Hiran Perinpanayagam

Implant Surface Roughness Affects Osteoblast Gene Expression

The transcription factor Cbfa1 regulates osteoblast differentiation and expression of genes necessary for the development of a mineralized phenotype. The purpose of this study was to determine if Cbfa1 and BSPII gene expression are influenced by implant surface microtopography. Osteoblasts were cultured on 600-grit (grooved) or sandblasted (roughened) cpTi implant discs. Mineralization was evaluated by Alizarin-Red-S staining. Real Time PCR was used for quantitative analysis of Cbfa1 and BSPII gene expression. Enhanced mineralization was seen in osteoblasts grown on roughened implant surfaces relative to tissue culture plastic. Real Time PCR showed significant (P < 0.05) increases in Cbfa1 gene expression in cells grown on roughened, as compared with grooved, implant surfaces. BSPII gene expression was also increased on rough surfaces in the UMR cells, but was reduced in the rat calvarial osteoblast cultures. These results suggest that osteoblast gene expression and mineralization are affected by roughened implant surface microtopographies during osseointegration of dental implants.

Association of Free Arginine and Lysine Concentrations in Human Parotid Saliva with Caries Experience

We determined the free-amino acid content of stimulated parotid (ductal) saliva from two groups of adult subjects whose caries experiences were markedly different. The levels of free arginine and free lysine in the parotid saliva of caries-free adults were significantly higher than those found in the parotid saliva of individuals with a history of dental decay. There was no correlation, however, between the levels of these amino acids and the DMFS score within the caries-susceptible groups. Microbial catabolism of dibasic amino acids contributes to the neutralization of plaque acids and may partially account for the higher resting plaque pH observed in caries-free subjects. Alternatively, the elevations observed in free levels of arginine and lysine may reflect a systemic alteration in amino acid metabolism which is common to the caries-free group of subjects.

pH Regulation of Urease Levels in Streptococcus salivarius

Potential mechanisms for regulation of urease levels in Streptococcus salivarius were examined, including: induction by urea, nitrogen or carbon source repression, and effects of pH and CO2 (because CO2 enrichment enhanced urease detection on urea agar plates). Regulation by either pH or CO2 was confirmed by comparison of the urease accumulation pattern during anaerobic growth under CO2 with that under N2. Under CO2, there was an initial buffering plateau at pH 6.2 and a rate of Streptococcus salivarius urease accumulation three-fold that under N2, with a pH 7.6 plateau. With both gas phases there was also an increase in the rate of urease appearance coincident with the decrease in medium pH following the pH plateau. The effects of pH, CO2, and HCO3- on urease levels and on growth were separately assessed by culture in media containing 0, 25, 100 mmol/L KHCO3 buffered at different pH levels. There was an inverse relationship between the logarithm of the urease level after 24-hour growth and the pH during growth-the urease specific activity was 100-fold higher at pH 5.5, compared with pH 7.0 and above. HCO3-/CO2 (100 mmol/L) had little effect on urease levels, but was essential for growth at pH 5.5. There was no significant urease induction by urea, or repression by ammonia or glucose. There was also evidence of pH regulation of urease levels in some staphylococci, Klebsiella pneumonia, and Corynebacterium renale, but not in Actinomyces naeslundii and several other species. We conclude that the external pH is a major factor regulating urease levels in S. salivarius and possibly some other species-a mechanism equivalent to urease repression by OH-.

Characterization of Low-molecular-weight Peptides in Human Parotid Saliva

The low-molecular-weight components of human saliva remain poorly characterized. Therefore, low-molecular-weight peptides (Mr < 3000) have been purified from human parotid saliva and characterized with respect to their amino acid sequence. From the sequences obtained, it is likely that these peptides are derived from proteolysis of the hydroxyapatite-interactive human salivary proteins, histatins, proline-rich proteins, and statherins. Since human parotid saliva is an amicrobial fluid, much of the low-molecular-weight peptide fraction of this secretion appears to be derived from the proteolytic processing of the larger proteins. Because of their small size, these peptides are likely to be in exchange with dental plaque fluid and may therefore help modulate events such as demineralization/remineralization, microbial attachment, and dental plaque metabolism at the tooth-saliva interface.

