Early inhibitory effects of zoledronic acid in tooth extraction sockets in dogs are negated by recombinant human bone morphogenetic protein.

PURPOSE: This study was conducted with 2 purposes. The first was to determine the effect of a single dose of zoledronic acid (ZA) on the healing of a tooth extraction socket in dogs. The second was to determine if placement of recombinant human bone morphogenetic protein-2 (rhBMP-2)/absorbable collagen sponge (ACS) - INFUSE, (Medtronic, Memphis, TN) into these extraction sockets would inhibit the inhibition on bone healing and remodeling by ZA. MATERIALS AND METHODS: Nine adult female beagle dogs (2 to 3 yr old) were placed into 3 groups of 3 dogs each. Group I received 15 mL of sterile saline intravenously; group II received 2.5 mg of ZA intravenously; and group III received 5 mg of ZA intravenously. Forty-five days after treatment, all dogs underwent extraction of noncontiguous right and left mandibular first molars and second premolars. In group I, the right mandibular extraction sockets had nothing placed in them, whereas the left mandibular sockets had only ACS placed in them. In groups II and III, the right mandibular sockets had rhBMP-2/ACS placed in them, whereas the left mandibular sockets had only ACS placed. All extraction sockets were surgically closed. Tetracycline was given intravenously 5 and 12 days later, and all animals were euthanized 15 days after tooth extraction. The extraction sockets and rib and femur samples were harvested immediately after euthanasia, processed, and studied microscopically. RESULTS: A single dose of ZA significantly inhibited healing and bone remodeling in the area of the tooth extractions. The combination of rhBMP-2/ACS appeared to over-ride some of the bone remodeling inhibition of the ZA and increased bone fill in the extraction sites, and remodeling activity in the area was noted. The effects of rhBMP-2/ACS were confined to the area of the extraction sockets because bone activity at distant sites was not influenced. CONCLUSIONS: A single dose of ZA administered intravenously inhibits early healing of tooth extraction sockets and bone remodeling in this animal model. The combination of rhBMP-2/ACS significantly increased bone fill and bone remodeling in these areas, negating much of the effect of the ZA.

Characterization of gene expression profiles of 3 different human oral squamous cell carcinoma cell lines with different invasion and metastatic capacities.

PURPOSE: The gene expression of 3 oral squamous cell carcinoma (OSCC) human cell lines, BHY, HN, and HSC-3, were studied based on their reported ability to invade adjacent bone or metastasize to cervical lymph nodes and/or distant organs. MATERIALS AND METHODS: The characteristics of each cell line were confirmed on scid mice using micro-positron emission tomography (PET)/computerized tomography (CT) imaging techniques. Complimentary DNA (cDNA) microarray techniques were used to determine the gene expression profile differences between each of the three OSCC cell lines. RESULTS: BHY, HN, and HSC-3 cell lines expressed 139, 214, and 128 up-regulated genes; and 117, 262, and 117 down-regulated genes, respectively. The clusterization of data showed that there are 13 genes that are up-regulated and 83 genes that are down-regulated in all 3 OSCC cell lines. Collection of genes organized by pathway may cause aggregate evaluation of anomalies. Thus the pathway analysis performed for each cell line based on cDNA microarray results showed BHY, HN, and HSC-3 cell lines to have 8, 10, and 3 up-regulated pathways and 3, 9, and 6 down-regulated pathways, respectively. CONCLUSIONS: This study showed that cDNA microarray analysis is an effective tool for mapping molecular signatures. With this technique it is possible to observe the entire genome of a malignant tumor so as to appreciate the simultaneous interactions among thousands of genes.

Characterization of 3 oral squamous cell carcinoma cell lines with different invasion and/or metastatic potentials.

PURPOSE: Proteolytic enzymes may confer specific types of invasion and metastasis in patients with oral squamous cell carcinoma (OSCC). The purpose of this study was to determine if OSCC that invades adjacent bone has different proteolytic enzyme expression profiles than OSCC that metastasizes to lymph nodes or distant organs. Three OSCC cell lines, BHY, HSC-3, and HN, with known behavior regarding bone invasion and lymph node and distant metastatic profiles, were evaluated. The characteristics of a control, human normal nasal epithelial cell line (HNEC), and BHY, HSC-3 and HN were evaluated with regard to their expression of the matrix metalloproteinases and cathepsins. MATERIALS AND METHODS: Expressions of proteolytic enzymes including matrix metalloproteinase, MMP-1, MMP-2, MMP-3, MMP-9, extracellular matrix metalloproteinase inducer (EMMPRIN), cathepsin B, and cathepsin L were compared using immunocytochemistry and flow cytometry in 3 OSCC cell lines and HNEC. The cell morphologies of these 4 cell lines were compared using transmission electron microscopy (TEM). RESULTS: All OSCC cell lines showed higher expression of all the proteolytic proteins when compared with HNEC, except the HSC-3 cell line showed no difference in the expression of MMP-9. There was no detectable difference at the expression level of MMP-1, MMP-2, MMP-3, cathepsin B, and cathepsin L in any of the OSCC cell lines. However, MMP-9 and EMMPRIN levels were higher in the BHY cell line. According to electron microscopy, the cells of the HSC-3 cell line were the smallest and least differentiated among the 3 OSCC cell lines. The BHY cell line was the most highly differentiated showing interdigitation and numerous cell junctions. CONCLUSIONS: MMPs play an important role in the invasion and metastasis of oral cancer. MMP-9 might play a more important role than MMP-2 during invasion. Increased expression of MMP-1, MMP-9, and EMMPRIN proteins might be involved in invasion of OSCC to adjacent bone, as they are necessary for the collagen matrix degradation. Increased expression of MMP-3, cathepsin B and L in OSCC might be associated with both invasion and a high incidence of metastasis.

