Clark M. Stanford

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.

Calcium and phosphate supplementation promotes bone cell mineralization: Implications for hydroxyapatite (HA)-enhanced bone formation

Organic phosphate, in particular beta-glycerophosphate (beta-GP), has been used to induce mineralization in cell culture systems. It serves as a source of inorganic phosphate when hydrolyzed by alkaline phosphatase. This study examined the effect of supplemental calcium and phosphate as well as the influence of various metabolic inhibitors on mineralization in a rat osteoblast-like cell-culture system. Mineralization was induced by supplementation of 1.8 mM of Ca(+2) and 5 mM of beta-GP or Pi. Mineral deposits associated with in vitro mineralization were revealed under SEM and TEM. Levamisole (10-100 microM) inhibited alkaline phosphatase activity and effectively reduced mineral formation. Actinomycin (500 ng/mL) and cycloheximide (50 microg/mL) also reduced mineral depositions by blocking RNA synthesis and protein synthesis, respectively. Levamisole and beta-GP did not appear to influence DNA synthesis. Spontaneous precipitation of calcium phosphate mineral was not detected in the culture medium with calcium and phosphate supplements in the absence of cell culture. The findings suggest that an elevated concentration of calcium and phosphate is crucial for in vitro mineralization. Furthermore, the mineralization process is associated with biologic events rather than with a spontaneous precipitation of calcium phosphate mineral. In view of the degradation potential of hydroxyapatite (HA)-coated implants, these results may be a viable indication that HA enhances bone formation through a similar mechanism.

Osteoblast Integrin Adhesion and Signaling Regulate Mineralization

Integrin adhesion and signaling events may contribute to the progressive differentiation of the osteoblast and to the initiation of a mineralized matrix. The purpose of our study was to begin to analyze the role of integrin receptors, in particular α2β1, α5β1, and αVβ3, regarding mediation of the initiation of a mineralized matrix. Integrin-perturbation assays were conducted in microdot cultures of UMR-106-01 Bone Sialoprotein (BSP) osteoblast cells. For phenotypic analysis, we performed bright-field microscopy and Aliziran Red S staining to analyze effects on mineralization initiation. Mineralization was reduced significantly (P < 0.001) following the addition of a5- or β1-integrin subunit antibody by approximately 20% and 45%, respectively—αVβ3 integrin by nearly 65%, and α2β1 integrin by nearly 95%. This effect was reversible following the removal of the anti-integrin antibody. These results suggest that integrin adhesion and signaling events may contribute to the ability of this cell line to mediate the initiation of the mineralization phenotype biologically.

Bone Cell Expression on Titanium Surfaces is Altered by Sterilization Treatments

Phenotypic responses of rat calvarial osteoblast-like cells (RCOB) were evaluated on commercially pure titanium (cpTi) surfaces when cultured at high density (5100 cells/mm2). These surfaces were prepared to three different clinically relevant surface preparations (1-μm, 600-grit, and 50-μm-grit sand-blast), followed by sterilization with either ultraviolet light, ethylene oxide, argon plasma-cleaning, or routine clinical autoclaving. Osteocalcin and alkaline phosphatase, but not collagen expression, were significantly affected by surface roughness when these surfaces were altered by argon plasma-cleaning. In general, plasma-cleaned cpTi surfaces demonstrated an inverse relationship between surface roughness and phenotypic markers for a bone-like response. On a per-cell basis, levels of the bone-specific protein, osteocalcin, and the enzymatic activity of alkaline phosphatase were highest on the smooth 1-μm polished surface and lowest on the roughest surfaces for the plasma-cleaned cpTi. Detectable bone cell expression can be altered by clinically relevant surfaces prepared by standard dental implant preparation techniques.

Toward an understanding of implant occlusion and strain adaptive bone modeling and remodeling

STATEMENT OF PROBLEM Dental implant failure rates for osseointegration are greater in the highly atro-phic maxilla. Presuming higher failure rates relate to strain-driven adaptation, an enhanced understanding of formative bone response to loading (modeling) and maintenance of an integrated state (remodeling) should improve treatment. PURPOSE To understand the role of occlusal loading on long-term osseointegration in areas of compromised cancellous bone, a review of the salient features of adaptive bone modeling and remodeling is presented with an emphasis on cancellous bone responses. CONCLUSIONS The ability for dental implants to maintain a long-term stable interface in the maxilla lies in the ability of trabecular bone to maintain adequate local material (strength) and architectural (connectivity) properties. In this discussion, an emphasis has been placed on understanding how trabecular bone can respond to the mastication-induced loading environment on an implant.

Periodontal Disease and Atherosclerosis From Dental to Arterial Plaque

The conventional risk factors for atherosclerosis are well understood, but they can account for only about 50% to 70% of atherosclerotic events in the general population. Many other putative risk factors for atherosclerosis have been proposed, including traits related to obesity, inflammation, and infection. Periodontal disease is a candidate risk factor that shares many of these related traits. The periodontal diseases reflect a spectrum of oral pathology from gingivitis (gum inflammation) to severe periodontitis (progressive loss of gum attachment) with alveolar bone and tooth loss. See page 1405 The pathogenesis of periodontal disease is thought to be due to accumulation of dental plaque (bacteria in subgingival biofilms) with consequent mucosal infection and inflammation. Abnormal host responses, with upregulation of matrix metalloproteinases, contribute to a more rapid disease progression in some patients. Periodontal disease is more common with cigarette smoking, obesity, and diabetes, and it affects about 75% of the adult population in the United States, with about 20% to 30% of adults having severe forms.1,2⇓ Increasing evidence over the past decade suggests a link between periodontal disease and atherosclerosis. Multiple cross-sectional studies have demonstrated a higher incidence of atherosclerotic complications in patients with periodontal disease.3–6⇓⇓⇓ In the NHANES III cohort, severe periodontal disease was associated with an almost 4-fold higher incidence of myocardial infarction than found in patients without periodontal disease.6 In cross-sectional studies, the cardiovascular risk associated with periodontal disease appears dependent on severity of the disease, and it is independent of conventional risk factors (including smoking, poverty, diabetes, and body mass index).7 One problem with cross-sectional and case-control studies is that they do not tell us whether a disease is a cause or a consequence of another condition. For example, it is possible that atherosclerosis might exacerbate periodontal disease, by …

