Biomedical subjects

D A Puleo

Publications and source records attributed to D A Puleo.

28 records · Page 2Linked to original sources

PubMed1995

Effects of sublethal metal ion concentrations on osteogenic cells derived from bone marrow stromal cells.

Ions released from implant surfaces are suspected of playing some role in osteolysis surrounding metal prostheses. To understand how ions may affect osteogenesis, previous work exposed osteogenic cells to metal ions to study acute cytotoxic responses. The purpose of this study was to assess the long-term effects of sublethal ion concentrations on osteogenic cell proliferation and function. Bone marrow stromal cells were harvested from juvenile rats and exposed to solutions of ions associated with Co-Cr-Mo and Ti-6Al-4V implants. Cells were cultured for up to 4 weeks and assayed for total protein, alkaline phosphatase, osteocalcin, and calcium. Other than V+5, none of the ions affected cell proliferation, indicating that the chosen concentrations were sublethal as desired. V+5 elicited delayed gross toxicity not previously observed during acute experiments. At the chosen concentrations, Co+2, Cr+6, Mo+6, and Co-Cr-Mo alloy elicited little effect on cell proliferation and moderate effects on matrix mineralization. Cultures exposed to Ti+4, Al+3, and Ti-6Al-4V alloy also showed no decrease in cell number, but did show near total suppression of osteocalcin secretion and matrix mineralization. These results suggest that ions released from Ti alloy implants may interfere with osteoblastic cell differentiation, contributing to periprosthetic osteolysis by impairing normal osteogenesis.

Alkaline Phosphatase↗

PubMed1995

Activity of enzyme immobilized on silanized Co-Cr-Mo.

The surface of an orthopedic biomaterial was modified by the covalent immobilization of biomolecules. Derivatization of Co-Cr-Mo samples with organic and aqueous solutions of gamma-aminopropyltriethoxysilane (APS) resulted in a concentration-dependent number of reactive NH2 groups on the surface available for coupling to protein. The enzyme trypsin was used as a model biomolecule to investigate the effect of immobilization on proteolytic activity. Trypsin was coupled to the silanized samples by formation of Schiff's base linkages via glutaraldehyde. The nature of the interaction between trypsin and biomaterial was then probed by treatment with concentrated guanidine hydrochloride (GuHCl) and urea. Residual activity (following treatment with chaotropic agents) of trypsin immobilized on silanized Co-Cr-Mo was dependent both on the nature of the silane solution and on the type of chaotropic agent. Organic silanization with APS required a minimum density of approximately 49 NH2 per nm2 of nominal surface area (> 0.021 M APS) for residual activity of immobilized trypsin. For aqueous silanization, approximately 5.4 NH2/nm2 (0.51 M APS) resulted in maximal residual trypsin activity. Treatment with GuHCl removed more trypsin activity from Co-Cr-Mo samples silanized with organic solutions of APS than did treatment with urea. On the contrary, with aqueous silanization the samples possessed greater residual activity following treatment with GuHCl than following urea. Compared to simple adsorption with protein onto Co-Cr-Mo, both methods of silanization with APS resulted in superior residual immobilized enzyme activity.

Amines↗

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

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

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↗

PubMed1991

RGDS tetrapeptide binds to osteoblasts and inhibits fibronectin-mediated adhesion.

The mechanisms of osteoblast attachment to surfaces were probed using the adhesive tetrapeptide RGDS (Arg-Gly-Asp-Ser) and the related but non-adhesive RGES (Arg-Gly-Glu-Ser). Specifically, RGDS and RGES were investigated for their ability both to bind to a suspension of well-characterized neonatal rat calvarial osteoblasts and to inhibit cell attachment to fibronectin-coated microtiter plates. RGDS bound to the cells with an average Kd approximately 9.4 x 10(-4) M, and RGES bound with an average Kd approximately 3.0 x 10(-4) M; at saturation, the osteoblasts bound almost twice as much RGDS as RGES. RGDS partially inhibited cell adhesion (55% to 60%) in a competitive, dose-dependent manner. In contrast, RGES had minimal effect on cell attachment. Since complete inhibition of attachment was not observed, it is likely that a synergistic adhesion site in the fibronectin molecule and/or cell surface molecules such as proteoglycans are active in mediating osteoblast/substrate adhesion.

Amino Acid Sequence↗

PubMedDate not supplied

Biomaterials and biomechanics of oral and maxillofacial implants: current status and future developments.

Research in biomaterials and biomechanics has fueled a large part of the significant revolution associated with osseointegrated implants. Additional key areas that may become even more important--such as guided tissue regeneration, growth factors, and tissue engineering--could not be included in this review because of space limitations. All of this work will no doubt continue unabated; indeed, it is probably even accelerating as more clinical applications are found for implant technology and related therapies. An excellent overall summary of oral biology and dental implants recently appeared in a dedicated issue of Advances in Dental Research. Many advances have been made in the understanding of events at the interface between bone and implants and in developing methods for controlling these events. However, several important questions still remain. What is the relationship between tissue structure, matrix composition, and biomechanical properties of the interface? Do surface modifications alter the interfacial tissue structure and composition and the rate at which it forms? If surface modifications change the initial interface structure and composition, are these changes retained? Do surface modifications enhance biomechanical properties of the interface? As current understanding of the bone-implant interface progresses, so will development of proactive implants that can help promote desired outcomes. However, in the midst of the excitement born out of this activity, it is necessary to remember that the needs of the patient must remain paramount. It is also worth noting another as-yet unsatisfied need. With all of the new developments, continuing education of clinicians in the expert use of all of these research advances is needed. For example, in the area of biomechanical treatment planning, there are still no well-accepted biomaterials/biomechanics "building codes" that can be passed on to clinicians. Also, there are no readily available treatment-planning tools that clinicians can use to explore "what-if" scenarios and other design calculations of the sort done in modern engineering. No doubt such approaches could be developed based on materials already in the literature, but unfortunately much of what is done now by clinicians remains empirical. A worthwhile task for the future is to find ways to more effectively deliver products of research into the hands of clinicians.

Biocompatible Materials↗