scholarly journals Bone Matrix Proteins: Isolation and Characterization of a Novel Cell-binding Keratan Sulfate Proteoglycan (Osteoadherin) from Bovine Bone

1998 ◽  
Vol 141 (3) ◽  
pp. 839-847 ◽  
Author(s):  
Mikael Wendel ◽  
Yngve Sommarin ◽  
Dick Heinegård
Keyword(s):  
Guanidine Hydrochloride ◽  
Bone Matrix ◽  
Keratan Sulfate ◽  
Apparent Size ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Bone ◽  
Metabolic Labeling ◽  
Cell Binding ◽  
Isolation And Characterization

A small cell-binding proteoglycan for which we propose the name osteoadherin was extracted from bovine bone with guanidine hydrochloride–containing EDTA. It was purified to homogeneity using a combination of ion-exchange chromatography, hydroxyapatite chromatography, and gel filtration. The Mr of the proteoglycan was 85,000 as determined by SDS-PAGE. The protein is rich in aspartic acid, glutamic acid, and leucine. Two internal octapeptides from the proteoglycan contained the sequences Glu-Ile-Asn-Leu-Ser-His-Asn-Lys and Arg-Asp-Leu-Tyr-Phe-Asn-Lys-Ile. These sequences are not previously described, and support the notion that osteoadherin belongs to the family of leucine-rich repeat proteins. A monospecific antiserum was raised in rabbits. An enzyme-linked immunosorbent assay was developed, and showed the osteoadherin content of bone extracts to be 0.4 mg/g of tissue wet weight, whereas none was found in extracts of various other bovine tissues. Metabolic labeling of primary bovine osteoblasts followed by immunoprecipitation showed the cells to synthesize and secrete the proteoglycan. Digesting the immunoprecipitated osteoadherin with N-glycosidase reduced its apparent size to 47 kD, thus showing the presence of several N-linked oligosaccharides. Digestion with keratanase indicated some of the oligosaccharides to be extended to keratan sulfate chains. In immunohistochemical studies of the bovine fetal rib growth plate, osteoadherin was exclusively identified in the primary bone spongiosa. Osteoadherin binds to hydroxyapatite. A potential function of this proteoglycan is to bind cells, since we showed it to be as efficient as fibronectin in promoting osteoblast attachment in vitro. The binding appears to be mediated by the integrin αvβ3, since this was the only integrin isolated by osteoadherin affinity chromatography of surface-iodinated osteoblast extracts.

scholarly journals In vitro induction of cartilage-specific macromolecules by a bone extract.

1983 ◽  
Vol 97 (6) ◽  
pp. 1950-1953 ◽  
Author(s):  
S M Seyedin ◽  
A Y Thompson ◽  
D M Rosen ◽  
K A Piez
Keyword(s):  
Morphological Changes ◽  
Bone Matrix ◽  
Type Ii Collagen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Bone ◽  
In Vitro System ◽  
In Vitro Induction ◽  
Inhibition Technique

An in vitro system has been developed to study the onset of chondrogenesis. Embryonic rat muscle mesenchymal cells, when treated in suspension culture with an extract of bovine bone matrix, synthesized cartilage-specific proteoglycan and type II collagen. The synthesis of these two macromolecules was assayed by the enzyme-linked immunosorbent assay inhibition technique. Further evidence of chondrogenesis was demonstrated by morphological changes of treated cells when cultured in firm agarose and stained for metachromatic matrix. Even with crude bone matrix extracts, the assay was sensitive at the microgram level and significant differences in cartilage macromolecules compared with controls were observed in 2-3 d. In vivo the same extract induced first cartilage and then bone.

Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion

Blood ◽  
2003 ◽  
Vol 102 (2) ◽  
pp. 718-724 ◽  
Author(s):  
Nicholas A. Watkins ◽  
Lily M. Du ◽  
J. Paul Scott ◽  
Willem H. Ouwehand ◽  
Cheryl A. Hillery
Keyword(s):  
Sickle Cell ◽  
Matrix Protein ◽  
Binding Domain ◽  
Extracellular Matrix Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Adhesion Assay ◽  
Single Chain ◽  
Cell Binding

AbstractThe enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.

scholarly journals Isolation and characterization of two sialoproteins present only in bone calcified matrix

1985 ◽  
Vol 232 (3) ◽  
pp. 715-724 ◽  
Author(s):  
A Franzén ◽  
D Heinegård
Keyword(s):  
Sialic Acid ◽  
Core Protein ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Dry Weight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Bone ◽  
Guanidinium Chloride ◽  
The Core ◽  
Isolation And Characterization

Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.

