In vivo evaluation of micro-rough and bioactive titanium dental implants using histometry and pull-out tests

We report on the in vivo histological and mechanical performance of titanium dental implants with a new surface treatment (2Step) consisting of an initial grit-blasting process to produce a micro-rough surface, followed by a combined chemical and thermal treatment that produces a potentially bioactive surface, i.e., that can form an apatitic layer when exposed to biomimetic conditions in vitro. Our aim was to assess the short- and mid-term bone regenerative potential and mechanical retention of 2Step implants in mandible and maxilla of minipigs and compare them with micro-rough grit-blasted, micro-rough acid-etched, and smooth as-machined titanium implants. The percent of bone-to-implant contact after 2, 4, 6, and 10 weeks of implantation as well as the mechanical retention after 4, and 6 weeks of implantation were evaluated with histometric and pull-out tests, respectively, as a measure of the osseointegration of the implants. We also aimed to assess the bioactive nature of 2Step surfaces in vivo. Our results demonstrated that the 2Step treatment produced micro-rough and bioactive implants that accelerated bone tissue regeneration and increased mechanical retention in the bone bed at short periods of implantation in comparison with all other implants tested. This was mostly attributed to the ability of 2Step implants to form in vivo a layer of apatitic mineral that coated the implant and could rapidly stimulate (a) bone nucleation directly on the implant surface, and (b) bone growing from the implant surface. We also proved that roughness values of Ra≈4.5 μm favoured osseointegration of dental implants at short- and mid-term healing periods, as grit-blasted implants and 2Step implants had higher retention values than as machined and acid-etched implants. The surface quality resulting from the 2Step treatment applied on cpTi provided dental implants with a unique combination of rapid bone regeneration and high mechanical retention.     

Pulsed laser deposition of pseudowollastonite coatings

Pseudowollastonite (alpha-CaSiO3) is a bioactive ceramic material that induces direct bone growth. A process to obtain pseudowollastonite coatings that may be applied to implants is described and evaluated in this work. The coatings were first deposited on titanium alloy by laser ablation with a pulsed Nd:YAG laser tripled in frequency. After deposition, they were submitted to a soft laser treatment with a continuous wave Nd:YAG infrared laser. Coatings were characterised by X-ray diffractometry, Raman spectroscopy, scanning electron microscopy and energy dispersive spectroscopy before and after the laser treatment. As-deposited coatings are composed of pseudowollastonite and amorphous material. They have a porous structure of gathered grains and poor cohesion. After the laser treatment the coatings crystallinity and cohesion are improved. The laser treatment also makes the coatings dense and well adhered to the substrate. Therefore, this two-step process has been demonstrated as a valuable method to coat titanium implants with pseudowollastonite.     

Surface modifications induced by ns and sub-ps excimer laser pulses on titanium implant material

Medical implants used in oral and orthopaedic surgery are mainly produced from titanium. Their biological behaviour, e.g. osseointegration, essentially depends on both the chemical composition and the morphology of the surface. Modifications achieved by excimer laser irradiation of titanium samples were investigated in order to improve their surface characteristics so as to facilitate biointegration. To enlarge the effective interfacial area of bone-implant contact, holes were ablated by laser pulses of ns or sub-ps length. During ns ablation, crown-like projecting rims formed around the borders of the holes. Ultra-short (0.5ps) KrF excimer laser pulses were successfully applied to avoid these undesirable formations. Since a smooth dental implant surface is necessary to maintain a healthy connection with the soft tissues, laser polishing of samples was investigated, too. Irradiation with a series of ns laser pulses resulted in effective smoothing, as measured with atomic force microscope. X-ray photoelectron spectroscopy analysis of the laser-polished titanium surface revealed that laser treatment led to a decrease of the surface contamination and in thickening of the oxide layer. X-ray diffraction measurements demonstrated that the original alpha-titanium crystal structure was preserved.     

