Biomedical subjects

R Bizios

Publications and source records attributed to R Bizios.

At least 19 recordsLinked to original sources

PubMed1999

Exposure of human vascular endothelial cells to sustained hydrostatic pressure stimulates proliferation. Involvement of the alphaV integrins.

The present study investigated the effects of sustained hydrostatic pressure (SHP; up to 4 cm H2O) on human umbilical vein endothelial cell (HUVEC) proliferation, focal adhesion plaque (FAP) organization, and integrin expression. Exposure of HUVECs to SHP stimulated cell proliferation and a selective increase in the expression of integrin subunit alphaV. The increase in alphaV was observed as early as 4 hours after exposure to pressure and preceded detectable increases in the bromodeoxyuridine labeling index. Laser confocal microscopy studies demonstrated colocalization of the alphaV integrin to FAPs. The individual FAPs in pressure-treated cells demonstrated a reduced area and increased aspect ratio and were localized to both peripheral and more central regions of the cells, in contrast to the predilection for the cell periphery in cells maintained under control pressure conditions. The pressure-induced changes in alphaV distribution had functional consequences on the cells: adhesivity of the cells to vitronectin was increased, and alphaV antagonists blocked the pressure-induced proliferative response. Thus, the present study suggests a role for alphaV integrins in the mechanotransduction of pressure by endothelial cells.

Antigens, CD↗

PubMed1999

Osteoblast population migration characteristics on substrates modified with immobilized adhesive peptides.

The process of cell migration is inextricably linked with the process of cell adhesion and, therefore, with cell/substrate adhesiveness. The present study adapted an under-agarose cell migration assay to quantitatively examine population migration characteristics of osteoblasts, on substrates modified with adhesive peptides, in the absence and presence of growth factors. Short-term, that is, 48 h osteoblast migration distances on substrates modified with adhesive Arg-Gly-Asp-Ser peptides were significantly (P < 0.05) less than migration distances on substrates modified with non-adhesive Arg-Asp-Gly-Ser peptides, demonstrating that osteoblast population haptokinesis was significantly decreased on substrates modified with adhesive peptides. Random motility coefficients calculated in the present study for osteoblast populations were an order of magnitude lower than a published random motility coefficient for leukocytes, proving quantitatively that, compared to leukocytes, osteoblasts migrate via haptokinesis more slowly. The 48 and 72 h osteoblast population migration differentials in the presence of an initial mass of 60 ng of basic Fibroblast Growth Factor, on substrates modified with Arg-Gly-Asp-Ser or with Arg-Asp-Gly-Ser, were larger than all other chemotactic differentials on these substrates. Quantitative investigations (such as the present study) of cell population migration characteristics on model biomaterial surfaces will become increasingly necessary as the discipline of cell/tissue engineering matures.

Adhesiveness↗

PubMed1998

Design and function of novel osteoblast-adhesive peptides for chemical modification of biomaterials.

Proactive, "next generation" dental/orthopedic biomaterials must be designed rationally to elicit specific, timely, and desirable responses from surrounding cells/tissues; for example, such biomaterials should support and enhance osteoblast adhesion (a crucial function for anchorage-dependent cells). In the past, integrin-binding peptides have been immobilized on substrates to partially control osteoblast adhesion; the present study focused on the design, synthesis, and bioactivity of the novel peptide sequence Lys-Arg-Ser-Arg that selectively enhances heparan sulfate-mediated osteoblast adhesion mechanisms. Osteoblast, but not endothelial cell or fibroblast, adhesion was enhanced significantly (p < 0.05) on substrates modified with Lys-Arg-Ser-Arg peptides, indicating that these peptides may be osteoblast- or bone cell specific. Blocking osteoblast cell-membrane receptors with various concentrations of soluble Arg-Gly-Asp-Ser peptides did not inhibit subsequent cell adhesion on substrates modified with Lys-Arg-Ser-Arg peptides, providing evidence that osteoblasts interact with Arg-Gly-Asp-Ser and with Lys-Arg-Ser-Arg peptides via distinct (i.e., integrin- and proteoglycan-mediated) mechanisms, each uniquely necessary for osteoblast adhesion. The present study constitutes an example of rational design/selection of bioactive peptides, confirms that osteoblast adhesion to substrates can be controlled selectively and significantly by immobilized peptides, and elucidates criteria and strategies for the design of proactive dental/orthopedic implant biomaterials.