The bacteria responsible for ureolysis in artificial dental plaque.

The origin of ureolytic activity in artificial-mouth plaques was established by assessing the contribution to plaque ureolytic activity of the isolated bacteria. To overcome losses of ureolytic activity caused by the unstable presence of urease in oral bacteria, ureolytic bacteria were isolated from an exceptionally active plaque (1 mumol NH3/min per mg protein) in which 63 per cent of the flora was ureolytic. After their ability to metabolize urea was stabilized, 13 ureolytic bacteria remained: seven strains of Streptococcus salivarius, one Streptococcus bovis, two Staphylococcus epidermidis and three Staphylococcus haemolyticus. Their urease activity, measured after growth into stationary phase, was reproducible and strain specific with a 20-fold range within each genus. The mean ureolytic activity of each species, when weighted by its calculated incidence in the original plaque, accounted for 40 per cent of the total plaque ureolytic activity. However, these values for urease levels were only a small fraction of the bacterial ureolytic potential. Urease per mg cell protein measured during the growth cycle of a selected Strep. salivarius, and Staph. epidermidis, varied 10-fold, and reached much higher activities (i.e. 6-8 mumol NH3/min per mg of cell protein) than under the growth conditions that were used to assess the contribution of these species to total plaque ureolysis. Thus urea metabolism in artificial plaque was due mainly to Strep. salivarius, with a small contribution from Staph. epidermidis. The presence of further unidentified species of ureolytic oral bacteria need not be invoked.

Osteoblasts interact with MTA surfaces and express Runx2

OBJECTIVE Periradicular healing involves osteoblasts that are dependent on the Runt-related transcription factor 2 (Runx2). The purpose of this study was to determine if mineral trioxide aggregate (MTA) root-end filling materials support Runx2 expression in osteoblasts. STUDY DESIGN Human alveolar bone cells were grown on alternative formulations of MTA. Cell-surface interactions were visualized by scanning electron microscopy. Gene expression was examined by reverse-transcription polymerase chain reaction and Western blot analysis. RESULTS Cells attached to and spread out on MTA surfaces within 24 hours and formed a collagenous matrix overlay within 1 week of growth. Runx2 expression increased from low levels in the 24-hour cultures to an abundance during 2 weeks of growth and differentiation on MTA surfaces and on tissue culture plastic controls. The cells responded similarly to ProRoot, Tooth-Colored MTA, and MTA mixed with local anesthetic solution. CONCLUSION Mineral trioxide aggregate materials support cell attachment and Runx2 expression in osteoblasts.

Three-dimensional analysis of maxillary first molar mesiobuccal root canal configuration and curvature using micro-computed tomography.

OBJECTIVE The complex anatomic configuration of the maxillary first molar mesiobuccal (MB) root canal system has been the subject of several studies. The purpose of this investigation was to analyze the 3-dimensional (3D) characteristics of the maxillary first molar MB canal system using micro-computed tomography (microCT). STUDY DESIGN Extracted maxillary first molars (46) were scanned by microCT and their canals reconstructed by 3D modeling software. RESULTS In these MB roots, nearly two-thirds (65.2%) had 2 canals, fewer than one-third (28.3%) had only 1 canal, and a few (6.5%) had 3 canals. The most common root canal configuration was 2 distinct canals (type III: 37.0%), followed by 1 single canal (type I: 28.3%), 2 canals that joined together (type II: 17.4%), 1 canal that split into 2 (type IV: 10.9%), and 3 canals (type V: 6.5%). CONCLUSION MicroCT provided an in-depth analysis of canal configurations, as well as length, curvature, and location of calcified segments.

Early cell adhesion events differ between osteoporotic and non-osteoporotic osteoblasts.