"Burnishing" demineralization of root surfaces: ultrastructural surface characteristics.

PURPOSE: The purpose of this study was to compare the surface topography of roots treated with a resin bonding demineralizing agent using either a "placed" or "burnished" application technique. MATERIALS AND METHODS: Fifteen roots of human teeth were sectioned in half and a treatment area prepared on the coronal portion of each proximal section. This area was root planed to expose dentin. Treatment areas were demineralized with (1) a commercially available demineralizing agent (10% citric acid with 3% ferric chloride) (Amalgambond; Parkell) or (2) 30% citric acid solution. Cotton pellets saturated in either solution were placed or burnished (vigorously rubbed) on the treatment area for 3 min. Sections were prepared for SEM analysis using liquid CO2 dehydration. RESULTS: Areas of cementum and dentin were evident on most treatment areas. Specimens of both placed groups lacked a smear layer and exhibited a cracked-eroded, flat surface of matted or ridged fibrous material. Specimens in both burnished groups also lacked a smear layer, yet in stark contrast, exhibited an abundant array of deeply tufted fibril material similar to that of a "shag carpet". Two types of tufted fibril patterns were present: a lace-like array of shorter fibrils seen on dentin, and a voluminous mass of longer fibrils seen on cementum. CONCLUSION: Root cementum and dentin, treated with either demineralizing agent using the burnishing application technique, were ultrastructurally similar in that both displayed an abundant array of deeply tufted fibril material. This differed from the flat/matted fibril material seen using the placed application technique.

Giant platelet disorder in the Cavalier King Charles Spaniel.

OBJECTIVE: The aim of this study was to describe the clinical, functional, and morphologic characteristics of platelets in Cavalier King Charles Spaniel dogs (Cavaliers). MATERIALS AND METHODS: Blood from 69 clinically normal Cavaliers was collected and anticoagulated with ethylenediamine-tetraacetic acid (EDTA) and citrate. Automated and manual platelet counts were obtained. Percent platelet aggregation in response to ADP (2, 4, 8, 16, and 32 microM) was determined. Electron microscopy was performed to examine platelet internal morphology and dense granule distribution. A cardiologist recorded the quality of murmurs. RESULTS: Thrombocytopenia (<100,000/microL) was present in 51.43% (36/69) of Cavaliers. Macrothrombocytes (>3 microm) were present in 33.33% (22/69). Mean manual platelet count was 118,770/microL. Manual (EDTA blood) and automated (EDTA and citrated blood) methods of platelet counting were correlated. Prevalence of cardiac murmurs was 38% (26/69). There was no association between affected dogs and murmur, signalment, or coat color. Mean percent platelet aggregation was significantly higher in controls than in Cavaliers (79% vs 38%, p=0.001). Response to ADP was unaffected by thrombocytopenia, macrothrombocytes, murmur, or any combination thereof. Platelet electron microscopy showed normal and giant sized platelets with normal internal morphology. CONCLUSIONS: A benign inherited giant platelet disorder affects approximately 50% of Cavalier King Charles Spaniels. It is characterized by thrombocytopenia, macrothrombocytes, or decreased platelet aggregation in response to ADP. Platelet ultrastructure is normal. Citrated or EDTA blood provides accurate platelet counts. Further studies are indicated to determine platelet glycoprotein structure and any association with mitral endocardiosis. Cavaliers may be useful models of inherited giant platelet disorders.

HL-60 cell growth-conditioned medium is an effective inducer of myeloperoxidase expression in K-562 human leukemia cells.

K-562 cells were cultured in HL-60 cell growth-conditioned medium (GCM) for up to 96h. Myeloperoxidase (MPO) mRNA was transiently detected by reverse transcription-polymerase chain reaction (RT-PCR) techniques at 12, 24, and 48h. The de novo expression of MPO protein was subsequently detectable by intracellular flow cytometry at 24, 48, 72 and 96h. Immunogold staining and cytochemical analysis demonstrated granularly-sequestered MPO in approximately 40% of HL-60 GCM-cultured cells after 48h of culture. The sequential detection of MPO mRNA and MPO biosynthesis is considered an indicator of serial maturation evocative of myeloblastic cells, and suggest that K-562 cells maintain the ability to differentiate along this lineage.