Adhesiveness↗

PubMed1997

Quantitative characterization of vascular endothelial cell morphology and orientation using Fourier transform analysis.

Fourier Transform methods were used to quantify mean elongation, mean orientation, and standard deviation of orientations of cultured vascular endothelial cells. Images of cell populations, which had been subjected to 11 and 20 hours of shear stress at 30 dynes/cm2 and 20 hours of no shear, were analyzed by Fourier Transform methods. Measurements of cell morphology and orientation characteristics were also obtained using a manual method for comparison purposes. The results of the study showed that mean cell orientation can be determined accurately with the Fourier Transform methods. Attempts to determine the standard deviation of cell orientations, however, resulted in poorer estimates of mean elongation and standard deviation of orientations except in the case of exposure of endothelial cells to 20 hours of shear, where the actual standard deviation of orientations was low. When the value for standard deviation of orientations was constrained to zero, a minimum possible mean elongation was determined reliably using the Fourier Transform methods. Use of the Fourier Transform methods in determining morphological and orientation characteristics of cell monolayers is fast and objective and may provide a basis for identifying other characteristics of cell shape.

Animals↗

PubMed1997

Analysis and modeling of the primary cilium bending response to fluid shear.

Since a nonmotile, primary (9 + 0) cilium projects from most mammalian kidney epithelial cells into the tubule lumen, where it is exposed to fluid motion, the present study examined primary cilium response to fluid shear stress. The reversible, large-angle bending of the primary cilium upon exposure to fluid shear forces (10(-11)-10(-10) N.m2 = 10(-8)-10(-7) dyn/cm) was characterized in vitro using videomicroscopic side views of PtK1 cells, and the cilium was then mathematically modeled as a cantilevered beam. The flexural rigidity of the primary cilium was calculated to be 3.1 +/- 0.8 x 10(-23) N.m2 with a corrected quadruple integration approach and 1.4-1.6 x 10(-23) N.m2 with the "heavy elastica" theory. Comparison of theoretical profiles to the experimental bending responses of cilia established the validity of the "heavy elastica" model; this model, in turn, was used to predict primary cilium bending behavior under representative conditions in the rat nephron. The results of the study are consistent with the hypothesis that primary cilia serve a mechanosensory function in kidney epithelial cells.

Animals↗

PubMed1996

Surfaces modified with covalently-immobilized adhesive peptides affect fibroblast population motility.

Cell population motility and adhesion of rat skin fibroblasts were evaluated on aminophase glass modified with covalently-immobilized biologically active peptides, specifically, either arginine glycine-aspartic acid-serine (RGDS) or tyrosine-isoleucine-glycine-serine-arginine-glycine (YIGSRG). Fibroblast population motility was decreased and adhesion was increased on substrates modified with covalently immobilized RGDS peptide compared to substrates with the covalently immobilized non-adhesive peptides arginine-glycine-glutamic acid-serine and arginine-aspartic acid-glycine-serine. Fibroblast motility was not significantly changed on substrates modified with covalently-immobilized YIGSRG peptide; however, fibroblast adhesion was decreased on that substrate.

Amines↗

PubMed1996

Conditions which promote mineralization at the bone-implant interface: a model in vitro study.

This in vitro study was an investigation of osteoblast functions on glass substrates modified with the bioactive peptide Arg-Gly-Asp-Ser (RGDS) in the absence and presence of recombinant human Osteogenic Protein-1 (OP-1); control substrates were plain glass, glass modified with amine groups, and glass modified with the non-adhesive peptide Arg-Asp-Gly-Ser. In serum-free cell culture medium, osteoblasts adhered in greater numbers (P < 0.1) to glass modified with RGDS, compared to adhesion on all other substrate types tested in the present study. In the presence of serum proteins, osteoblasts adhered similarly to all substrate types examined, in the absence or presence of 100 ng ml-1 OP-1. The presence of 100 ng ml-1 OP-1 inhibited (P < 0.1) 72 h proliferation of sparsely seeded (2500 cells cm-2) cultures on all substrates examined in the present study. OP-1 (100 ng ml-1) promoted 21 day mineralization on all substrates examined; in addition, mineralization was further enhanced in osteoblast cultures grown on glass modified with the adhesive peptide RGDS. The present study establishes conditions which can be utilized in the design of dental/orthopaedic biomaterials which elicit timely, specific responses from surrounding bone tissue.