In osteoporosis, the regenerative capacity of bone is compromised, which may involve altered osteoblast (OB) activity. This could be attributed to an inappropriate synthesis and assembly of an extracellular matrix (ECM), altered cell adhesion to the ECM, or be due to inappropriate downstream activation of adhesion‐mediated signaling cascades through proteins such as focal adhesion kinase (FAK). The purpose of our study was to compare early adhesion‐mediated events using previously described and characterized clinically derived OBs obtained from human patients undergoing major joint arthroplasty for osteoporosis or osteoarthritis. The presence or absence of osteoporosis was established with a radiographic index. Using light microscopy and crystal violet staining, we show that OB cells derived from sites of osteoporosis do not attach and spread as well as non‐osteoporotic (OP) OB cells. OP cells initially have a more rounded morphology, and show significantly less (P < 0.001) attachment to serum‐coated tissue culture plastic over a 24 h time period. Immunofluorescent labeling after 24 h of attachment showed that OP OB focal adhesions (FAs) and stress fibers were less defined, and that the OP cells were smaller and had a more motile phenotype. When normalized protein lysates were Western blotted for phosphotyrosine (PY) a band corresponding to pp125FAK was identified. FAK tyrosine phosphorylation was evident at 6 h in both the OP and non‐OP OBs, but decreased or was absent through 24 h in OP OBs. These results suggest early adhesion‐mediated events, such as cell adhesion, attachment, and FAK signaling via PY may be altered in OP OBs.

Comparison of Alternative Image Reformatting Techniques in Micro–Computed Tomography and Tooth Clearing for Detailed Canal Morphology

INTRODUCTION Micro-computed tomography (MCT) shows detailed root canal morphology that is not seen with traditional tooth clearing. However, alternative image reformatting techniques in MCT involving 2-dimensional (2D) minimum intensity projection (MinIP) and 3-dimensional (3D) volume-rendering reconstruction have not been directly compared with clearing. The aim was to compare alternative image reformatting techniques in MCT with tooth clearing on the mesiobuccal (MB) root of maxillary first molars. METHODS Eighteen maxillary first molar MB roots were scanned, and 2D MinIP and 3D volume-rendered images were reconstructed. Subsequently, the same MB roots were processed by traditional tooth clearing. Images from 2D, 3D, 2D + 3D, and clearing techniques were assessed by 4 endodontists to classify canal configuration and to identify fine anatomic structures such as accessory canals, intercanal communications, and loops. RESULTS All image reformatting techniques in MCT showed detailed configurations and numerous fine structures, such that none were classified as simple type I or II canals; several were classified as types III and IV according to Weine classification or types IV, V, and VI according to Vertucci; and most were nonclassifiable because of their complexity. The clearing images showed less detail, few fine structures, and numerous type I canals. Classification of canal configuration was in 100% intraobserver agreement for all 18 roots visualized by any of the image reformatting techniques in MCT but for only 4 roots (22.2%) classified according to Weine and 6 (33.3%) classified according to Vertucci, when using the clearing technique. CONCLUSIONS The combination of 2D MinIP and 3D volume-rendered images showed the most detailed canal morphology and fine anatomic structures.

Titania-polymeric powder coatings with nano-topography support enhanced human mesenchymal cell responses

Titanium implant osseointegration is dependent on the cellular response to surface modifications and coatings. Titania-enriched nanocomposite polymeric resin coatings were prepared through the application of advanced ultrafine powder coating technology. Their surfaces were readily modified to create nano-rough (<100 nm) surface nano-topographies that supported human embryonic palatal mesenchymal cell responses. Energy dispersive x-ray spectroscopy confirmed continuous and homogenous coatings with a similar composition and even distribution of titanium. Scanning electron microscopy (SEM) showed complex micro-topographies, and atomic force microscopy revealed intricate nanofeatures and surface roughness. Cell counts, mitochondrial enzyme activity reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to dark purple, SEM, and inverted fluorescence microscopy showed a marked increase in cell attachment, spreading, proliferation, and metabolic activity on the nanostructured surfaces. Reverse Transcription- Polymerase Chain Reaction (RT-PCR) analysis showed that type I collagen and Runx2 expression were induced, and Alizarin red staining showed that mineral deposits were abundant in the cell cultures grown on nanosurfaces. This enhancement in human mesenchymal cell attachment, growth, and osteogenesis were attributed to the nanosized surface topographies, roughness, and moderate wetting characteristics of the coatings. Their dimensional similarity to naturally occurring matrix proteins and crystals, coupled with their increased surface area for protein adsorption, may have facilitated the response. Therefore, this application of ultrafine powder coating technology affords highly biocompatible surfaces that can be readily modified to accentuate the cellular response.