface. Biochemical assays, alizarin red S staining, and scanning electron microscopy (SEM) with elemental analysis of calcium and phosphorus were consistent in that they confirmed that PCL scaffolds containing 35 wt% DBM in SBF at 14 days post-immersion showed signs of early apatite formation.

scholarly journals Osteoclast-Derived Extracellular miR-106a-5p Promotes Osteogenic Differentiation and Facilitates Bone Defect Healing

2021 ◽  
Author(s):  
Yutong Wu ◽  
Hongbo Ai ◽  
Yuchi Zou ◽  
Jianzhong Xu
Keyword(s):  
Demineralized Bone Matrix ◽  
Bone Matrix ◽  
Signal Molecules ◽  
Calvarial Defect ◽  
Communication Mode ◽  
Defect Healing ◽  
Bone Defect Healing ◽  
Intercellular Communications

Abstract Small extracellular vesicles (sEVs) are considered to play critical roles in intercellular communications during normal and pathological processes since they are enriched with miRNAs and other signal molecules. In bone remodeling, osteoclasts generate large amounts of sEVs. However, there is very little research about whether and how osteoclast-derived sEVs (OC-sEVs) affect surrounding cells. In our study, microarray analysis identified miR-106a-5p highly enriched in OC-sEV. Further experiments confirmed that OC-sEVs inhibited Fam134a through miR-106a-5p and significantly promoted bone mesenchymal stem cell (BMSC) osteogenic mineralization in vitro. Next, we prepared sEV-modified demineralized bone matrix (DBM) as a repair scaffold, and used a calvarial defect mouse model to evaluate the pro-osteogenic activities of the scaffold. In vivo result indicated DBM modified with miR-106a-5p-sEVs showed an enhanced capacity of bone regeneration. This important finding further emphasizes that sEV-mediated miR-106a-5p transfer play critical roles in osteogenesis and indicate a novel communication mode between osteoclasts and BMSCs.

scholarly journals LOSS OF PROTEINPOLYSACCHARIDES AT SITES WHERE BONE MINERALIZATION IS INITIATED

1972 ◽  
Vol 20 (4) ◽  
pp. 279-292 ◽  
Author(s):  
D. BAYLINK ◽  
J. WERGEDAL ◽  
E. THOMPSON
Keyword(s):  
Calcium Concentration ◽  
Bone Mineralization ◽  
Bone Matrix ◽  
Indirect Evidence ◽  
Experimental Conditions ◽  
Acid Polysaccharide ◽  
Major Acid ◽  
Tibial Cortex

In both ground sections and demineralized frozen sections of the rat tibial cortex, osteoid but not mature bone matrix stained for proteinpolysaccharides with the Alcian Blue and toluidine blue techniques. The loss of proteinpolysaccharide staining occurred precisely at the mineralizing front, which was identified by in vivo lead or procion markers, not only in normal animals but also in animals in which osteoid width was either increasing or decreasing. In vitro, both proteases and saccharidases abolished proteinpolysaccharide staining of osteoid. Critical electrolyte concentration and other procedures indicated that the major acid polysaccharide component in osteoid is chondroitin sulfate. Consistent with these findings, electron microprobe analyses revealed that sulfur concentration was high in osteoid but dropped abruptly as calcium concentration increased at the mineralizing front. The precise synchronization between loss of proteinpolysaccharides and onset of mineralization under various experimental conditions provides strong indirect evidence that the loss of these macromolecules is somehow involved in initiation of mineralization in bone.

scholarly journals Biofunctionalization of porcine-derived collagen matrices with platelet rich fibrin: influence on angiogenesis in vitro and in vivo

2020 ◽  
Vol 24 (10) ◽  
pp. 3425-3436 ◽  
Author(s):  
Sebastian Blatt ◽  
Valentin Burkhardt ◽  
Peer W. Kämmerer ◽  
Andreas M. Pabst ◽  
Keyvan Sagheb ◽  
...  
Keyword(s):  
Growth Factor ◽  
Immunohistochemical Staining ◽  
Collagen Matrices ◽  
Platelet Rich Fibrin ◽  
Angiogenic Potential ◽  
Branching Points ◽  
Growth Factor Release ◽  
Factor Release