Amino Acid Sequence↗

PubMed1996

Analysis of osteoblast mineral deposits on orthopaedic/dental implant metals.

Neonatal rat calvarial osteoblasts were cultured on Ti-6Al-4V, Co-Cr-Mo alloy, 316L stainless steel and polystyrene (reference substrate) in the presence of ascorbic acid and 10 mM beta-glycerophosphate for 16, 17, 18, 19, 20, 21, 24 and 28 d. Scanning electron microscopy examination revealed that osteoblasts cultured on these orthopaedic/dental implant metals synthesized and deposited an extracellular matrix containing collagenous and non-collagenous components, as well as mineral nodules of various morphologies. Energy dispersive spectrometry revealed that the mineral deposits consisted of three distinct chemical compositions: calcium phosphate, calcium-sulphur-phosphorus, and calcium only. Backscattered electron imaging demonstrated that both the calcium phosphate and calcium-only deposits were electron dense, while the calcium-sulphur-phosphorus deposits were electron translucent. X-ray diffraction analysis indicated that the bulk of the osteoblast mineral deposits was amorphous hydroxyapatite; in addition, electron diffraction analysis revealed small regions of crystalline hydroxyapatite.

Alloys↗

PubMed1995

A transmission electron microscopy examination of the interface between osteoblasts and metal biomaterials.

Transmission electron microscopy was used to examine the interface between metal implant materials and bone cells. Specifically, neonatal rat calvaria osteoblasts were cultured on CoCrMo alloy and on 316L stainless steel discs (mechanically polished to a 0.3 micron finish) in Dulbecco's Modified Eagle Medium (supplemented with 10% fetal bovine serum, 50 micrograms/mL ascorbic acid, and 10 mM beta-glycerophosphate) under standard, sterile, cell culture conditions for 14 to 28 days. At the end of the prescribed time periods, the cells were fixed and embedded in resin before removing the metal substrates using an electrolytic dissolution technique and a 7% NaCl solution. Transmission electron microscopic examination of stained, ultrathin sections of the biological samples revealed an intact interface with microscopic details characteristic to the cell line and similar to those reported in the literature for animal and explant studies. The osteoblasts exhibited continuous contact and intimate apposition to both the CoCrMo and stainless steel substrate surfaces and grew in multilayered structures; an electron dense layer (composed of mucopolysaccharides and proteins) was observed at the surface of both substrates; collagen fibrils and mineralized foci were observed in the extracellular matrix interspersed among the multilayered osteoblasts.

Animals↗

PubMed1994

Osteoblast function on synthetic biodegradable polymers.

Rat osteoblasts were cultured on films of biodegradable poly(L-lactic acid) (PLLA), 75:25 poly(DL-lactic-co-glycolic acid) (PLGA), 50:50 PLGA, and poly(glycolic acid) (PGA) for up to 14 days. Osteoblasts attached equally well to all the polymer substrates after 8 h in culture. By day 4 in culture, osteoblasts had exceeded confluency numbers, and their proliferation leveled off by day 7. An increase in alkaline phosphatase (ALP) activity from 1.92 (+/- 0.47) x 10(-7) for day 7 to 5.75 (+/- 0.12) x 10(-7) mumol/cell per min for day 14 was reported for osteoblasts cultured on 75:25 PLGA, which was comparable to that observed for tissue culture polystyrene (TCPS) controls. The ALP activities expressed by osteoblasts cultured on PLLA, 50:50 PLGA, and PGA films did not significantly increase over time. Collagen synthesis for osteoblasts cultured on all polymer substrates was similar to that of TCPS and did not vary with time. The morphology of cultured osteoblasts was not affected by the continuous degradation of the polymer substrates. These results demonstrate that poly(alpha-hydroxy esters) can provide a suitable substrate for osteoblast culture and hold promise in bone regeneration by osteoblast transplantation.

Alkaline Phosphatase↗

PubMed1993

Osteoblast attachment monitored with a quartz crystal microbalance.

