Treatment of peri-implant defects with combination growth factor cement.

BACKGROUND: The use of growth factor agents in the regeneration of oral tissues is an area of current investigation. Combinations of growth factors have been used synergistically to improve tissue regeneration. The aim of this study was to determine the effects of a combination growth factor cement (GFC) on guided bone regeneration around dental implants. METHODS: A combination of bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) was used in a bioabsorbable, non-hydroxyapatite, calcium phosphate cement. Five adult hound dogs were used to compare the effects of GFC, plain cement, and control (no cement). The right and left second, third, and fourth mandibular premolar teeth were extracted; the implant osteotomies were prepared; and a uniform circumferential gap was prepared 1.5 mm beyond the width of the implant in the coronal half of the osteotomy for cement placement. Titanium machine-polished dental implants were placed in the prepared sites, and coronal defects were treated according to previously randomized, assigned modality. A bioabsorbable collagen membrane was secured over the control site, and the flaps were closed primarily. The dogs were maintained on a soft diet to avoid soft tissue trauma. The dogs were sacrificed at 3 months. The specimens were sectioned, mounted, and stained with Stevenel's blue and van Gieson's picric fuchsin. The bone-to-implant contact and bone 1 mm peripheral to the implant surface were recorded with a computerized microscopic digitizer. RESULTS: The findings of this study indicate a significant effect of GFC on increased bone-to-implant contact and amount of bone per surface area compared with the other treatment modalities (P <0.0009). Plain cement demonstrated slight but nonsignificant increases compared with the control (P>0.05). CONCLUSIONS: GFC increases bone-to-implant contact and bone surface area within peri-implant defects. Further studies may be beneficial to determine the feasibility of its use for other regenerative applications.

Pathogenesis of ascites tumor growth: vascular permeability factor, vascular hyperpermeability, and ascites fluid accumulation.

Previous studies have shown that accumulation of tumor ascites fluid results in large part from increased permeability of peritoneal lining vessels (Nagy et al., Cancer Res., 49: 5449-5458, 1989; Nagy et al., Cancer Res., 53: 2631-2643, 1993). However, the specific microvessels rendered hyperpermeable have not been identified nor has the basis of peritoneal vascular hyperpermeability been established. To address these questions, TA3/St and MOT carcinomas, well-characterized transplantable murine tumors that grow in both solid and ascites form, were studied as model systems. Ascites tumor cells of either type were injected i.p. into syngeneic A/Jax and C3Heb/FeJ mice, and ascites fluid and plasma were collected at intervals thereafter up to 8 and 28 days, respectively. Beginning several days after tumor cell injection, small blood vessels located in tissues lining the peritoneal cavity (mesentery, peritoneal wall, and diaphragm) became hyperpermeable to several macromolecular tracers (125I-human serum albumin, FITC-dextran, colloidal carbon, and Monastral Blue B). Increased microvascular permeability correlated with the appearance in ascites fluid of vascular permeability factor (VPF), a tumor cell-secreted mediator that potently enhances vascular permeability to circulating macromolecules. VPF was measured in peritoneal fluid by both a functional bioassay and a sensitive immunofluorometric assay. The VPF concentration, total peritoneal VPF, ascites fluid volume, tumor cell number, and hyperpermeability of peritoneal lining microvessels were found to increase in parallel over time. The close correlation of peritoneal fluid VPF concentration with the development of hyperpermeable peritoneal microvessels in these two well-defined ascites tumors suggests that VPF secretion by tumor cells is responsible, in whole or in part, for initiating and maintaining the ascites pattern of tumor growth.

Vascular permeability factor (vascular endothelial growth factor) in guinea pig and human tumor and inflammatory effusions.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor, is a dimeric M(r) 34,000-42,000 glycoprotein that possesses potent vascular permeability-enhancing and endothelial cell-specific mitogenic activities. It is synthesized by many rodent and human tumor cells and also by some normal cells. Recently we developed a sensitive and specific time-resolved immunofluorometric assay for quantifying VPF in biological fluids. We here report findings with this assay in guinea pigs and patients with both malignant and nonmalignant effusions. Line 1 and line 10 tumor cells were injected into the peritoneal cavities of syngeneic strain 2 guinea pigs, and ascitic fluid, plasma, and urine were collected at various intervals. Within 2 to 4 days, we observed a time-dependent, parallel increase in VPF, ascitic fluid volume, and tumor cell numbers in animals bearing either tumor line; in contrast, VPF was not detected in plasma or urine, even in animals with extensive tumor burdens. However, low levels of VPF were detected in the inflammatory ascites induced by i.p. oil injection. In human studies, high levels of VPF (> 10 pM) were measured in 21 of 32 effusions with cytology-documented malignant cells and in only seven of 35 effusions without cytological evidence of malignancy. Thus, VPF levels in human effusions provided a diagnostic test for malignancy with a sensitivity of 66% and a specificity of 80% (perhaps as high as 97% in that six of the seven cytology-negative patients with VPF levels > 10 pM had cancer as determined by other criteria). As in the animal tumor models, VPF was not detected in serum or urine obtained from patients with or without malignant ascites. Many nonmalignant effusions contained measurable VPF but, on average, in significantly smaller amounts than were found in malignant effusions. VPF levels in such fluids correlated strongly (p = 0.59, P < 0.001) with monocyte and macrophage content. Taken together, these data relate ascitic fluid accumulation to VPF concentration in a well-defined animal tumor system and demonstrate, for the first time, the presence of VPF in human malignant effusions. It is likely that VPF expression by tumor and mononuclear cells contributes to the plasma exudation and fluid accumulation associated with malignant and certain inflammatory effusions. The VPF assay may prove useful for cancer diagnosis as a supplement to cytology, especially in tumors that grow in the pleural lining but not as a suspension in the effusions that they induce.

