Effect of three distinct treatments of titanium surface on osteoblast attachment, proliferation, and differentiation

Cell-titanium interactions are crucial to the clinical success of bone and dental implants. The physico-chemical characteristics of the substrates surface influence osteoblast proliferation, differentiation, and activity as well. The osteoblast behavior was analyzed on three different titanium surfaces: ground with an abrasive 600 grit SiC paper, blasted with alumina particles (65 microm diameter) and alumina blasted followed by a double chemical etch (4% HF+4% HF/8% H2O2). Scanning electron microscopy (SEM) and profilometry showed distinct microtopographies. Ground samples showed parallel-groove orientation. The Al2O3-blasted surface presented the roughest microtopography with aluminum-rich particles incrusted in the titanium surface. Osteoblasts cells from femora of Balb/c mice were seeded onto the substrates tested. Cell morphology and initial attachment were evaluated by SEM. Osteoblasts adhered to and spread on all samples tested. However, on rough surfaces, osteoblasts did not spread completely and acquired a polygonal morphology. Besides, the cell proliferation rate was diminished at the beginning of incubation on rough surfaces. Our results suggest a delay, rather than an impairment, in osteoblast viability and alkaline phosphatase activity when cells are cultured on rough surfaces, inducing a distinct osteoblast phenotype, rather than blocking its activity. At least in the culture conditions used in this work, alumina particles did not affect osteoblast behavior.     

In vitro periodontal ligament fibroblast attachment to plasma-cleaned titanium surfaces.

The objective of this research was to characterize the in vitro cellular behavior of fibroblast-like cells derived from rat periodontal ligament on commercially pure titanium surfaces which were sterilized by a variety of treatments. Following standard surface preparation protocols, the Ti specimens were sterilized by either steam autoclaving, exposure to ethylene oxide gas, exposure to ultraviolet light, or plasma-cleaning in argon for either one min or five min. Fibroblast-like cells in serum-supplemented media were incubated on the various Ti specimens for up to two h. In general, the levels of cell attachment for plasma-cleaned surfaces were significantly higher than those for steam-sterilized surfaces, but were significantly lower than the attachment levels for both the ultraviolet-treated surfaces and the tissue culture plastic control. The duration of plasma cleaning itself did not have a significant effect on the percentage of cell attachment at any time period. SEM evaluations indicated that by two h, the cellular morphology was different on the variously treated specimens. These studies indicate that the method of sterilization following implant surface preparation can affect the initial in vitro biological events of cell attachment and spreading.     

The attachment mechanism of epithelial cells to titanium in vitro

It has previously been difficult to examine the nature of the attachment between tissues and metal implants. To resolve this problem, a technique has been developed in which oral epithelial cells are grown on very thin films of titanium on epoxy resin. Discs of epon/araldite were formed by polymerizing the liquid resin in 60 mm tissue culture dishes. Discs were then placed in a vacuum deposition chamber and titanium wire vaporized onto the surface to a final thickness of about 300Å. Discs were cleaned in acetone and 70 % alcohol before being placed in plastic culture dishes and plated with epithelial cells cultured from porcine periodontal ligament in MEM plus 15 % calf serum and antibiotics for fourteen days. Cultures were fixed in situ in 2.5 % glutaraldehyde, post-fixed in OsO₄ and epon/ araldite polymerized on the surface of the discs. Thin sections were cut perpendicular to the surface and examined in the electron microscope. Results show that the epithelial cells attach to the titanium surface by means of basal lamina and hemidesmosomes, in much the same manner in which the epithelial attachment is applied to the surface of a tooth.     

Improvement of in vitro titanium bioactivity by three different surface treatments.

OBJECTIVE: Dental implants are usually made from commercially pure titanium or titanium alloys. The aim of this investigation was to determine the influence of surface treatments of commercially pure titanium samples on in vitro bioactivity. METHODS: Commercially pure (cp) titanium (Ti) sheets were submitted to three different surface treatments, including, for all samples, etching with an HCl/H(2)SO(4) solution. Part of each etched sample was further submitted either to anodic oxidation by using an H(3)PO(4) solution or to thermal oxidation. Treated and non-treated samples were analyzed by using scanning electron microscopy (SEM), profilometry and photoelectron X-ray spectroscopy (XPS). The in vitro assessment was carried out through the immersion of samples in simulated body fluid (SBF). In vitro testing was carried out by SEM and by the determination of calcium (Ca) content in solution by atomic absorption spectrometry (AAS). The non-treated titanium samples were used as the control group. RESULTS: This study has shown that, after up to 7-day exposure, a calcium phosphate layer precipitated only on samples submitted to at least one of the three treatments used. This result, based on SEM images, is in good agreement with Ca content and XPS analysis, in which remarkable effects of surface modifications on Ti samples are highlighted. SIGNIFICANCE: These results suggest that suitable surface treatments, such as employed here, may improve in vitro titanium bioactivity in a SBF solution at 37 degrees C. This behavior suggests a possibility of a further favorable in vivo response.     

