Bone Formation by Human Oseoblasts on Implant Materials

1998 ◽  
Vol 4 (S2) ◽  
pp. 938-939
Author(s):  
G. Gronowicz ◽  
M. Ahmad
Keyword(s):  
In Vitro System ◽  
Implant Materials ◽  
Sequence Of Events ◽  
Ecm Proteins ◽  
Transmission Electron ◽  
Fluorescence Light ◽  
Primary Human Osteoblasts ◽  
Fetal Bovine

We have developed an in vitro system to study the process by which osteoblasts attach and secrete extracellular matrix (ECM) proteins that subsequently mineralize in response to implant materials. In vivo mineralization is characterized by the sequential expression by osteoblasts of specific ECM proteins that are capable of calcifying in an orderly manner. A similar sequence of events is shown to occur in this in vitro system. The extent and pattern of mineralization was visualized by fluorescence light microscopy and transmission electron microscopy (TEM), and was dependent on the substrate to which osteoblasts adhered.Materials and MethodsDisks (22 mm diameter) composed of Tivanium (Tiv) (Ti6AI4V, ASTM F-136) and Zimaloy (Zim)(CoCrMo, ASTM F-75), prepared identically to clinical orthopaedic implants, were supplied by Zimmer, Inc. Glass disks were used as controls. Osteoblast-like cells (Saos-2), derived from a human osteosarcoma, or primary human osteoblasts from a 41 year-old male patient were added to the disks in a-MEM medium with 10% fetal bovine serum,

78 EFFICACY OF USING IN VITRO-PRODUCED CATTLE EMBRYOS TO DEVELOP AN EQUINE EMBRYO CRYOPRESERVATION SYSTEM

2009 ◽  
Vol 21 (1) ◽  
pp. 139
Author(s):  
C. M. Syverson ◽  
A. M. Paprocki ◽  
R. W. Koppang ◽  
J. R. Dobrinsky
Keyword(s):  
The United States ◽  
Blastocyst Stage ◽  
Embryo Cryopreservation ◽  
State University ◽  
Pump System ◽  
In Vitro System ◽  
Colorado State University ◽  
Fetal Bovine

Unfortunately, there is no reliable in vitro system available to provide a routine and economical source of equine embryos for research and commercial use. We explore the use of standard in vitro-produced (IVP) cattle embryos for the development of a direct transfer (dt) equine embryo cryopreservation system. Ovaries were collected from mature females at an abattoir and transported to our laboratory. Cumulus–oocyte complexes (COC) were aspirated from 2- to 6-mm follicles with an 18-gauge needle fixed to a vacuum pump system. Only COC surrounded by 2 or more layers of compact cumulus investment and containing oocytes of equal size were placed into a commercial TCM-199-based IVM system (Minitube of America Inc., Verona, WI). After 22 h of IVM, mature COC were placed into standard IVF. Prospective embryos were cultured for 120 h in CR-1/BSA (Minitube of America Inc.), and then supplemented with 10% fetal bovine serum (FBS) and cultured for an additional 48 h. Only excellent to good morula-blastocyst stage IVP cattle embryos were used in this study. As a control, the Colorado State University equine embryo dt-vitrification system (CSU; Carnevale EM Vet. Clin. North Am. Equine Pract 22, 831–841) was used on IVP cattle blastocysts. Two different Minitube EquiPRO-based dt-media with either 10% FBS or Minitube BSA-V (Fraction-V; Minitube of America Inc.) were tested on IVP cattle embryos. Minitube BSA-V is highly defined and internationally compliant BSA approved for use in raw form or in culture medium in the United States and European Union. Of 103 IVP cattle embryos cryopreserved by the CSU method, 45 (43.7%) embryos were viable after 24 h of culture. Of 121 IVP cattle embryos cryopreserved with Minitube EquiPRO + 10% FBS, 52 (42.9%) embryos were viable after 24 h of culture. Of 90 IVP cattle embryos cryopreserved with Minitube EquiPRO + BSA-V, 40 (44.4%) embryos were viable after 24 h of culture. Minitube EquiPRO + BSA-V was used with in vivo-produced equine morulae (no capsule). A total of 9 mares were flushed on Day 6.5, and 8 excellent to good morulae were recovered. All 8 embryos were dt-vitrified in the Minitube EquiPRO + BSA-V system. All 8 embryos were later warmed and transferred to synchronous recipient mares. On Day 14 of presumptive pregnancy, 7 (87.5%) mares were confirmed pregnant by real-time ultrasound examination. Not all presumptive foals were needed, so 4 recipient mares were randomly selected and intentionally aborted. Three mares were selected to carry presumptive foals to term. All 3 mares produced live and healthy foals at the term of gestation. Standard IVP cattle embryos can serve as a relatively inexpensive model for the development and testing of equine embryo cryopreservation systems.

