Effect of glucocorticoid-induced osteoporotic-like conditions on osteoblast cell attachment to implant surface microtopographies

OBJECTIVES: The objectives of this work were to: (1) establish methodology for pretreating osteoblast-like cells in vitro with dexamethasone to cause glucocorticoid-induced osteoporosis, (2) perform quantitative and qualitative assessments of cellular attachment of osteoporosis-like osteoblasts when grown on implant surfaces of differing roughness, (3) and explore the hypothesis that dexamethasone-treated osteoblasts have altered cell attachment properties by focal adhesion disassembly and decreased tyrosine phosphorylation of the focal adhesion tyrosine kinase. METHODS: Osteoblasts were cultured with dexamethasone (10(-7) and 10(-6) M) for up to 4 days of incubation to induce osteoporosis-like conditions. Cellular attachment assays demonstrated the effect of dexamethasone treatments on cellular attachment properties of osteoblasts. Qualitative data were obtained utilizing immunofluorescent microscopy and Western blotting. Focal adhesion kinase (FAK) immunoprecipitation and tyrosine-phosphorylation Western blots were obtained from dexamethasone-treated human embryonic palatal mesenchymal- 1486 osteoblast cultures supplemented with ascorbate and beta-glycerol phosphate medium. RESULTS: Cellular attachment was significantly greater (P < 0.05) with non-dexamethasone-treated osteoblasts (92%) as compared to dexamethasone-treated osteoblasts after 1 (72%), 2 (63%), and 4 days (53%) of exposure. Dexamethasone-treated osteoblasts were viable and capable of proliferation, suggesting that the reduction of cellular attachment may be related to these cell adhesion processes. Immunofluorescent microscopy of both dexamethasone-treated osteoblasts and non-dexamethasone-treated osteoblasts failed to show any relative difference in the disassembly of focal adhesions and actin filaments. Extended dexamethasone treatment periods (up to 3 weeks) showed changes in the levels of FAK and FAK-phosphotyrosine in human embryonic palatal mesenchymal-1486 osteoblasts. CONCLUSIONS: The protocol used in this study demonstrated a glucocorticoid-induced osteoporosis-like suppression of osteoblasts. FAK disassembly was not a significant factor in short period; however, FAK protein levels and phosphotyrosine signaling on FAK were affected after 1-week exposure to dexamethasone. Phosphorylated FAK was not associated with the rise in the level of FAK, further indicating the possibility of FAK involvement in reduced cell attachment.     

Effect of osteoblast cell culture on the bone implant contact

Abstract

Purpose. The aim of this study is to acquire an ideal bone implant contact under the cover of osteogenic effect of osteoblasts derived from Mesenchymal Stem Cells (MSCs). Materials and methods. Thirty dental implants were used for this study. Implants were placed in sheep mandibles and defects were created 4 mm coronally in the dental implants. These defects were filled with Platelet Rich Plasma (PRP) in one group and with PRP + Osteoblast Cell Culture (OCC) in another group. No procedure was conducted on the control group defects (empty defect group). Eight weeks later, osseointegration was investigated with Bone Implant Contact (BIC) measurements histomorphologically. Data were checked statistically. Results. The variation of BIC rates between Empty Defect Group and PRP groups was significant (p <0.05). The BIC rate of the PRP group was higher than that of the Empty Defect Group. The variation of BIC rates between Empty Defect Group and PRP + OCC groups was significant (p <0.05). The BIC rate of the PRP + OCC group was higher than that of the Empty Defect Group. The variation of BIC rates between PRP and PRP + MSC groups was significant (p<0.05). The BIC rate of the PRP + OCC group was higher than that of the PRP group. At the end of the 8-week healing period, it was observed that the percentage of BIC was highest in the PRP + OCC group. Conclusions. Implant–bone connection was better in the OCC?PRP group compared with the PRP group and the empty defect group. The use of OCC-PRP combination was effective on healing. The BIC value was increased significantly by OCC.     