Inhibition of vascular permeability factor (vascular endothelial growth factor) with antipeptide antibodies.

Vascular permeability factor (VPF), also known as vascular endothelial cell growth factor (VEGF), is a 34- to 43-kDa dimeric protein synthesized and secreted by a variety of tumor and normal cells. At nanomolar concentrations, VPF causes an increase in microvascular permeability and is thought to be responsible for enhanced permeability of tumor blood vessels and for the fluid accumulation associated with solid and ascites tumors. In addition, VPF/VEGF is a mitogen for endothelial cells and may play an important role in maintaining vascular endothelium and in promoting tumor angiogenesis. Antibodies were raised against a series of synthetic peptides derived from the predicted human VPF amino acid sequence. The antibodies were assayed for their ability to bind native and denatured/reduced VPF. Antibodies to peptides from the N- and C-termini bound both denatured/reduced and native VPF; antibodies directed to internal segments (e.g., amino acids 27-48 and 85-101) strongly bound denatured/reduced VPF but were substantially less effective at binding native VPF. These results suggest that the N- and C-termini are exposed regions of the protein in solution. Individually, antibodies to the N- and C-termini each partially blocked VPF permeability activity, and, in combination, blocked nearly 100% of this activity. Also, the N- and C-terminal antibodies blocked the VPF-mediated stimulation of both endothelial cell growth and increase in free cytosolic calcium.

Development of time-resolved immunofluorometric assay of vascular permeability factor.

We describe a two-site time-resolved immunofluorometric assay for guinea pig vascular permeability factor (VPF) for quantifying VPF in different biological fluids. Antibody against the carboxy terminus (C-IgG) is immobilized on microtiter wells, and antibody against the amino terminus (N-IgG) is labeled with Eu(3+)-chelate. Line 10 tumor culture medium, known to be rich in VPF, is assayed in a two-step incubation. Bound Eu3+ is then quantified by dissociation into a fluorescent enhancement solution, with measurement of the time-resolved fluorescence. The analytical sensitivity is 0.35 VPF unit, and the intra-assay CV is about 20%. The assay is specific for VPF, because pre-treatment with the appropriate C- or N-peptide, or pre-extraction of VPF, greatly decreases fluorescence. The VPF immunoassay is highly correlated (r2 = 0.94) with the Miles permeability assay, the classical bioassay of VPF. In addition, the immunofluorometric assay is about 30-fold more sensitive than the Miles assay.

Distribution of vascular permeability factor (vascular endothelial growth factor) in tumors: concentration in tumor blood vessels.

Vascular permeability factor (VPF) is a highly conserved 34-42-kD protein secreted by many tumor cells. Among the most potent vascular permeability-enhancing factors known, VPF is also a selective vascular endothelial cell mitogen, and therefore has been called vascular endothelial cell growth factor (VEGF). Our goal was to define the cellular sites of VPF (VEGF) synthesis and accumulation in tumors in vivo. Immunohistochemical studies were performed on solid and ascites guinea pig line 1 and line 10 bile duct carcinomas using antibodies directed against peptides synthesized to represent the NH2-terminal and internal sequences of VPF. These antibodies stained tumor cells and, uniformly and most intensely, the endothelium of immediately adjacent blood vessels, both preexisting and those newly induced by tumor angiogenesis. A similar pattern of VPF staining was observed in autochthonous human lymphoma. In situ hybridization demonstrated VPF mRNA in nearly all line 10 tumor cells but not in tumor blood vessels, indicating that immunohistochemical labeling of tumor vessels with antibodies to VPF peptides reflects uptake of VPF, not endogenous synthesis. VPF protein staining was evident in adjacent preexisting venules and small veins as early as 5 h after tumor transplant and plateaued at maximally intense levels in newly induced tumor vessels by approximately 5 d. VPF-stained vessels were also hyperpermeable to macromolecules as judged by their capacity to accumulate circulating colloidal carbon. In contrast, vessels more than approximately 0.5 mm distant from tumors were not hyperpermeable and did not exhibit immunohistochemical staining for VPF. Vessel staining disappeared within 24-48 h of tumor rejection. These studies indicate that VPF is synthesized by tumor cells in vivo and accumulates in nearby blood vessels, its target of action. Because leaky tumor vessels initiate a cascade of events, which include plasma extravasation and which lead ultimately to angiogenesis and tumor stroma formation, VPF may have a pivotal role in promoting tumor growth. Also, VPF immunostaining provides a new marker for tumor blood vessels that may be exploitable for tumor imaging or therapy.