Fibroblast growth on surface-modified dental implants: an in vitro study

A major consideration in designing dental implants is the creation of a surface that provides strong attachment between the implant and bone, connective tissue, or epithelium. In addition, it is important to inhibit the adherence of oral bacteria on titanium surfaces exposed to the oral cavity to maintain plaque-free implants. Previous in vitro studies have shown that titanium implant surfaces coated with titanium nitride (TiN) reduced bacterial colonization compared to other clinically used implant surfaces. The aim of the present study was to examine the support of fibroblast growth by a TiN surface that has antimicrobial characteristics. Mouse fibroblasts were cultured on smooth titanium discs that were either magnetron-sputtered with a thin layer of titanium nitride, thermal oxidized, or modified with laser radiation (using a Nd-YAG laser). The resulting surface topography was examined by scanning electron microscopy (SEM), and surface roughness was estimated using a two-dimensional contact stylus profilometer. A protein assay (BCA assay) and a colorimetric assay to examine fibroblast metabolism (MTT) were used. Cellular morphology and cell spreading were analyzed using SEM and fluorescence microscopy. Fibroblasts on oxidized titanium surfaces showed a more spherical shape, whereas cells on laser-treated titanium and on TiN appeared intimately adherent to the surface. The MTT activity and total protein were significantly increased in fibroblasts cultured on titanium surfaces coated with TiN compared to all other surface modifications tested. This study suggests that a titanium nitride coating might be suitable to support tissue growth on implant surfaces.     

Three-dimensional topographic scanning electron microscope and Raman spectroscopic analyses of the irradiation effect on teeth by Nd:YAG, Er: YAG, and CO(2) lasers

A three-dimensional analyzer installed in a scanning electron microscope was used to evaluate the morphology and surface roughness using noncontact profilometry. Observations were carried out on the enamel and dentin surface irradiated by three different lasers: Nd:YAG (wavelength 1.06 microm), Er:YAG (2.94 microm), and CO(2) (10.6 microm). Spectroscopic analysis was done by Raman spectroscopy for nonirradiated and laser-irradiated surfaces. The lasers were applied perpendicularly to vertically sectioned and polished human extracted caries-free molars. The tooth was sectioned at each cavity for cross-section analysis after laser irradiation. Irradiation by Nd:YAG and CO(2) lasers of the enamel surface showed an opaque white color, different from dentin where the surface turned black. The Er:YAG laser induced no changes in color of the dentin. Numerous cracks associated with thermal stress were observed in the CO(2) laser-irradiated dentin. Noncontact surface profile analysis of Er:YAG laser-irradiated enamel and dentin showed the deepest cavities, and direct cross-sectional observations of them showed similar cavity outlines. The CO(2) laser-irradiated dentin had the least surface roughness. Raman spectroscopic analysis showed that fluorescence from the laser-irradiated tooth was generally greater than from nonirradiated teeth. Bands in dentin attributed to organic collagen matrix were lost after Nd:YAG and CO(2) laser irradiation, and a broad peak due to amorphous carbon appeared. The Er:YAG laser-irradiated dentin showed no sign of a carbon band and had more suitable results for dental ablation. Noncontact surface profile analysis was effective to evaluate the structural change in the tooth in the microarea of study after laser irradiation.     

Scanning electron microscopical analysis of laser-treated titanium implant surfaces--a comparative study

Design and surface qualities of titanium implants are of vital importance for long-term stability following implantation. Four different implant surfaces treated individually were analyzed with special attention focused on laser surface treatment. Surfaces with machine roughness, titanium spray coating, treated by aluminum oxide and treated by laser were examined individually. Evaluation of the surface was carried out by electron microscope examination and mechanical profilometry. The EDS analysis determined the degree of contamination of the implant surface. Electron microscope examination showed that the titanium plasma spray as well as the laser-treated implants have optimum surface qualities: a secondary and tertiary structure with micro-roughness of 10 mm and roughness ranging from 0.5 to 4 mm. The least contamination was found for machine rough surfaces as well as those treated by laser. The other implants showed contamination corresponding to the method of surface treatment. In summary the optimal surface structure with the least contamination was found for the laser-treated titanium surface. Similar surface purity was found for the machine rough surfaces. An optimal structure was also achieved by the titanium plasma spray method, however, at the cost of surface purity.     