碱性成纤维细胞生长因子对成骨细胞粘附的调控作用

目的探讨碱性成纤维细胞生长因子(bFGF)对成骨细胞在钛片上粘附的调控作用。方法体外培养大鼠成骨细胞,利用存活细胞检测(MTT)法以及扫描电镜(SEM)观察bFGF对成骨细胞在钛片上粘附情况的影响。结果在0~27 ng/ml浓度之间,bFGF对促进成骨细胞在钛片上的粘附作用具有剂量依赖效应,27 ng/ml bFGF能促进成骨细胞在粘附初期的形态的改变以及在材料上的铺展。在高浓度的环境下,bFGF对促进成骨细胞在钛片上的粘附作用有一定的抑制作用。结论一定浓度的bFGF能促进成骨细胞在钛片上的粘附。     

Surface microtopography regulates osteointegration: the role of implant surface microtopography in osteointegration

Increased surface roughness of dental implants enhances the process of osseointegration. It increases bone conduction and increases BIC in all types of bone, resulting in elevated removal torque values. Surface roughness elevated the CSR of implants implanted in adverse conditions as augmented ridges and sinuses and areas of poor bone, such as the posterior maxilla, and in some cases abolished the deleterious effect of smoking. A growing number of clinical studies suggest that early and immediate loading of rough-surfaced implants may lead to predictable osseointegration. However, it is important to note that these studies provide short-term results based on radiographic observation and clinical mobility only. Before we adopt new surgical and prosthetic guidelines, longer and broader studies are needed. Most recent research has examined the effect of surface roughness on bone healing around implants in vivo and the influence of surface roughness on osteoblasts in vitro. In a study just published, it was found that changing the surface chemistry by submerging an implant in an isotonic sodium chloride solution following acid etching to avoid contamination with molecules from the atmosphere significantly increased osteoblast differentiation in vitro and BIC in vivo. This finding may lead us to a new era in dental implants.     

Ultrastructural study of epithelial cell attachment to implant materials

Attachment of gingival tissues to the protruding post of an endosseous dental implant is of great importance for the prognosis of its clinical success. Little is known about the requirements an implant material must meet to secure a durable permucosal seal. The objective of this study, therefore, was to gain insight into the morphology of epithelial cell-implant interfaces for a number of well-known or potential implant materials. Guinea pig epithelial cells were cultured on gold, titanium, carbon, hydroxylapatite, carbonate apatite, and modified polystyrene substrates. The metallic and carbon substrates were obtained by vapor-phase deposition of thin films on polystyrene carriers. This technique allowed for the preparation of ultra-thin sections for transmission electron microscopy containing substrate as well as the cells adhering to it. The cultures on apatite were decalcified prior to sectioning. The results revealed tht as attachment structures, focal contacts, extracellular matrix contacts, and hemidesmosome-like contracts could be distinguished. Hemidesmosome-like contacts were only observed on apatite and polystyrene and not on the metallic or carbon surfaces. The results of this study suggest the existence of some, as yet unknown, property of the substrate that determines the nature and the structure of the contact site with epithelial cells.     

Osteoblastic cell attachment to hydroxyapatite-coated implant surfaces in vitro

Hydroxyapatite (HA) used as a coating for implants can exhibit varying levels of interaction with the biologic environment. The crystallinity of the HA-based coating has been shown to control the rate of dissolution and appears to play a role in the initial cellular interaction with the implant surfaces. An osteoblastic cell attachment assay was employed to examine the cell attachment to untreated and pretreated (pH 5.2, 24 hours) titanium and HA coatings of low (50%), medium (75%), and high (90%) crystallinity. A slightly higher percentage of cell attachment (%CA) was found on untreated and pretreated HA surfaces as compared to the titanium surface. No significant difference could be found in the %CA between the 3 levels of crystallinity. However, higher levels of %CA were observed on pretreated HA surfaces than on untreated HA surfaces (t test, P < .05). Elevated calcium and phosphate levels in culture medium did not have any effect on cell attachment. Scanning electron microscopic examinations revealed surface degradation of the HA coating following pretreatment in the simulated inflammatory media (pH 5.2, 24 hours). The results suggest that the altered surface topography may influence the initial cell attachment to HA surfaces.     

