scholarly journals Evidence of ectokinase-mediated phosphorylation of osteopontin and bone sialoprotein by osteoblasts during bone formation in vitro

1997 ◽  
Vol 323 (3) ◽  
pp. 637-643 ◽  
Author(s):  
XinLi ZHU ◽  
Chun LUO ◽  
Jack M. FERRIER ◽  
Jaro SODEK
Keyword(s):  
Cell Surface ◽  
Bone Cells ◽  
Cell Attachment ◽  
Biological Properties ◽  
Bone Cell ◽  
Bone Sialoprotein ◽  
Surface Proteins ◽  
Pulse Labelling ◽  
Mediate Cell

Osteopontin (OPN) and bone sialoprotein (BSP) are phosphorylated glycoproteins that, together with osteonectin/secreted protein, acidic, rich in cysteine (SPARC) and osteocalcin, comprise the major non-collagen proteins of bone. Although phosphorylation of OPN and BSP, which is known to influence the biological properties of these proteins, has been shown to occur intracellularly, recent studies have demonstrated ectokinase activity in bone cell populations [Mikuni-Takagaki, Kakai, Satoyoshi, Kawano, Suzuki, Kawase and Saito (1995) J. Bone Miner. Res. 10, 231–241]. To determine whether OPN and BSP are phosphorylated by ectokinase activity we have used [γ-32P]ATP and [γ-32P]GTP as cell-impenetrable phosphate donors to analyse for ectokinase activity in osteoblastic UMR106.06 cells and fetal rat calvarial cells (FRCCs). By pulse-labelling confluent cells with radiolabelled nucleotides, the phosphorylation of endogenous and exogenously added OPN and BSP was demonstrated together with the labelling of a number of cell surface proteins. These phosphorylation reactions were inhibited by a cell-impermeable ectokinase inhibitor, K252b, and cell surface phosphorylation was also inhibited by exogenously added OPN and BSP substrates, indicating competition for the ectokinase enzyme. However, phosphorylation of OPN and BSP, both of which can mediate cell attachment through Arg-Gly-Asp (RGD) motifs, was not inhibited by an RGD peptide, suggesting that binding of OPN and BSP to cell surface integrins is not required. In similar experiments, ectokinase-mediated phosphorylation of OPN and BSP was demonstrated during mineralized tissue formation by FRCCs in vitro. These studies demonstrate that OPN and BSP secreted by bone cells are phosphorylated by a casein kinase II-like ectokinase present on the surface of osteoblastic cells.

Biological evaluation of micro-patterned hyaluronic acid hydrogel for bone tissue engineering

2014 ◽  
Vol 86 (12) ◽  
pp. 1911-1922 ◽  
Author(s):  
Hyo Seung Park ◽  
Su Yeon Lee ◽  
Hyunsik Yoon ◽  
Insup Noh
Keyword(s):  
Tissue Engineering ◽  
Hyaluronic Acid ◽  
Bone Tissue Engineering ◽  
Bone Tissue ◽  
Bone Cells ◽  
Cell Attachment ◽  
Biological Properties ◽  
Biological Evaluation ◽  
Micro Patterning

Abstract Design of micro-patterning of hydrogel is of critical importance in both understanding cellular behaviors and mimicking controlled microenvironments and architectures of diverse well-organized tissues. After micro-patterning of hyaluronic acid (HA) hydrogel on a poly(dimethyl siloxane) substrate, its physical and biological properties have been compared with those of a non-patterned hydrogel for its possible applications in bone tissue engineering. The micro-patterned morphologies of HA hydrogel in both swollen and dehydrated forms have been observed with light microscope and scanning electron microscope, respectively, before and after in vitro cell culture. When MC3T3 bone cells were in vitro cultured on both HA hydrogels, the micro-patterned one shows excellence in cell proliferation and lining for 7 days along the micro-pattern paths over those of the non-patterned one, which have shown less cell-adhesiveness. The cytotoxicity of the micro-patterned HA hydrogels was in vitro evaluated by the assays of MTT, BrdU and Neutral red. The viability and morphology of MC3T3 cells on both HA hydrogels were observed with a fluorescence microscope by the live & dead assay, where their viability was confirmed by staining of F-actin development. The results of their H&E staining showed that both micro-patterned and non-patterned hydrogels induced development of tissue regeneration as observed by cell attachment, proliferation, and survivability, but the micro-patterned one induced distinctive patterning of both better initial cells adhesion on the micro-patterns and subsequently development of their proliferation and extracellular matrix, which were considered as important characteristics in their applications to tissue engineering.

scholarly journals Functionally distinct laminin receptors mediate cell adhesion and spreading: the requirement for surface galactosyltransferase in cell spreading.