Factors influencing synthesis and mineralization of bone matrix from fetal bovine bone cells grown in vitro

2009 ◽  
Vol 7 (7) ◽  
pp. 727-741 ◽  
Author(s):  
S. William Whitson ◽  
Marcia A. Whitson ◽  
Daniel E. Bowers ◽  
Michael C. Falk
Keyword(s):  
Bone Cells ◽  
Bone Matrix ◽  
Bovine Bone ◽  
Factors Influencing ◽  
Fetal Bovine

scholarly journals Dynamics of reparative histogenesis of bone tissue in presence of some osteoplastic materials in vitro

2020 ◽  
Vol 4 (34) ◽  
pp. 46-50
Author(s):  
S. Yu. Ivanov ◽  
A. V. Volkov ◽  
D. A. De
Keyword(s):  
Bone Regeneration ◽  
Bone Tissue ◽  
Bone Matrix ◽  
Three Dimensional ◽  
Bone Defects ◽  
Control Group ◽  
Bovine Bone ◽  
Reparative Osteogenesis ◽  
The Impact

Currently, to solve the bone deficiency problem in the maxillofacial region, osteoplastic materials based on allogeneic and xenogenic collagen bone matrix are used, both in pure and in activated forms, by adding growth factors. It is impossible to determine the effectiveness and mechanisms of the osteoplastic materials effect on bone regeneration without a comprehensive study, including not only histological, but also morphometric studies of the structural components and cellular reactions in the impact area. Such studies provide reliable and objective information on the main processes taking place in bone regeneration.Purpose. To determine the spatial distribution of reparative osteogenesis in the presence of some osteoplastic materials in vitro.Materials and methods. Svetlogorsk breed pigs were used as a biomodel. Depending on the osteoplastic preparations used, the animals were divided into four groups of the two in each: 1st — a preparation based on a natural bovine bone graft was injected into bone defects. 2nd — a preparation based on collagenized porcine transplant was injected into bone defects. 3rd — a preparation consisting of 60 % hydroxyapatite (HA) and 40 % beta-tri-calcium phosphate; 4th — control group — the bone defect healed under a blood clot. Animals were removed from the experiment on the 45th day. We examined sections with a thickness of 20 μm using the method of light and fluorescence microscopy.Results. The results indicate different dynamics of the reparative osteogenesis in the presence of osteoplastic materials of different classes. In group 1, the filling of the defect with newly formed bone tissue is not uniform; in group 2, the filling of the defect with newly formed bone tissue is uniform; in group 3 the filling of the defect with non-formed bone tissue is uneven due to the pronounced hyperostosis; in the control group, the filling of the defect with newly formed bone tissue is not happening.Conclusion. Stimulation, the dynamics of reparative osteogenesis and the three-dimensional organization of bone regenerate depend on the osteoplastic material class, which requires further study of the dynamics and three-dimensional organization of bone regenerate to select the optimal bone-replacing agent.

scholarly journals Evaluation of the cytocompatibility of mixed bovine bone

2007 ◽  
Vol 18 (3) ◽  
pp. 179-184 ◽  
Author(s):  
Esther Rieko Takamori ◽  
Eduardo Aleixo Figueira ◽  
Rumio Taga ◽  
Mari Cleide Sogayar ◽  
José Mauro Granjeiro
Keyword(s):  
Cytotoxic Effect ◽  
Bone Matrix ◽  
Neutral Red ◽  
Water Soluble ◽  
Mechanical Resistance ◽  
Bovine Bone ◽  
3T3 Cells ◽  
Cell Based Therapy ◽  
Viable Cells