scholarly journals Mechanism of CK2.3, a Novel Mimetic Peptide of Bone Morphogenetic Protein Receptor Type IA, Mediated Osteogenesis

2019 ◽  
Vol 20 (10) ◽  
pp. 2500 ◽  
Author(s):  
Vrathasha Vrathasha ◽  
Hilary Weidner ◽  
Anja Nohe
Keyword(s):  
Signaling Pathways ◽  
Treatment Options ◽  
Time Frame ◽  
Bmp Signaling ◽  
C2c12 Cells ◽  
Molecular Sequence ◽  
Sequence Of Events ◽  
Protein Receptor

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

The Use of the Chorioallantoic Membrane (CAM) of the Embryonic Chick for the Direct Assessment of Implant Toxicity

1985 ◽  
Vol 13 (4) ◽  
pp. 261-266
Author(s):  
P.P. Monro ◽  
D.P. Knight ◽  
W.S. Pringle ◽  
D.M. Fyfe ◽  
J.R. Shearer
Keyword(s):  
Chorioallantoic Membrane ◽  
In Vitro Techniques ◽  
Implant Materials ◽  
Metal Foils ◽  
Complex Interactions ◽  
Cobalt Nickel ◽  
Histological Criteria ◽  
Fluid Environment

The toxicity of implant materials requires investigation prior to clinical use. We have developed a method where materials are directly applied to the chorioallantoic membrane (CAM) of 9-day-old chick embryos and toxicity is assessed using histological criteria. We evaluated the method using metal foils. The number and organisation of fibroblasts seemed to be the most useful criteria for assessing metal toxicity. Differences were greatest after 10 days of culture on the CAM. The method is sensitive enough to enable us to discriminate between the less toxic aluminium and titanium and the highly toxic cobalt, nickel and tungsten. The proposed method has advantages over in vitro techniques which provide an abnormal fluid environment and in which the more complex interactions that are possible between implant materials and tissue in vivo cannot be modelled.

scholarly journals Cemp1-p3 Peptide Promotes the Transformation of Octacalcium Phosphate into Hydroxyapatite Crystals

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1131
Author(s):  
Maricela Santana ◽  
Gonzalo Montoya ◽  
Raúl Herrera ◽  
Lía Hoz ◽  
Enrique Romo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Octacalcium Phosphate ◽  
Sequence Motifs ◽  
Simple Method ◽  
Transmission Electron ◽  
Precursor Phase ◽  
Mineralized Tissues ◽  
Potent Growth

Dental cementum contains unique molecules that regulate the mineralization process in vitro and in vivo, such as cementum protein 1 (CEMP1). This protein possesses amino acid sequence motifs like the human recombinant CEMP1 with biological activity. This novel cementum protein 1-derived peptide (CEMP1-p3, from the CEMP1’s N-terminal domain: (QPLPKGCAAVKAEVGIPAPH), consists of 20 amino acids. Hydroxyapatite (HA) crystals could be obtained through the combination of the amorphous precursor phase and macromolecules such as proteins and peptides. We used a simple method to synthesize peptide/hydroxyapatite nanocomposites using OCP and CEMP1-p3. The characterization of the crystals through scanning electron microscopy (SEM), powder X-ray diffraction (XRD), high--resolution transmission electron microscopy (HRTEM), and Raman spectroscopy revealed that CEMP1-p3 transformed OCP into hydroxyapatite (HA) under constant ionic strength and in a buffered solution. CEMP1-p3 binds and highly adsorbs to OCP and is a potent growth stimulator of OCP crystals. CEMP1-p3 fosters the transformation of OCP into HA crystals with crystalline planes (300) and (004) that correspond to the cell of hexagonal HA. Octacalcium phosphate crystals treated with CEMP1-p3 grown in simulated physiological buffer acquired hexagonal arrangement corresponding to HA. These findings provide new insights into the potential application of CEMP1-p3 on possible biomimetic approaches to generate materials for the repair and regeneration of mineralized tissues, or restorative materials in the orthopedic field.

scholarly journals Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+Signaling

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Nahed El-Najjar ◽  
Rashmi P. Kulkarni ◽  
Nancy Nader ◽  
Rawad Hodeify ◽  
Khaled Machaca
Keyword(s):  
Insulin Resistance ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Complex Disease ◽  
Prolonged Exposure ◽  
In Vitro System ◽  
A7r5 Cells

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+signaling, including most prominently an inhibition of the passive ER Ca2+leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+signaling machinery are different.