飞秒激光处理促进人牙周成纤维细胞在纯钛材料表面的增殖和分化

背景：目前对种植体表面优化处理促进人工牙种植体骨整合的形成是近年来这一领域的研究热点,而通过飞秒激光这种表面处理钛金属表面改性促进成纤维细胞的增殖和分化的相关性研究报道较少。目的：观察飞秒激光处理对于人牙周膜成纤维细胞在钛金属表面增殖和分化的影响。方法：将人牙周膜成纤维细胞接种在分别经过飞秒激光处理、TiO₂颗粒喷砂和砂纸顺向抛光的纯钛片表面(飞秒激光组,TiO₂颗粒喷砂组和对照组),观察细胞增殖和分化情况。结果与结论：与对照组相比,飞秒激光组和TiO₂颗粒喷砂组钛片表面粗糙度明显增加,表面细胞密度明显增加,RUNX2和OSX mRNA表达水平增加;且飞秒激光组上述指标高于TiO₂颗粒喷砂组。提示飞秒激光处理的纯钛表面更加粗糙,更有利于人牙周膜成纤维细胞的增殖和分化。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

The effect of microstructured surfaces and laminin-derived peptide coatings on soft tissue interactions with titanium dental implants

In the present study, we investigated the dental implant protection from peri-implant inflammation by improving the soft tissue adhesion on the titanium surface. Porous titanium was used to create, at the level of the transmucosal part of the implants (the “neck”), a microstructured 3-dimensional surface that would tightly seal the interface between the implant and soft tissue. Cell-specific adhesion properties were induced via an adhesion peptide derived from laminin-5 coupled to native or cross-linked PLL/PGA multilayered polyelectrolyte films (MPFs), which are used for biomedical device coatings. Porous titanium exhibited good cell-adhesion properties, but the colonisation of the material was further improved by a coating with laminin-5 functionalised MPFs and especially with (PLL/PGA)_6,5–PGA–peptide film. Focal contact formation was observed on cross-linked architectures, reflecting cell anchorage on these surfaces. In contrast, when seeded on laminin-5-functionalised native films, epithelial cells formed only very diffuse focal contacts, but adhered via hemidesmosome formation. In vivo experiments confirmed that the porous titanium was colonised by cells of soft tissue. Altogether, the results indicate that the microstructure of the implant neck combined with a specific bioactive coating could constitute efficient routes to improve the integration of soft tissue on titanium dental implants, which could significantly protect implants from peri-implant inflammation and enhance long-term implant stabilisation.     

Overexposure analysis of pulsed distributed feedback laser source

The goal of this study is to assess the risk of overexposure, when DFB dye laser is used for medical treatment in pulsed mode operation. Results of experimental study showing an unexpected rise of energy in pulses of distributed feedback dye laser (DFDL) output due to temperature phase gratings in dye cell during passively Q switched and mode-locked operation is reported. This unintended increase in the number of pulses, pulse duration, per pulse energy may cause side effects, when used for selective photothermolysis. To probe this phenomenon the most commonly used Rh6G dye was excited with 10-20 pulses of second harmonic of a passively Q switched and mode-locked Nd:yttrium-aluminum-garnet(YAG) laser. The outputs of DFDL and Nd:YAG laser were recorded by an Imacon-675 streak camera. The peak of DFDL output pulses was found delayed proportionally from the peak of the Nd:YAG pulses by more than an interpulse period of excitation laser. A computer program was used to simulate the experimentally measured results to estimate the thermal decay constants and energy retained by medium. The delay between peaks of Nd:YAG (input) and DFDL (output) pulses was found to vary from 10 to 14 ns for various cavity lengths. It was interesting to note that for smaller inter-pulse periods the effect of gradual gain buildup satisfied the threshold conditions for some of the pulses that otherwise cannot lase. This may lead to unintended increase in energy fluence causing overexposure-induced bio effects.     

Detachment of titanium and fluorohydroxyapatite particles in unloaded endosseous implants

The shape, surface composition and morphology of orthopaedic and endosseous dental titanium implants are key factors to achieve post-surgical and long-term mechanical stability and enhance implant osteointegration.In this study a comparison was made between 12 titanium screws, plasma-spray-coated with titanium powders (TPS), and 12 screws with an additional coating of fluorohydroxyapatite (FHA-Ti). Screws were implanted in the femoral and tibial diaphyses of two mongrel sheep and removed with peri-implant tissues 12 weeks after surgery.The vibrational spectroscopic, ultrastructural and morphological analyses showed good osteointegration for both types of implants in host cortical bone. The portion of the FHA-Ti implants in contact with the medullary canal showed a wider area of newly formed peri-implant bone than that of the TPS implants.Morphological and EDAX analyses demonstrated the presence of small titanium debris in the bone medullary spaces near the TPS surface, presumably due to the friction between the host bone and the implant during insertion. Few traces of titanium were detected around FHA-Ti implants, even if smaller FHA debris were present.The present findings suggest that the FHA coating may act as a barrier against the detachment of titanium debris stored in the medullary spaces near the implant surface.     