Treatment of bovine bone with peroxides and chaotropic agents aims to obtain an acellular bone matrix that is able to maintain the collagen-apatite complex and a higher mechanical resistance, a mixed biomaterial hereby named mixed bovine bone (MBB). The purpose of this study was to evaluate the cytocompatibility of MBB and cell-MBB interaction. Cell morphology, number of viable cells, ability to reduce methyltetrazolium and to incorporate neutral red upon exposure to different concentrations of the hydrosoluble extract of MBB were assessed in Balb-c 3T3 cells according to ISO 10993-5 standard. The interaction between cells and MBB surface was evaluated by scanning electron microscopy. The water-soluble MBB extracts were cytotoxic and led to cell death possibly due to its effect on mitochondrial function and membrane permeability. Cells plated directly onto the MBB did not survive, although after dialysis and material conditioning in DMEM + 10% FCS, the cells adhered and proliferated onto the material. It may be concluded that, in vitro, water-soluble MBB extracts were cytotoxic. Nevertheless, MBB cytotoxic effect was reverted by dialysis resulting in a material that is suitable for cell based-therapy in the bioengineering field.

scholarly journals Biohybrid Bovine Bone Matrix for Controlled Release of Mesenchymal Stem/Stromal Cell Lyosecretome: A Device for Bone Regeneration

2021 ◽  
Vol 22 (8) ◽  
pp. 4064
Author(s):  
Elia Bari ◽  
Ilaria Roato ◽  
Giuseppe Perale ◽  
Filippo Rossi ◽  
Tullio Genova ◽  
...  
Keyword(s):  
Bone Regeneration ◽  
Bone Matrix ◽  
Stromal Vascular Fraction ◽  
In Vitro Tests ◽  
Burst Release ◽  
Bovine Bone ◽  
Class Iii ◽  
Freeze Dried ◽  
Quantification Analysis

SmartBone® (SB) is a biohybrid bone substitute advantageously proposed as a class III medical device for bone regeneration in reconstructive surgeries (oral, maxillofacial, orthopedic, and oncology). In the present study, a new strategy to improve SB osteoinductivity was developed. SB scaffolds were loaded with lyosecretome, a freeze-dried formulation of mesenchymal stem cell (MSC)-secretome, containing proteins and extracellular vesicles (EVs). Lyosecretome-loaded SB scaffolds (SBlyo) were prepared using an absorption method. A burst release of proteins and EVs (38% and 50% after 30 min, respectively) was observed, and then proteins were released more slowly with respect to EVs, most likely because they more strongly adsorbed onto the SB surface. In vitro tests were conducted using adipose tissue-derived stromal vascular fraction (SVF) plated on SB or SBlyo. After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. On SB, on the other hand, the process was still present, but tissue formation was less organized at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralize after 60 days. Nonetheless, SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralized tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors present in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β.

Bioactivation of Bovine Bone Matrix and Collagen Scaffold Using Argon Plasma: In Vitro Study

2021 ◽  
Vol 36 (2) ◽  
pp. 242-247
Author(s):  
Luigi Canullo ◽  
Tullio Genova ◽  
Sara Petrillo ◽  
Katsuhiko Masuda ◽  
Kazushige Tanaka ◽  
...  
Keyword(s):  
Bone Matrix ◽  
Argon Plasma ◽  
Collagen Scaffold ◽  
In Vitro Study ◽  
Vitro Study ◽  
Bovine Bone

scholarly journals Isolation and Characterization of Human Monoclonal Antibodies from Individuals Infected with West Nile Virus

2006 ◽  
Vol 80 (14) ◽  
pp. 6982-6992 ◽  
Author(s):  
Mark Throsby ◽  
Cecile Geuijen ◽  
Jaap Goudsmit ◽  
Arjen Q. Bakker ◽  
Jehanara Korimbocus ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
West Nile Virus ◽  
Viral Envelope ◽  
Phage Library ◽  
Enzyme Linked Immunosorbent Assay ◽  
West Nile ◽  
Isolation And Characterization ◽  
E Protein ◽  
Domain Iii

ABSTRACT Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 μg/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.

In Vitro and in Vivo Comparison of Binding of 99m-Tc-Iabeled Anti-CEA MAb F33-104 with 99m-Tc-labeled Anti-CEA MAb BW431/26

1999 ◽  
Vol 38 (04) ◽  
pp. 115-119
Author(s):  
N. Oriuchi ◽  
S. Sugiyama ◽  
M. Kuroki ◽  
Y. Matsuoka ◽  
S. Tanada ◽  
...  
Keyword(s):  
Nude Mice ◽  
Binding Capacity ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Cell Binding ◽  
Vitro Binding ◽  
Labeling Method ◽  
Human Colon Adenocarcinoma

Summary Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tclabeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 107 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tcactivity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

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