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

scholarly journals Anti-CD63 antibodies suppress IgE-dependent allergic reactions in vitro and in vivo

2005 ◽  
Vol 201 (3) ◽  
pp. 385-396 ◽  
Author(s):  
Stefan Kraft ◽  
Tony Fleming ◽  
James M. Billingsley ◽  
Shih-Yao Lin ◽  
Marie-Hélène Jouvin ◽  
...  
Keyword(s):  
Pi3k Pathway ◽  
Therapeutic Agents ◽  
Allergic Reactions ◽  
Ecm Proteins ◽  
Broad Array ◽  
High Affinity Ige Receptor ◽  
Nonadherent Cells ◽  
Tetraspanin Cd63

High-affinity IgE receptor (FcεRI) cross-linking on mast cells (MCs) induces secretion of preformed allergy mediators (degranulation) and synthesis of lipid mediators and cytokines. Degranulation produces many symptoms of immediate-type allergic reactions and is modulated by adhesion to surfaces coated with specific extracellular matrix (ECM) proteins. The signals involved in this modulation are mostly unknown and their contribution to allergic reactions in vivo is unclear. Here we report the generation of monoclonal antibodies that potently suppress FcεRI-induced degranulation, but not leukotriene synthesis. We identified the antibody target as the tetraspanin CD63. Tetraspanins are membrane molecules that form multimolecular complexes with a broad array of molecules including ECM protein-binding β integrins. We found that anti-CD63 inhibits MC adhesion to fibronectin and vitronectin. Furthermore, anti-CD63 inhibits FcεRI-mediated degranulation in cells adherent to those ECM proteins but not in nonadherent cells. Thus the inhibition of degranulation by anti-CD63 correlates with its effect on adhesion. In support of a mechanistic linkage between the two types of inhibition, anti-CD63 had no effect on FcεRI-induced global tyrosine phosphorylation and calcium mobilization but impaired the Gab2–PI3K pathway that is known to be essential for both degranulation and adhesion. Finally, we showed that these antibodies inhibited FcεRI-mediated allergic reactions in vivo. These properties raise the possibility that anti-CD63 could be used as therapeutic agents in MC-dependent diseases.

scholarly journals Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

1989 ◽  
Vol 9 (11) ◽  
pp. 4746-4749 ◽  
Author(s):  
D I Chasman ◽  
J Leatherwood ◽  
M Carey ◽  
M Ptashne ◽  
R D Kornberg
Keyword(s):  
Rna Polymerase Ii ◽  
Fusion Proteins ◽  
In Vitro System ◽  
Transcription System ◽  
Natural Mechanism ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription ◽  
Fold Stimulation

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

Accurate transcription of a plant mitochondrial gene in vitro

1991 ◽  
Vol 11 (4) ◽  
pp. 2035-2039
Author(s):  
P J Hanic-Joyce ◽  
M W Gray
Keyword(s):  
Mitochondrial Gene ◽  
Plant Mitochondria ◽  
In Vitro Transcription ◽  
Dna Templates ◽  
In Vitro System ◽  
Transcription System ◽  
Translation Initiation Codon ◽  
Mitochondrial Extract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

Abstract 531: Tie-2/Cre--Mediated Conditional Deletion of Lipid Phosphate Phosphatase-3 Reveals Its Role in Endothelial Cell Apoptosis

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Ishita Chatterjee ◽  
Kishore K Wary
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Genome Wide Association Study ◽  
Vascular Endothelial Cell ◽  
Protein Levels ◽  
Conditional Deletion ◽  
Transmission Electron ◽  
Increased Risk

Rationale: A recent genome-wide association study (GWAS) has linked a frequently occurring variation in the LPP3 (also known as PPAP2b) loci to increased risk of coronary heart disease (CAD). However, the in vivo function of LPP3 in vascular endothelial cell is incompletely understood. Goal: To address the endothelial cell (EC) specific function of Lpp3 in mice. Results: Tie-2/Cre mediated Lpp3 deletion did not affect normal vasculogenesis in early embryonic development, in contrast, in late embryonic stages it led to impaired angiogenesis associated with hemorrhage, edema and late embryonic lethal phenotype. Immunohistochemical staining followed by microscopic analyses of mutant embryos revealed reduced fibronectin and VE-cadherin expression throughout different vascular bed, and increased apoptosis in CD31+ vascular structures. Transmission electron microscopy (TEM) showed the presence of apoptotic endothelial cells and disruption of adherens junctions in mutant embryos. LPP3-knockdown in vitro showed an increase in p53 and p21 protein levels, with concomitant decrease in cell proliferation. LPP3-knockdown also decreased transendothelial electrical resistance (TER), interestingly re-expression of ß-catenin cDNA into LPP3-depleted endothelial cells partially restored the effect of loss of LPP3. Conclusion: These results suggest the ability of LPP3 to regulate survival and apoptotic activities of endothelial cells during patho/physiological angiogenesis.

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