1988 ◽  
Vol 107 (5) ◽  
pp. 1863-1871 ◽  
Author(s):  
R B Runyan ◽  
J Versalovic ◽  
B D Shur
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Molecular Mechanisms ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Biological Properties ◽  
Initial Cell ◽  
Laminin Receptors ◽  
Mediate Cell ◽  
A Chain

The molecular mechanisms underlying cell attachment and subsequent cell spreading on laminin are shown to be distinct form one another. Cell spreading is dependent upon the binding of cell surface galactosyltransferase (GalTase) to laminin oligosaccharides, while initial cell attachment to laminin occurs independent of GalTase activity. Anti-GalTase IgG, as well as the GalTase modifier protein, alpha-lactalbumin, both block GalTase activity and inhibited B16-F10 melanoma cell spreading on laminin, but not initial attachment. On the other hand, the addition of UDP galactose, which increases the catalytic turnover of GalTase, slightly increased cell spreading. None of these reagents had any effect on cell spreading on fibronectin. When GalTase substrates within laminin were either blocked by affinity-purified GalTase or eliminated by prior galactosylation, cell attachment appeared normal, but subsequent cell spreading was totally inhibited. The laminin substrate for GalTase was identified as N-linked oligosaccharides primarily on the A chain, and to a lesser extent on B chains. That N-linked oligosaccharides are necessary for cell spreading was shown by the inability of cells to spread on laminin surfaces pretreated with N-glycanase, even though cell attachment was normal. Cell surface GalTase was distinguished from other reported laminin binding proteins, most notably the 68-kD receptor, since they were differentially eluted from laminin affinity columns. These data show that surface GalTase does not participate during initial cell adhesion to laminin, but mediates subsequent cell spreading by binding to its appropriate N-linked oligosaccharide substrate. These results also emphasize that some of laminin's biological properties can be attributed to its oligosaccharide residues.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

scholarly journals Cell Surface Expression of Nrg1 Protein in Candida auris

2021 ◽  
Vol 7 (4) ◽  
pp. 262
Author(s):  
Anuja Paudyal ◽  
Govindsamy Vediyappan
Keyword(s):  
Cell Surface ◽  
Growth Conditions ◽  
Zinc Ion ◽  
Surface Proteins ◽  
Cell Surface Expression ◽  
Unexpected Finding ◽  
Candida Auris ◽  
Cell Surface Proteins ◽  
Biofilm Growth

Candida auris is an emerging antifungal resistant human fungal pathogen increasingly reported in healthcare facilities. It persists in hospital environments, and on skin surfaces, and can form biofilms readily. Here, we investigated the cell surface proteins from C. auris biofilms grown in a synthetic sweat medium mimicking human skin conditions. Cell surface proteins from both biofilm and planktonic control cells were extracted with a buffer containing β-mercaptoethanol and resolved by 2-D gel electrophoresis. Some of the differentially expressed proteins were excised and identified by mass spectrometry. C. albicans orthologs Spe3p, Tdh3p, Sod2p, Ywp1p, and Mdh1p were overexpressed in biofilm cells when compared to the planktonic cells of C. auris. Interestingly, several proteins with zinc ion binding activity were detected. Nrg1p is a zinc-binding transcription factor that negatively regulates hyphal growth in C. albicans. C. auris does not produce true hypha under standard in vitro growth conditions, and the role of Nrg1p in C. auris is currently unknown. Western blot analyses of cell surface and cytosolic proteins of C. auris against anti-CalNrg1 antibody revealed the Nrg1p in both locations. Cell surface localization of Nrg1p in C. auris, an unexpected finding, was further confirmed by immunofluorescence microscopy. Nrg1p expression is uniform across all four clades of C. auris and is dependent on growth conditions. Taken together, the data indicate that C. auris produces several unique proteins during its biofilm growth, which may assist in the skin-colonizing lifestyle of the fungus during its pathogenesis.

scholarly journals Osteosphere Model to Evaluate Cell–Surface Interactions of Implantable Biomaterials

Materials ◽  
2021 ◽  
Vol 14 (19) ◽  
pp. 5858
Author(s):  
Ana Carolina Batista Brochado ◽  
Victor Hugo de Souza ◽  
Joice Correa ◽  
Suzana Azevedo dos Anjos ◽  
Carlos Fernando de Almeida Barros Mourão ◽  
...  
Keyword(s):  
Cell Surface ◽  
3D Model ◽  
Bone Cells ◽  
Three Dimensional ◽  
Surface Interactions ◽  
Force Microscopy ◽  
Tissue Therapy ◽  
Cell Surface Interactions ◽  
Titanium Surfaces