A quartz crystal microbalance is used in aqueous solutions to monitor the rate of attachment of osteoblasts, bone-forming cells, to the surface of the crystal. Changes in resonant frequency of the crystal are measured for various surface coverages by osteoblasts. Crystal surface coverages are determined by digital image processing of scanning electron micrographs. A linear relationship is established between the surface coverages and the changes in resonant frequency of the crystal. The osteoblasts are observed to behave viscoelastically. Hence, the Sauerbrey equation can not be used to describe the relationship between the change in mass of osteoblasts on the surface and the change in resonant frequency of the crystal. Apparent viscosities at 5.0 MHz are also determined for osteoblasts.

Animals↗

PubMed1993

Morphological and proliferative responses of endothelial cells to hydrostatic pressure: role of fibroblast growth factor.

Subconfluent bovine pulmonary artery endothelial cells on rigid substrates were exposed to 1.5-15 cm H2O sustained hydrostatic pressure for up to 7 days and exhibited elongation, cytoskeletal rearrangement, increased cell proliferation, and bilayering. The role of basic fibroblast growth factor (bFGF) in the mechanism(s) of these endothelial cell responses to sustained hydrostatic pressure was investigated. Evidence that bFGF was released from endothelial cells exposed to sustained hydrostatic pressure or compression was provided by the following experimental results: 1) Cells exposed to control (3 mm H2O) pressure displayed intense nuclear and cytoplasmic bFGF staining by immunocytochemical techniques; this staining was absent in cells exposed to 10 cm H2O for 7 days. 2) Conditioned medium from endothelial cells exposed to 10 cm H2O for 7 days contained a transferable, growth-promoting activity exhibiting heparin-Sepharose affinity, lability to both heat and freeze/thawing, and neutralization by anti-bovine bFGF. 3) Suramin (0.1 mM), a growth-factor receptor inhibitor, abrogated the proliferative and morphological responses of endothelial cells exposed to sustained hydrostatic pressure. Endothelial cells exposed to elevated hydrostatic pressure demonstrated no detectable decrement in cell viability as assessed by Trypan blue exclusion. The results of the present study indicate that hydrostatic pressure or compression can induce bFGF release from endothelial cells independent of cell injury or death; bFGF is subsequently responsible for the morphological, proliferative, and bilayering responses of endothelial cells to hydrostatic pressure.

Analysis of Variance↗

PubMed1993

Examination of osteoblast-orthopaedic biomaterial interactions using molecular techniques.

Molecular techniques can be used to elucidate the effects of extended periods of cell-biomaterial interactions on the time-course and level of expression of particular genes which determine cellular phenotype. We used the polymerase chain reaction to demonstrate the expression of genes for the bone-related proteins osteocalcin, osteonectin and osteopontin by neonatal rat calvarial osteoblasts. In addition, Northern blotting was subsequently used to show that messenger RNAs encoding osteonectin and osteopontin were consistently expressed during a 5 wk period of interaction of osteoblasts with Ti-6Al-4V, a commercial brand of hydroxyapatite, and tissue culture polystyrene.

Alloys↗

PubMed1992

Collagen-induced rat platelet reactivity is enhanced in whole blood in both the presence and absence of dense granule secretion.

Collagen induced aggregation, ATP secretion and thromboxane (TxB2) generation of storage pool deficient platelets were compared to normal platelets of closely related rat strains. Platelet function was monitored in citrated-platelet-rich-plasma (PRP) and citrated whole blood. Wistar (W) and fawn-hooded (FH) rat strains and their F2 hybrids were utilized. The W strain, which is ancestral to the FH strain, is not storage pool deficient while the FH strain is. This was manifested by the total lack of collagen induced ATP secretion from platelets of the FH strain while the platelets of the W strain secreted normally. Utilizing platelets from the F2 generation of WxFH matings, the absence of dense granule secretion (ATP) from the FH platelets, as well as other platelet defects of FH rats, were shown to be associated with homozygosity for the red-eyed dilution gene [r]. The non-secreting FH platelets were utilized to determine the effects of secreted dense granule constituents upon collagen induced aggregation and TxB2 generation. The non-secreting storage pool deficient platelets did aggregate and did generate TxB2 upon stimulation with collagen; however, the storage pool deficient FH platelets demonstrated less TxB2 generation and did not aggregate as effectively as the normally secreting platelets of the W strain. When evaluating collagen induced platelet function in whole blood as compared to PRP, the storage pool deficient platelets remained less reactive than normally secreting platelets, but both platelet types demonstrated enhanced aggregation and increased TxB2 generation in whole blood.

