John C. Keller

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.

The influence of water storage and C-factor on the dentin–resin composite microtensile bond strength and debond pathway utilizing a filled and unfilled adhesive resin

OBJECTIVE To test the elastic wall concept utilizing adhesive resins of varying stiffness in a low- and high-C-factor cavity design after short- and long-term water storage. METHODS A flat and box-shaped cavity was restored on occlusal dentin with a resin composite using a filled and unfilled adhesive resin from which microtensile specimens with a 0.5mm(2) cross-sectional area were formed. After storage for 30- and 150-days the microtensile bond strength (muTBS) was determined in a Zwick materials testing machine and the subsequent debond pathway was examined under scanning electron microscopy. Fishers exact test was used to determine differences in joint and substrate failure modes and a Weibull regression model with gamma frailties was used to test for differences between failure distributions. Tests for three-way and two-way interactions were also completed for storage time, C-factor and adhesive. All tests were at 95% confidence levels. RESULTS The characteristic strength (TBS degrees ) for the Optibond FL adhesive applied on a flat cavity was 47.57 and 20.90MPa and a box-shaped cavity was 49.26 and 17.49MPa for short- and long-term storage, respectively, while the corresponding TBS degrees for the unfilled Optibond adhesive on the flat cavity design was 36.93 and 32.68MPa and in a box-shaped cavity was 32.84 and 15.46MPa. Combining all groups according to storage time revealed a three-fold increase in the debond pathway including the bottom of the hybrid layer. SIGNIFICANCE Evidence suggests that the durability of the bonded joint is threatened by hydrolysis and the most susceptible region is the bottom half of the hybrid layer and in low C-factor cavity designs a more flexible adhesive resin liner was more durable.

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.

Biomechanical and morphometric analysis of hydroxyapatite-coated implants with varying crystallinity

PURPOSE The level of crystallinity in hydroxyapatite (HA) is thought to be responsible for its degradation in the physiologic milieu. The purpose of this study was to compare the in vivo bony response to HA coatings of varying levels of crystallinity and determine the optimum composition for promoting osseointegration. MATERIALS AND METHODS Cylindrical implants of sand-blasted CP titanium and HA-coated titanium of 50% (low), 70% (medium), and 90% (high) crystallinity were inserted into the canine femur for 1, 4, 12, and 26 weeks. Morphometric analysis of undecalcified sections determined the percentage of bone contact with the implant surface. A pullout test was used to measure the interfacial attachment strength of the bone-implant interface. Scanning electron microscope (SEM) examination of the implant surface aided in identifying the failure mode. Coating thickness was measured under light microscopy to determine whether degradation occurred. RESULTS No significant differences could be found in the percentage of bone contact and interfacial attachment strength between the three types of HA-coated implants throughout the four implantation periods. A significantly higher percentage of bone contact on HA-coated implants than on uncoated titanium implants was noted at 4 weeks. (ANOVA, P<.05). HA-coated implants were also found to have significantly higher interfacial attachment strength than titanium implants at 4, 12, and 26 weeks. Coating thickness decreased gradually with time. The most noticeable reduction was found on the low-crystallinity coatings during the first 4 weeks. Failure of the bone-coating-implant complex occurred mostly within the coating or near the coating-implant interface. CONCLUSIONS HA coatings on metal implants enhance osseointegration in the early stage of bone healing and provide strong bone-bonding capability, although titanium implants had about the same level of bone contact in the later stage of healing. Crystallinity of HA coatings has no significant influence over the bone formation capacity and the bone bonding strength. However, an HA coating of higher crystallinity is more desirable in providing durability and maintaining osteoconductive properties.

Mode of failure in the dentin-adhesive resin–resin composite bonded joint as determined by strength-based (μTBS) and fracture-based (CNSB) mechanical testing

OBJECTIVE To determine the failure mode between dentin-adhesive resin-resin composite bonded joint produced with a chevron-notch short-bar (CNSB) and microtensile test methods. METHODS Forty teeth were randomly selected for microtensile and forty for CNSB specimen fabrication and stored in 0.5% chloramine T at 37 degrees C until respective static load to failure testing at 30 and 180days. Failure modes were categorized by SEM and tested with Fishers exact test. Within respective mechanical testing methods the probability of failure curve distributions being significantly different were analyzed by the Wald chi-square statistic. RESULTS The characteristic fracture toughness at 30- and 180-day storage was 0.82 and 0.87MPam(1/2), while the Weibull Modulus (m) for the failure distributions, was 4.60 and 4.56, respectively. No significant difference was demonstrated in the failure distributions between these groups (p=0.45). The characteristic tensile strength (muTBS(o)) at 30- and 180-day storage was 52.53 and 14.71MPa with an m of 3.04 and 1.56, respectively. Failure distributions for muTBS groups were significantly different (p<0.001). K(IvM) failure modes, regardless of storage time, were within the adhesive joint with 30-day debonds primarily through the top region of the hybrid layer (THL) and after 180-days involving the bottom of the hybrid layer (BHL). The 30-day muTBS group demonstrated a propensity to debond in dentin or resin composite substrates but after 180-days storage debonds again involved the BHL. SIGNIFICANCE The weak links in the dentin-adhesive resin-resin composite bonded joint may be the interphase regions between the THL and the adhesive resin and the BHL and dentin.