Successful biomaterials for bone tissue therapy must present different biocompatible properties, such as the ability to stimulate the migration and proliferation of osteogenic cells on the implantable surface, to increase attachment and avoid the risks of implant movement after surgery. The present work investigates the applicability of a three-dimensional (3D) model of bone cells (osteospheres) in the evaluation of osteoconductive properties of different implant surfaces. Three different titanium surface treatments were tested: machined (MA), sandblasting and acid etching (BE), and Hydroxyapatite coating by plasma spray (PSHA). The surfaces were characterized by Scanning Electron Microscopy (SEM) and atomic force microscopy (AFM), confirming that they present very distinct roughness. After seeding the osteospheres, cell–surface interactions were studied in relation to cell proliferation, migration, and spreading. The results show that BE surfaces present higher densities of cells, leaving the aggregates towards than titanium surfaces, providing more evidence of migration. The PSHA surface presented the lowest performance in all analyses. The results indicate that the 3D model allows the focal analysis of an in vitro cell/surfaces interaction of cells and surfaces. Moreover, by demonstrating the agreement with the clinical data observed in the literature, they suggest a potential use as a predictive preclinical tool for investigating osteoconductive properties of novel biomaterials for bone therapy.

scholarly journals Protocol of Co-Culture of Human Osteoblasts and Osteoclasts to Test Biomaterials for Bone Tissue Engineering

2022 ◽  
Vol 5 (1) ◽  
pp. 8
Author(s):  
Giorgia Borciani ◽  
Giorgia Montalbano ◽  
Nicola Baldini ◽  
Chiara Vitale-Brovarone ◽  
Gabriela Ciapetti
Keyword(s):  
Tissue Engineering ◽  
Bone Tissue Engineering ◽  
Bone Tissue ◽  
Bone Cells ◽  
Bone Cell ◽  
Seeding Density ◽  
Bone Homeostasis ◽  
Bone Microenvironment

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.

scholarly journals The Anchorage of Bone Cells onto an Yttria-Stabilized Zirconia Surface with Mild Nano-Micro Curved Profiles

2020 ◽  
Vol 8 (4) ◽  
pp. 127
Author(s):  
Susanne Staehlke ◽  
Armin Springer ◽  
Thomas Freitag ◽  
Jakob Brief ◽  
J. Barbara Nebe
Keyword(s):  
Matrix Protein ◽  
Bone Cells ◽  
Cell Attachment ◽  
Yttria Stabilized Zirconia ◽  
Extracellular Matrix Protein ◽  
Cell Phenotype ◽  
Ros Generation ◽  
Stabilized Zirconia ◽  
Dental Surgery

The high biocompatibility, good mechanical properties, and perfect esthetics of ceramic dental materials motivate investigation into their suitability as an endosseous implant. Osseointegration at the interface between bone and implant surface, which is a criterion for dental implant success, is dependent on surface chemistry and topography. We found out earlier that osteoblasts on sharp-edged micro-topographies revealed an impaired cell phenotype and function and the cells attempted to phagocytize these spiky elevations in vitro. Therefore, micro-structured implants used in dental surgery should avoid any spiky topography on their surface. The sandblasted, acid-etched, and heat-treated yttria-stabilized zirconia (cer.face®14) surface was characterized by scanning electron microscopy and energy dispersive X-ray. In vitro studies with human MG-63 osteoblasts focused on cell attachment and intracellular stress level. The cer.face 14 surface featured a landscape with nano-micro hills that was most sinusoidal-shaped. The mildly curved profile proved to be a suitable material for cell anchorage. MG-63 cells on cer.face 14 showed a very low reactive oxygen species (ROS) generation similar to that on the extracellular matrix protein collagen I (Col). Intracellular adenosine triphosphate (ATP) levels were comparable to Col. Ceramic cer.face 14, with its sinusoidal-shaped surface structure, facilitates cell anchorage and prevents cell stress.

Laminin E8 alveolarization site: heparin sensitivity, cell surface receptors, and role in cell spreading

1997 ◽  
Vol 272 (3) ◽  
pp. L494-L503
Author(s):  
L. Chen ◽  
V. Shick ◽  
M. L. Matter ◽  
S. M. Laurie ◽  
R. C. Ogle ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Surface Proteins ◽  
Cell Surface Proteins ◽  
Surface Receptors ◽  
Beta1 Integrin ◽  
Alpha Dystroglycan ◽  
Alveolar Formation ◽  
Laminin 1