Abstract Objectives Porcine-derived collagen matrices (CM) can be used for oral tissue regeneration, but sufficient revascularization is crucial. The aim of this study was to analyze the influence of platelet-rich fibrin (PRF) on angiogenesis of different CM in vitro and in vivo. Materials and methods Three different CM (mucoderm, jason, collprotect) were combined with PRF in a plotting process. Growth factor release (VEGF, TGF-β) was measured in vitro via ELISA quantification after 1,4 and 7 days in comparison to PRF alone. In ovo yolk sac (YSM) and chorion allantois membrane (CAM) model, angiogenic potential were analyzed in vivo with light- and intravital fluorescence microscopy after 24 h, then verified with immunohistochemical staining for CD105 and αSMA. Results Highest growth factor release was seen after 24 h for all three activated membranes in comparison to the native CM (VEGF 24 h: each p < 0.05; TGF-β: each p < 0.001) and the PRF (no significant difference). All activated membranes revealed a significantly increased angiogenic potential in vivo after 24 h (vessels per mm2: each p < 0.05; branching points per mm2: each p < 0.01; vessel density: each p < 0.05) and with immunohistochemical staining for CD105 (each p < 0.01) and αSMA (each p < 0.05). Conclusions PRF improved the angiogenesis of CM in vitro and in vivo. Clinical relevance Bio-functionalization of CM with PRF could easily implemented in the clinical pathway and may lead to advanced soft tissue healing.

scholarly journals The Antitumor Effect of Gekko Sulfated Glycopeptide by Inhibiting bFGF-Induced Lymphangiogenesis

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Xiu-Li Ding ◽  
Ya-Nan Man ◽  
Jian Hao ◽  
Cui-Hong Zhu ◽  
Chang Liu ◽  
...  
Keyword(s):  
Immunohistochemical Staining ◽  
Antitumor Effect ◽  
Inhibitory Effect ◽  
Tube Formation ◽  
Fibroblast Growth ◽  
Lymphatic Endothelial Cells ◽  
Tumor Lymphangiogenesis ◽  
Extracellular Signal

Objective. To study the antilymphangiogenesis effect of Gekko Sulfated Glycopeptide (GSPP) on human lymphatic endothelial cells (hLECs).Methods. MTS was conducted to confirm the antiproliferation effect of GSPP on hLECs; flow cytometry was employed to detect hLECs cycle distribution; the antimigration effect of GSPP on hLECs was investigated by wound healing experiment and transwell experiment; tube formation assay was used to examine its inhibitory effect on the lymphangiogenesis; western blotting was conducted to detect the expression of extracellular signal-regulated kinase1/2 (Erk1/2) and p-Erk1/2 after GSPP and basic fibroblast growth factor (bFGF) treatment. Nude mice models were established to investigate the antitumor effect of GSPP in vivo. Decreased lymphangiogenesis caused by GSPP in vivo was verified by immunohistochemical staining.Results. In vitro, GSPP (10 μg/mL, 100 μg/mL) significantly inhibited bFGF-induced hLECs proliferation, migration, and tube-like structure formation (P<0.05) and antagonized the phosphorylation activation of Erk1/2 induced by bFGF. In vivo, GSPP treatment (200 mg/kg/d) not only inhibited the growth of colon carcinoma, but also inhibited the tumor lymphangiogenesis.Conclusion. GSPP possesses the antitumor ability by inhibiting bFGF-inducing lymphangiogenesis in vitro and in vivo, which may further inhibit tumor lymphatic metastasis.

Osseoinduction Evaluation of Hydroxyapatite and Zinc Containing Hydroxyapatite Granules in Rabbits

2011 ◽  
Vol 493-494 ◽  
pp. 252-257 ◽  
Author(s):  
L. Nascimento ◽  
M. Medeiros ◽  
J. Calasans-Maia ◽  
A. Alves ◽  
Antonella M. Rossi ◽  
...  
Keyword(s):  
Histological Analysis ◽  
Bone Matrix ◽  
Fibrous Tissue ◽  
Particle Diameter ◽  
Inflammatory Cells ◽  
Giant Cells ◽  
Multinucleated Giant Cells ◽  
Ethics Commission

This study investigated the osteoinductive potential of granules of stoichiometric hydroxyapatite (HA) and 0.5% zinc containing hydroxyapatite (ZnHA) in intramuscular (IM) site of rabbit’s abdomen. The biomaterials were both used in granular form, with 75% porosity and particle diameter between 450 and 500μm, sintered at 1100°C. Both materials performed adequately on a multiparametric in vitro cytocompatibility assay, indicating their suitability for in vivo testing. After approval by the Ethics Commission on Teaching and Research in Animals, fifteen rabbits were submitted to general anesthesia, incision and tissue dilatation, and a small site was created for HA (right incision) and ZnHA (left incision) intramuscular implantation. The animals were killed after 2, 4 and 12 weeks for biomaterials and surrounding tissues removal. Histological analysis after 2 weeks revealed the presence of granulation tissue surrounding biomaterials with multinucleated giant cells and no newly formed bone for both materials. After 4 weeks there was fibrous tissue involving the material and few inflammatory cells. Following 12 weeks it was observed the presence of connective tissue surrounding the biomaterial, cellularized enough for the two experimental groups, but it was not observed the presence of bone matrix associated with the biomaterials. We conclude that both biomaterials are cytocompatible and did not present the property of osseoinduction after 12 weeks of implantation.

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