Effects of multiple sterilization on surface characteristics and in vitro biologic responses to titanium.

PURPOSE This study evaluates the surface changes and effects on in vitro cell attachment and spreading brought about on prepared commercially pure titanium by multiple exposures to common sterilization methods. MATERIALS AND METHODS Discs of commercially pure titanium were prepared to approximate the surface roughness of commercially available bone miniplates. Samples underwent sterilization by exposure to ultraviolet light; ethylene oxide sterilization (1, 5, or 10 cycles); or by steam autoclaving (1, 5, or 10 cycles). Representative surfaces from these sterilization groups were examined using a series of surface analytical techniques including scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), auger electron spectroscopy (AES), and contact angle measurements. Cell attachment assays using murine fibroblasts were then performed on titanium surfaces from each sterilization group and on tissue culture plastic controls. Sterilized surfaces contained O, C, and N contaminants, which affected surface energetics. Mean percent cell attachment values for each group were obtained for periods of up to 1 hour. Representative samples from each group were examined using SEM to ascertain cell spreading and morphology for each sterilization group. RESULTS Ultraviolet (UV) sterilized surfaces showed no changes from the unsterilized state macroscopically or under SEM. UV surfaces showed cell attachment levels similar to control surfaces at all intervals, and a chronologic progression of cell spreading. Ethylene oxide-sterilized surfaces showed occasional bluish discoloration and a microscopic particulate contaminant, resulting in modest decreases in cell attachment levels without strong correlation to numbers of sterilization cycles. Autoclaved surfaces generally showed the greatest discoloration and heaviest particulate contamination. Cell attachment levels were lower, and cell spreading was diminished compared with the ethylene-oxide-treated group. CONCLUSIONS Both ethylene oxide and steam autoclave sterilization contaminated and altered the titanium surface, resulting in decreased levels of cell attachment and spreading in vitro. Although corroborative in vivo experiments should be conducted, the results of this study indicate that some multiple sterilization regimens for metallic materials may pose serious biologic concerns.

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.

Repair of craniofacial defects with hydroxyapatite cement

PURPOSE The objective of this study was to evaluate the course of healing of craniofacial bone defects when filled with hydroxyapatite cement and to determine whether adding various percentages by weight of demineralized bone powder to the cement will result in enhanced bone formation. MATERIALS AND METHODS The model for the study was the canine calvarium. The implants were placed into cranial defects and harvested at 3 or 6 months for qualitative evaluation by light microscopy, microradiography, and quantitative histomorphometry. RESULTS The implantation of hydroxyapatite cement resulted in characteristic replacement of the material with new bone ingrowth. The addition of demineralized bone powder to the hydroxyapatite cement appeared to improve the handling characteristics of the cement; however, improvement in the replacement of the material by bone was not observed. The implantation of only allogeneic demineralized bone showed limited new bone formation within the defect site. CONCLUSIONS Hydroxyapatite cement formed an effective osseoconductive scaffold for bone replacement. The addition of demineralized bone powder to the cement to serve as a carrier of osseoinductive factors did not result in additional bone being formed.

In vitro attachment of osteoblast-like cells to osteoceramic materials

OBJECTIVE The objective of this work was to examine osteoblast-like cell attachment and morphology in vitro to osteoceramic materials with three different surface morphologies. METHODS Osteoceramic composite disks were fabricated from tricalcium phosphate and magnesium-aluminate spinel (MgAl2O4) in a 50 vol% ratio. The disks were prepared with three different surface morphologies, including as-fired (irregular), etched (rough), or polished through 1 mm diamond paste (smooth). Osteoblast-like cell cultures were plated onto the prepared disks for 2 h, and the number of attached cells was determined. ANOVA and Student Newman-Kuels tests were used to test for significant differences in cell attachment (p < 0.05). SEM was used to visually evaluate the nature of the cellular adaptation on the osteoceramic surfaces. RESULTS Some additional surface roughening resulted from the interaction between the osteoceramic disks and the biological culture media during the attachment assay. A statistically larger number of cells was found to be attached to the etched osteoceramic surfaces compared to the as-fired and polished osteoceramic surfaces or the tissue culture plastic control. Cellular adaptation was extensive on all three osteoceramic surfaces at 2 h. SIGNIFICANCE These results are consistent with previous in vivo work and continue to support the hypothesis that osteoceramic materials have potential for implants and bone substitute materials.