Adenosine Triphosphate↗

PubMed1992

Formation of focal contacts by osteoblasts cultured on orthopedic biomaterials.

The nature of the contact sites formed during the adhesion of osteoblasts to orthopedic implant materials was investigated by fluorescence microscopy. More specifically, the cytoskeletal organization of and the focal contact formation by neonatal rat calvarial osteoblasts attaching to and spreading on 316L stainless steel, Ti-6Al-4V, Co-Cr-Mo, Synamel (hydroxyapatite), alumina, and borosilicate glass were examined. Focal contacts are regions where the plasma membrane approaches the substrate to within 10-15 nm and where bundles of cytoskeletal microfilaments terminate. Fluorescent-labeling of F-actin-containing microfilaments demonstrated a typical sequence of events as rounded, suspended osteoblasts spread onto the substrates. Immunofluorescent-labeling of the protein vinculin, which is found at the cytoplasmic face of focal contacts, initially showed the formation of streak-like focal patches. On the biomaterials, the vinculin staining subsequently extended up and along, but ventral to, the microfilament bundles. The fibrillar patterns observed at later times may evidence the formation of extracellular matrix contacts.

Actin Cytoskeleton↗

PubMed1992

Osteoblasts on hydroxyapatite, alumina and bone surfaces in vitro: morphology during the first 2 h of attachment.

The morphological responses of individual osteoblasts as they attached and spread on hydroxyapatite, bovine bone, alumina with rough and polished surfaces, and tissue culture polystyrene in vitro were examined with scanning electron microscopy. Depending on the surface tested two different morphological sequences were observed during 2 h of adhesion. On alumina, both rough and smooth, bone, and tissue culture polystyrene the cells were round after 0.5 h, and spread radially during the next 1.5 h until they were almost flat, with a nuclear bulge. On hydroxyapatite, however, the cells were flat and circular at 0.5 h, and the edge of the cytoplasm was hardly discernable. This morphology did not change much during the subsequent 1.5 h. The observed cellular morphological response may be related to the bioreactivity of hydroxyapatite.

Aluminum Oxide↗

PubMed1992

Mechanisms of fibronectin-mediated attachment of osteoblasts to substrates in vitro.

Adhesive proteins of plasma and the extracellular matrix, such as fibronectin, adsorbed onto surfaces mediate cell/substrate adhesion. In a series of experiments, the roles of the type III connecting segment (IIICS) adhesion sites (specifically, CS1 and CS5 peptides) of fibronectin, heparan sulfate proteoglycan, endogenous proteins, and passive attachment in fibronectin-mediated osteoblast attachment were examined in vitro. The CS1 and CS5 peptides of the IIICS of fibronectin had no effect on osteoblast attachment. Blocking the heparin-binding domains of fibronectin inhibited osteoblast attachment by 40-45%, which is complementary to inhibition results previously obtained with the RGDS tetrapeptide. Endogenously synthesized and secreted proteins played a role in maintaining and repairing the osteoblast surface. Osteoblast attachment to fibronectin, but not to the nonadhesive protein albumin, occurred via active mechanisms in that the process was dependent on free sulfhydryl groups, divalent cations and temperature.

Animals↗

PubMed1991

Osteoblast responses to orthopedic implant materials in vitro.

Responses of neonatal rat calvarial osteoblasts to a variety of orthopedic implant materials were examined in vitro. Attachment, proliferation, and collagen synthesis of a well-characterized line of osteoblasts with 316L stainless steel, Ti-6Al-4V, Co-Cr-Mo, PMMA, hydroxyapatite, borosilicate glass, and tissue culture polystyrene were studied. Cell adhesion and growth were similar on nonapatitic materials. In contrast, attachment and growth of osteoblasts were significantly lower and slower, respectively, on hydroxyapatite. Collagen synthesis per cell and relative collagen synthesis, however, were comparable on all the materials tested.

Animals↗