Effect of lipopolysaccharide contamination on the attachment of osteoblast-like cells to titanium and titanium alloy in vitro

Failing implants with loss of alveolar bone are associated with gram-negative bacteria that carry lipopolysaccharide (LPS) in the bacterial cell wall. Bony regeneration around these implants is still an unpredictable procedure due to the many clinical factors involved. One important factor is the presence of contaminants such as LPS on the implant surface. The effect of implant-associated LPS on the attachment of bone cells to the implant surface is unknown. This project investigated the effect of LPS on the attachment of osteoblast-like cells (MC3T3-E1) to titanium and titanium alloy surfaces in vitro. We hypothesized that LPS would inhibit bone cell attachment either through loss of cellular attachment sites or alteration of cellular function. Three experimental approaches were used. First, alloy surfaces were exposed to LPS (100 microgram/mL) before the cells were allowed to attach. In the second approach, the cells were exposed to the LPS before they were allowed to attach. Last, the cells were allowed to attach before exposure to LPS. Cellular attachment to implant materials was measured by using a histochemical stain (MTT). The results indicated that LPS presence did not significantly (P > .05) alter osteoblast attachment to titanium or titanium alloy surfaces whether the exposure occurred before or after cellular adherence. It was concluded that LPS did not directly effect the attachment of the MC3T3-E1 osteoblasts to these implant surfaces in vitro. Further research is needed to define the clinical liabilities of LPS during implant placement and maintenance.     

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.     

A study of in vitro attachment of Streptococcus sanguis and Actinomyces viscosus to saliva-treated titanium

This study examined the initial attachment of Streptococcus sanguis G9-B and Actinomyces viscosus T14V to saliva-treated powdered enamel and titanium surfaces. Using an in vitro adherence model, significantly lower numbers of Actinomyces viscosus T14V bound to the saliva-treated titanium surface when compared to that of the similarly treated enamel. The binding of Streptococcus sanguis G9-B to titanium or enamel did not vary significantly. A comparison of the percentage of cells bound to the titanium surface revealed that S sanguis cells attached in significantly higher numbers when compared to the A viscosus cells.     

三种太金属表面处理方法对牙周膜成纤维细胞附着和生长的影响

目的：比较三种不同表面处理方法对牙周膜细胞在钛金属表面附着和生长影响。方法：将三种经不同方法表面处理的钛金属（纯钛，钛75，钛表面氮化处理）试件放在12孔培养板内，取生长良好的第5代人牙周膜成纤维细胞（PDLF）接种在试件表面，分别在接种后24h和72h进行贴壁细胞计数，并在扫描电镜下观察细胞在金属表面附着情况.结果：接种24h后纯钛表面的贴壁细胞数多于钛75金属表面的细胞数（P＜0．05），但在接种后72h时这种差异消失，不论接种后24h或72h钛表面氮化处理金属表面的细胞数都明显少于另外两种金属表面的细胞数（P＜0．05），扫描电镜结果表明PDLF在纯钛和钛75表面伸展，而在钛表面氮化处理的金属表面细胞基本不伸展。结论：人PDLF在纯钛，钛75表面的生物性附着优于在表面氮化处理的钛金属表面。     

Protein adsorption and osteoblast responses to heat-treated titanium surfaces

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 (Ti) surfaces on protein adsorption and osteoblast responses in vitro. The passivated Ti samples used in this study were either nonheat-treated or heat-treated at 750 degrees C for 90 minutes. Using x-ray diffraction analyses, no oxide peaks were observed on the nonheat-treated surfaces, suggesting an amorphous oxide. Crystalline rutile TiO2 peaks were observed on the heat-treated Ti surfaces. The contact angles of water on heat-treated Ti surfaces (32.0 +/- 2.5 degrees) were statistically lower compared with the nonheat-treated Ti surfaces (47.7 +/- 2.3 degrees). In addition, the mean albumin concentration on the nonheat-treated Ti surfaces (3.57 +/- 0.33 micrograms/mL) was observed to be significantly different from the mean albumin concentration on heat-treated Ti surfaces (2.25 +/- 0.26 micrograms/mL). In the presence of an osteoblast precursor cell line, significantly different hexosaminidase activity, protein production, and alkaline phosphatase activity were observed for cells grown on heat-treated Ti surfaces compared with nonheat-treated Ti surfaces.     