Effect of heat-treated titanium surfaces on protein adsorption and osteoblast precursor cell initial attachment

The clinical success of dental implants is governed in part by surface properties of implants and their interactions with the surrounding tissues. The objective of this study was to investigate the effect of heat-treated titanium surfaces on protein adsorption and osteoblast precursor cell attachment in vitro. Passivated titanium samples used in this study were either non heat treated or heat treated at 750 degrees C for 90 minutes. It was observed that the contact angle on heat-treated titanium surfaces was statistically lower compared with the non-heat-treated titanium surfaces. The non-heat-treated titanium surface was also observed to be amorphous oxide, whereas heat treatment of titanium resulted in the conversion of amorphous oxide to crystalline anatase oxide. No significant difference in albumin and fibronectin adsorption was observed between the heat-treated and non-heat-treated titanium surfaces. In addition, no significant difference in initial cell attachment was observed between the two groups. It was concluded that heat treatment of titanium resulted in significantly more hydrophilic surfaces compared to non-heat-treated titanium surfaces. However, differences in oxide crystallinity and wettability were not observed to affect protein adsorption and initial osteoblast precursor cell attachment.     

Protein adsorption and osteoblast precursor cell attachment to hydroxyapatite of different crystallinities

PURPOSE: The effect of hydroxyapatite (HA) crystallinity on protein adsorption and osteoblast precursor cell attachment to HA was investigated. MATERIALS AND METHODS: Different weight ratios of 100% crystalline HA and 100% amorphous calcium phosphate powders were mixed and pressed into disks (0.5 g) of different crystallinities--either 0% (HAO), 30% (HA30), 50% (HA50), 70% (HA70), or 100% (HA100). RESULTS: X-ray diffraction indicated differences in HA crystallinities. In addition, dissolution of the HA was dependent on its crystallinity, with an increase in phosphorus dissolution as the degree of crystallinity was decreased. No significant difference in albumin adsorption and initial osteoblast precursor cell attachment was observed in the range of HA0 to HA70 surfaces. However, a significantly lower albumin adsorption and initial osteoblast precursor cell attachment were observed on HA100. DISCUSSION: It was suggested that changes in ionic interactions as a result of a change in crystallinity affect the amount of calcium ion ligands readily available to electrostatically bind to proteins. CONCLUSION: It was thus concluded from this study that HA crystallinity affects the amount of albumin adsorbed and initial osteoblast attachment.     

Effects of chlorhexidine and stannous fluoride on fibroblast attachment to different implant surfaces.

This study evaluated, in vitro, the attachment of gingival fibroblasts to dental implants of differing surface character treated with 0.12% chlorhexidine, 1.64% stannous fluoride, or sterile saline. Specimens were divided into three groups, each composed of 18 dental implants. Each group featured a different implant surface texture, ie, smooth titanium, hydroxylapatite plasma-spray, or titanium plasma-spray. Following adsorption of salivary pellicle, six implants from each group were treated with chlorhexidine, stannous fluoride, or sterile saline. They were then cultured with fibroblasts for 24 hours and photographed by scanning electron microscopy. Cell counts were then performed. Analysis of results showed a significantly greater number of fibroblasts attached to specimens treated with saline or chlorhexidine than to those treated with stannous fluoride. Furthermore, fibroblasts were more likely to attach to rough-surfaced than to smooth-surfaced specimens.     

A study on the attachment of human gingival cell structures to oral implant materials.

A technique enabling the study of adherence occurring among implant materials and epithelial and connective cells cultured in vitro and freshly obtained from explants of human gingival tissue is described. The data show that cell adherence to titanium alloy is influenced by the modality of implant surface preparation.     