Cell adhesion to amino acids 2179-2198 (SN-peptide) of the laminin-1 alpha1-chain is required for lung alveolar formation in vitro (M. L. Matter and G. W. Laurie. J. Cell Biol. 124: 1083-1090, 1994). The nature of the SN-peptide receptor(s) was probed with neutralizing anti-integrin monoclonal antibodies (MAb), cells lacking integrin subunits, soluble heparin, and SN-peptide columns. Cell adhesion and spreading studies confirmed the specificity of SN-peptide and revealed adhesion to be unaffected by inclusion of anti-beta1-, anti-alpha(2-6)- or anti-alpha(V)beta5-integrin MAb. Cells lacking beta1- or alpha6-integrin subunits were fully adherent. Adhesion was heparin, but not chondroitin sulfate or heparinase, sensitive, much as is alpha-dystroglycan-laminin-1 binding. Heparin eluted approximately 155- and 180-kDa cell-surface proteins from SN-peptide columns. An additional approximately 91-kDa protein was eluted by EDTA. All were unrecognized by anti-beta1-integrin MAb. SN-peptide therefore interacts with three cell-surface proteins for which the identity remains to be determined.

scholarly journals Fetal bovine bone cells synthesize bone-specific matrix proteins.

1984 ◽  
Vol 99 (2) ◽  
pp. 607-614 ◽  
Author(s):  
S W Whitson ◽  
W Harrison ◽  
M K Dunlap ◽  
D E Bowers ◽  
L W Fisher ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Bone Cells ◽  
Bone Sialoprotein ◽  
Cell System ◽  
Matrix Proteins ◽  
Type I ◽  
Bovine Bone ◽  
Isolated Cells ◽  
Noncollagenous Proteins

We isolated cells from both calvaria and the outer cortices of long bones from 3- to 5-mo bovine fetuses. The cells were identified as functional osteoblasts by indirect immunofluorescence using antibodies against three bone-specific, noncollagenous matrix proteins (osteonectin, the bone proteoglycan, and the bone sialoprotein) and against type 1 collagen. In separate experiments, confluent cultures of the cells were radiolabeled and shown to synthesize and secrete osteonectin, the bone proteoglycan and the bone sialoprotein by immunoprecipitation and fluorography of SDS polyacrylamide gels. Analysis of the radiolabeled collagens synthesized by the cultures showed that they produced predominantly (approximately 94%) type I collagen, with small amounts of types III and V collagens. In agreement with previous investigators who have employed the rodent bone cell system, we confirmed in bovine bone cells that (a) there was a typical cyclic AMP response to parathyroid hormone, (b) freshly isolated cells possessed high levels of alkaline phosphatase, which diminished during culture but returned to normal levels in mineralizing cultures, and (c) cells grown in the presence of ascorbic acid and beta-glycerophosphate rapidly produced and mineralized an extracellular matrix containing largely type I collagen. These results show that antibodies directed against bone-specific, noncollagenous proteins can be used to clearly identify bone cells in vitro.

Control of Oriented Extracellular Matrix Similar to Anisotropic Bone Microstructure

2014 ◽  
Vol 783-786 ◽  
pp. 72-77 ◽  
Author(s):  
Takayoshi Nakano ◽  
Aira Matsugaki ◽  
Takuya Ishimoto ◽  
Mitsuharu Todai ◽  
Ai Serizawa ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Bone Tissue ◽  
Bone Cells ◽  
Crystal Surface ◽  
Early Stage ◽  
Bone Microstructure ◽  
Bone Cell ◽  
Collagen Fibers

Bone microstructure is dominantly composed of anisotropic extracellular matrix (ECM) in which collagen fibers and epitaxially-oriented biological apatite (BAp) crystals are preferentially aligned depending on the bone anatomical position, resulting in exerting appropriate mechanical function. The regenerative bone in bony defects is however produced without the preferential alignment of collagen fibers and the c-axis of BAp crystals, and subsequently reproduced to recover toward intact alignment. Thus, it is necessary to produce the anisotropic bone-mimetic tissue for the quick recovery of original bone tissue and the related mechanical ability in the early stage of bone regeneration. Our group is focusing on the methodology for regulating the arrangement of bone cells, the following secretion of collagen and the self-assembled mineralization by oriented BAp crystallites. Cyclic stretching in vitro to bone cells, principal-stress loading in vivo on scaffolds, step formation by slip traces on Ti single crystal, surface modification by laser induced periodic surface structure (LIPSS), anisotropic collagen substrate with the different degree of orientation, etc. can dominate bone cell arrangement and lead to the construction of the oriented ECM similar to the bone tissue architecture. This suggests that stress/strain loading, surface topography and chemical anisotropy are useful to produce bone-like microstructure in order to promote the regeneration of anisotropic bone tissue and to understand the controlling parameters for anisotropic osteogenesis induction.

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