Effects of the Nd:YAG laser and combined treatments on in vitro fibroblast attachment to root surfaces

Abstract The purpose of this in vitro study was to evaluate the effects of the Nd:YAG laser either alone or in combination with root planning or air-powder abrasive treatment on fibroblast attachment to non-diseased root surfaces. 28. 4x4 mm root specimens and four disc-shaped root specimens 6 mm in diameter were obtained from unreputed 3rd molars. The root segments were randomly assigned to 4 treatment groups: (1) control: (2) laser-only treated: (3) laser treated followed by root planning; (4) laser treated followed by air-powder abrasive treatment. Laser-treated root specimens were exposed for 1 min with the Nd:YAG laser calibrated at an energy setting of 75 mJ at 20 pulses/s using a 320 μm contact fiber. The contact fiber was held parallel to the root segments and the root segments were kept moist with distilled water. Following the prescribed treatments, the root specimens were incubated with fibroblast cultures and then prepared for SEM examination. Results of cell counts of fibroblasts attached to specimens within each treatment group yielded the following means and standard deviations: control groups. 181.64 ± 44.74; lased only, 78.57 ± 21.35; lased and root planed 125.35 ± 26.13: and lased followed by an air-powder abrasive, 177.28 ± 55.71. Application of ANOVA followed by the Dunn Multiple Comparison test revealed significant differences (p<0.01) in the number of attached cells between the control and laser-only treated groups: and between the laser-only and laser/air-powder abrasive treated groups. The decreased fibroblast attachment observed in the laser-only treated group suggests a laser-induced bio in compatibility of the root surface. Several surface alterations including ablation of cementum with exposure of dentinal tubules and crater formation were observed. Increased numbers of fibroblasts were seen attached to the lased root segments after root planning or after exposure to an air-powder abrasive, indicating that the laser-induced bioincompatibility is reversible and most likely a surface phenomena. A pilot study using pholoacoustic Fourier transform infrared spectroscopy revealed reductions in the intensity of the Amide II band between 1500–1550 cm-1, suggesting the laser exposure denatures surface protein which, in turn, may contribute to inhibition of fibroblast attachment.     

In vitro study of collagen coating of titanium implants for initial cell attachment

The aim of this study was to examine the influence of collagen coating on titanium on the initial attachment of human gingival fibroblasts for the development of the implant with periimplant soft tissue attachment. The morphological changes of cultured human gingival fibroblasts were investigated by scanning electron microcopy (SEM). Four different surfaces, i.e. non-coated mirror-polished titanium, collagen-coated titanium, non-coated tissue-culture polystyrene, and collagen-coated polystyrene were examined. Collagen coating of titanium was effective for enhancing the initial cell attachment. It is expected that collagen coating of titanium implants will improve the attachment of the peri-implant soft tissue to titanium at early stages after the implantation. SEM observation revealed the morphological effect of collagen coating on both titanium and polystyrene surfaces. Many lamellipodia and filopodia were recognized on collagen-coated titanium or polystyrene. Collagen coating improved the activity of human gingival fibroblasts.     

Integrin mediated attachment of periodontal ligament to titanium surfaces

ObjectiveReducing the force between the implant and the bone by recapitulating a similar matrix has the potential to reduce implant failure. To begin to pursue the goal of creating a periodontal ligament interface between a dental implant and bone, the mechanism of cellular attachment to dental implant surfaces must be characterized.MethodsIn this study we examined the role of integrin receptors in the attachment of periodontal ligament fibroblasts to titanium surfaces utilized on dental implants; those surfaces included smooth polished titanium, acid pickled titanium, ground titanium, sandblasted and acid etched titanium, non-oxidized titanium that has been sandblasted and acid etched, hydroxyapatite coated titanium, titanium plasma sprayed or uncoated titanium. For these studies integrin mediated fibroblast attachment was blocked by the integrin blocking peptide GRGDSP or anti-integrin β1 antibody or a combination of the two. Quantitation of periodontal ligament fibroblast attachment was completed by counting cells on the various implant surfaces after culturing in vitro for 24 h with and without the integrin receptor blockers.ResultsAntibody and peptide treatment significantly reduced the number of fibroblasts cells attached to the various implant surfaces but this effect varied significantly depending on the surface. Moreover, increased levels of peptide further decreased fibroblasts attachment in a dose dependent manner.SignificanceBlocking studies suggest first, that integrin receptors function in periodontal ligament attachment to titanium surfaces and second, that different integrin subunits are important in attachment to a particular surface.     

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.     

Magnesium vs. machined surfaced titanium - osteoblast and osteoclast differentiation

PURPOSE

This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source.

MATERIALS AND METHODS

40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs).

RESULTS

MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM.

CONCLUSION

Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.