The effects of scaling titanium implant surfaces with metal and plastic instruments on cell attachment

This study examined the ability of tissue culture fibroblasts to attach and colonize on the surface of pure titanium dental implants following instrumentation of the implant surface with curettes of dissimilar composition. Pure titanium dental implants were scaled with a plastic, titanium-alloy, or stainless steel curette and then immersed in a cell suspension of 3T3 fibroblasts. Counts of attached cells were made at 24 and 72 hours; the implants were then processed for scanning electron microscopy (SEM). At 24 hours, only surfaces scaled with a stainless steel curette showed a significant reduction in number of attached cells relative to untreated control surfaces. At 72 hours, both stainless steel and titanium-alloy curette instrumented surfaces showed significantly fewer attached cells than untreated control surfaces, with the greatest reduction in cell attachment observed on the stainless steel curette instrumented surfaces. SEM observations showed that fibroblasts on stainless steel instrumented surfaces tended to show a somewhat rounded morphology and a relatively reduced degree of spreading: while fibroblasts on untreated control, plastic, or titanium-alloy instrumented surfaces showed a well-spread, polygonal morphology, more typical of fibroblasts in favorable culture conditions. To the extent that such observations of cell attachment and morphology are indicative of in vivo biocompatibility, these findings could have clinical implications for the proper maintenance of titanium dental implants.     

Effect of bone sialoprotein and collagen coating on cell attachment to TICER and pure titanium implant surfaces

To improve integration between implants and biological tissues, this study compared bone sialoprotein (BSP) as a surface-coating material against the major organic and inorganic components of bone, collagen type I and hydroxyapatite (TICER). The expression of osteocalcin, osteonectin and transforming growth factor ss was evaluated using immunohistochemical staining procedures. The distribution patterns of osteoblasts on the surface of pure titanium with a smooth machined surface and a rough surface (TICER) were determined by image processing using confocal laser scanning microscopy. The results compared to uncoated control materials showed that, at all times investigated, the number of cells on the surface of the TICER and pure titanium samples differed significantly (P<0.1), demonstrating the superiority of TICER over pure titanium in this respect. For pure titanium implants, collagen-precoated surfaces were not beneficial for the attachment of bone-derived cells with the exception of day 3 in vitro (P<0.01). BSP-precoated implant surfaces displayed non-significantly higher numbers of settled cells. BSP-precoated implant surfaces were beneficial for osteoinduction as revealed by osteocalcin and osteonectin expression. BSP precoating of the rough TICER implant surface enhanced the osteoinductive effect much more than did collagen precoating. These results contribute to the consideration of at least two distinct pathways of osseointegration.     

Clip attachment for the mandibular staple implant

A technique for making the staple implant superstructure has been described, which is simple, economically reasonable, and readily replaced with a minimum of difficulty. The procedure reduces both clinical and laboratory time compared with the use of precision attachments and ceramometal restorations. The size of the Rider clip generally allows sufficient space for the proper arrangement of denture teeth and the development of acceptable external contours of the prosthesis.     

Comparative value of attachment measurements in implant dentistry

PURPOSE: In implant dentistry, the level of bone attachment is normally assessed by clinical and radiologic parameters. In the literature, however, the accuracy of these measurements has been a source of controversy. The purpose of this study was to assess the reliability of attachment measurements in the beagle dog model. MATERIALS AND METHODS: In 6 beagle dogs, a total of 60 implants were placed. Bony defects resulting from plaque accumulation were treated surgically. All defects were evaluated at the time of surgery (T3) and 4 months later (T4). Evaluation included standardized measurements on radiographs, pressure-forced implant probing, and histometry. Furthermore, both conventional and digital radiographic techniques were used. RESULTS: Both radiographic techniques showed very similar results at T3 and T4. At time T4, pressure-forced probing revealed statistically significantly different values than those obtained with radiography and histometry. When radiographic and histometric measurements were compared, no significant differences were found at either time T3 or time T4. DISCUSSION: In this study, histometry showed better accordance with radiography than with pressure-forced probing. These results support the hypothesis that peri-implant attachment should be evaluated with a combination of both clinical and radiologic parameters. CONCLUSION: The exclusive use of radiography cannot be recommended for the measurement of peri-implant attachment.