2,5-Hexanedione-treated tubulin microinjected into sea urchin zygotes induces mitotic abnormalities.

Zygotes of Lytechinus pictus and Lytechinus variegatus were microinjected with 2,5-hexanedione (2,5-HD)-treated tubulin prior to the first mitotic cycle. Mitotic spindles were small with a well-defined metaphase plate, but poor birefringence and poor astral development. Abnormalities were observed in chromosome movement at anaphase and cytokinesis. Neither microinjections of untreated tubulin or 3-acetyl-2,5-hexanedione-treated tubulin, nor incubation of zygotes in 2,5-HD-containing sea water produced abnormalities. The results can be explained in terms of the nondissociating properties of 2,5-HD-treated tubulin. 2,5-HD-treated tubulin dissociates slowly from microtubules, a property which, besides favoring the formation of stable microtubules, allows this tubulin to induce microtubule assembly when present in substoichiometric amounts. These characteristics have been implicated as a cause of 2,5-HD-induced Sertoli cell dysfunction. The effect of 2,5-HD-treated tubulin on microtubule dynamics in sea urchin zygotes may bear similarities to the effects of 2,5-HD treatment in vivo on Sertoli cell microtubules.

The Sertoli cell cytoskeleton: a target for toxicant-induced germ cell loss.

Numerous studies in recent years have elucidated fundamental properties of axoplasmic structure, biochemistry, and function. The structural role of the cytoskeletal elements, the orientation of MTs within the axon, the phenomenon of MT-dependent transport, and the identity and direction of movement of two MT motors--kinesin and MAP-1C--have been revealed. For many years to come, researchers investigating the structure and function of the Sertoli cell cytoskeleton will be able to adapt techniques gleaned from work on the axonal cytoskeleton. Innovative thinking will be required to apply these techniques to the special circumstances of the male reproductive system; however, the underlying questions are similar. For example, knowledge of several fundamental properties of transport processes in the Sertoli cell would facilitate the toxicologic evaluation of this system. What is the orientation of MTs within the Sertoli cell cytoplasm? Are the fast-growing (+) ends of all MTs in the Sertoli cell cytoplasm directed toward the lumen? This is an important question because the direction of MT-dependent transport involving known MT motors is dependent upon the MT orientation. Which of the Sertoli cell transport pathways are MT-dependent pathways? What are the MT motors involved in these pathways? Ultrastructural examination following exposure to specific cytoskeleton-disrupting agents has highlighted the importance of AFs, IFs, and MTs in the Sertoli cell. Future research will focus on the nature of those molecules which integrate these cytoskeletal components into a dynamic whole, the regulatory systems which control this integration, and the role of an integrated cytoskeleton in Sertoli cell function and testicular homeostasis. Toxicology will be an active participant in this process of scientific discovery. The selective nervous system and testicular toxicants may be useful tools in revealing similarities in the cytoskeletal organization of these apparently disparate organ systems. By searching for common targets in the testis and nervous system, the mechanisms of action of these agents may be more easily, and more confidently, determined.

Selection of a nucleation-promoting element following chemical modification of tubulin.

Following a 16-h incubation with a large excess of 2,5-hexanedione (2,5-HD) while in the assembled state, bovine brain tubulin contained a powerful nucleating component, the presence of which lowered the dissociation rate from 83 s-1 for untreated tubulin to 13 s-1 for 2,5-HD-treated tubulin. This nucleating component could be selectively concentrated by sequential stringent (conditions of low temperature and low tubulin concentration) cycles of assembly and disassembly. In 2-(N-morpholino)ethanesulfonic acid buffer without glycerol, the critical concentration of assembly of untreated tubulin (2.4 mg/mL) was 19 times higher than that of 2,5-HD-treated tubulin subjected to three sequential stringent cycles of assembly and disassembly (0.13 mg/mL). This highly nucleating 2,5-HD-treated tubulin preparation could both copolymerize with untreated tubulin and seed subcritical concentration assembly of untreated tubulin. Experiments to define the assembly-altering component have identified structural alterations to the alpha-tubulin monomer. While the alpha-tubulin subunit of native untreated tubulin dimer contained no chymotryptic cleavage sites, the native 2,5-HD-treated alpha-tubulin subunit was cleaved by chymotrypsin to yield a 37-kDa C-terminal fragment.