The Concept of Osseointegration and Bone Matrix Expression

Osseointegration has been defined as the direct structural and functional connection between ordered, living bone and the surface of a load-carrying implant. To date, this concept has been described by descriptive histological and ultrastructural criteria but not by biochemical means. This review evaluates the basic science work performed on this concept and then applies the concept to the principle of osseous healing. Specific studies are cited where alterations in the healing response are due to clinical management of implant placement and how studies of surface properties may lead to further insights on implant design and prognosis. In addition, a review of bone expression as a function of in vitro stress applications is given. This is followed by an indepth review of the collagens and noncollagenous proteins, described to date, within isolated bone matrix. It is this collagenous matrix (especially type I) that is described as being close to and oriented with a glycoprotein component next to the implant surface. In turn, the large family of noncollagenous proteins are important in mediating bone proliferation, matrix accumulation, orientation, mineralization, and turnover. This section is followed by a discussion of specific growth factors as they may relate to osseous healing around an implant.

The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor

Gene therapy using non-viral vectors that are safe and efficient in transfecting target cells is an effective approach to overcome the shortcomings of protein delivery of growth factors. The objective of this study was to develop and test a non-viral gene delivery system for bone regeneration utilizing a collagen scaffold to deliver polyethylenimine (PEI)-plasmid DNA (pDNA) [encoding platelet derived growth factor-B (PDGF-B)] complexes. The PEI-pPDGF-B complexes were fabricated at amine (N) to phosphate (P) ratio of 10 and characterized for size, surface charge, and in vitro cytotoxicity and transfection efficacy in human bone marrow stromal cells (BMSCs). The influence of the complex-loaded collagen scaffold on cellular attachment and recruitment was evaluated in vitro using microscopy techniques. The in vivo regenerative capacity of the gene delivery system was assessed in 5 mm diameter critical-sized calvarial defects in Fisher 344 rats. The complexes were ~100 nm in size with a positive surface charge. Complexes prepared at an N/P ratio of 10 displayed low cytotoxicity as assessed by a cell viability assay. Confocal microscopy revealed significant proliferation of BMSCs on complex-loaded collagen scaffolds compared to empty scaffolds. In vivo studies showed significantly higher new bone volume/total volume (BV/TV) % in calvarial defects treated with the complex-activated scaffolds following 4 weeks of implantation (14- and 44-fold higher) when compared to empty defects or empty scaffolds, respectively. Together, these findings suggest that non-viral PDGF-B gene-activated scaffolds are effective for bone regeneration and are an attractive gene delivery system with significant potential for clinical translation.

Biomechanical and functional behavior of implants.

The ability to achieve a long-term stable implant interface is not a significant clinical issue when sufficient uni- or bi-cortical stabilization is available. Clinical outcomes studies suggest that the higher-risk implants are those placed in compromised cortical bone (thin, porous, etc.) in anatomic sites with minimal existing trabecular bone (characterized as type IV bone). In establishing and maintaining an implant interface in such an environment, one needs to consider the impact of masticatory forces. These forces, in turn, have the potential to create localized changes in interfacial stiffness through the viscoelastic properties of bone. Changes in these properties will alter the communication between osteocytes and osteoblasts, leading to an increase in new bone growth, a maintenance of established bone, or a loss (potentially catastrophic) of either cortical or trabecular bone. Therefore, a key to understanding the biomechanical and functional behavior at an implant interface is to control the extent of anticipated modeling and remodeling behavior through an optimal implant design combined with a thorough understanding of how tissues respond to the mechanically active environment.

Effects of radiofrequency glow discharge on impression material surface wettability

STATEMENT OF PROBLEM Argon radiofrequency glow discharge (RGD) may simultaneously sterilize and improve surface wettability of impression materials. PURPOSE The purpose of this study was to define RGD technical parameters that influence the surface wettability of impression material (optimization phase). Definition of RGD was followed by an assessment of these optimized RGD parameters on the wettability of four impression materials either uncontaminated or contaminated with saliva, compared with conventional liquid disinfection (application phase). MATERIAL AND METHODS For the optimization phase, addition silicone samples were cast against glass with 10 samples per group/parameter (n = 210). Parameters evaluated were duration of exposure, sample shape and angle, position within the RGD chamber, and argon gas delivery pressure. Changes in surface wettability were determined with contact angle measurements. For the application phase, standardized RGD parameters (90 degrees to the plasma flow, flat, 60 seconds, 5 psi) were used on four groups of impression materials with (n = 120 samples, 30 per material) or without (n = 120 samples, 30 per material) prior saliva contamination. RESULTS RGD treatment of a polyvinyl siloxane impression material significantly (p < 0.0001) reduced contact angle measurements from 63 +/- 1 to 13 +/- 4 degrees, regardless of the parameter evaluated. For the application phase, results indicated different responses to RGD relative to nontreated controls. With all materials treated with RGD or disinfectant exposure, the finest 20 microns standard line was reproduced at x10 magnification with the American National Standards Institute/American Dental Association Specification 19 test die (Sabri Enterprise, Downers Grove, Ill.; n = 80, 10 samples per group). CONCLUSION These results suggest RGD selectively alters impression material surface wettability.