The effect of plasma chemical oxidation of titanium alloy on bone-implant contact in rats

Many different technologies have been used to enhance osseointegration in orthopaedic and dental implant surgery. Hydroxyapatite coatings, pure or in combination with growth factors or bisphosphonates, showed improved osseointegration of titanium alloy implants. We choose a different approach to enhance osseointegration: plasma chemical oxidation was used to modify the surface of titanium alloy implants. This technique converts the nm-thin natural occurring titanium oxide layer on an implant to a 4?μm thick ceramic coating (TiOB surface). Bioactive TiOB surfaces have a macroporous structure and were loaded with calcium and phosphorus, while bioinert TiOB surfaces are smooth. A rat tibial model with bilateral placement of titanium alloy implants was employed to analyze the bone response to TiOB surfaces in?vivo. 64 rats were randomly assigned to four groups of implants: (1) titanium alloy (control), (2) titanium alloy, type III anodization, (3) bioinert TiOB surface and (4) bioactive TiOB surface. Mechanical fixation, peri-implant-bone area and bone contact were evaluated by pull-out tests and histology at three and eight weeks. Shear strength and bone contact at eight weeks were significantly increased in the bioactive TiOB group compared to all other groups. The results of plasma chemical oxidation in a rat model showed that the bioactive TiOB surface has a positive effect on implant anchorage by enhancing the bone-implant contact in normal bone.     

Residual aluminum oxide on the surface of titanium implants has no effect on osseointegration

The cleanliness of titanium dental implants surfaces is considered to be an important requirement for achieving osseointegration, and it has been hypothesized that the presence of inorganic contaminants could lead to lack of clinical success. Aluminum ions are suspected to impair bone formation by a possible competitive action to calcium. The objective of the present study was to describe the effects of residual aluminum oxide particles on the implant surface on the integration of titanium dental implants as compared to decontaminated implants in a rabbit experimental model. Threaded screw-shaped machined grade 3 c.p. titanium dental implants, produced with high-precision equipment, were used in this study. The implants were sandblasted with 100-120 microm Al2O3 particles at a 5atm pressure for 1min, then 24 implants (control implants) underwent ASTM F 86-68 decontamination process in an ultrasonic bath. The other 24 implants (test implants) were washed in saline solution for 15min. Both test and control implants were air-dried and sterilized at 120 degrees C for 30min. After sterilization the implants were inserted into the tibiae (two test and two control implants in each rabbit). Twelve New Zealand white mature male rabbits were used in this study. The protocol of the study was approved by the Ethical Committee of our University. No complications or deaths occurred in the postoperative period. All animals were euthanized, with an overdose of intravenous pentobarbital, after 4 weeks. A total of 48 implants were retrieved. The images were analyzed for quantitation of percentage of surface covered by inorganic particles, bone-implant contact, multinucleated cells or osteoclasts in contact with the implant surface and multinucleated cells or osteoclasts found 3mm from the implant surface. The differences in the percentages between the two groups have been evaluated with the analysis of variance. The implant surface covered by inorganic particles on test implants was significantly higher than that of control implants (p=0.0000). No statistically significant differences were found in the bone-implant contact percentages of test and control implants (p=0.377). No statistically significant differences were found in the number of multinucleated cells and osteoclasts in contact with the implant surface (p=0.304), and at a distance of 3mm from the implant surface (p=0.362). In conclusion, our histological results do not provide evidence to support the hypothesis that residual aluminum oxide particles on the implant surface could affect the osseointegration of titanium dental implants.     

Optothermal transfer simulation in laser-irradiated human dentin

Laser technology has been studied as a potential replacement to the conventional dental drill. However, to prevent pulpal cell damage, information related to the safety parameters using high-power lasers in oral mineralized tissues is needed. In this study, the heat distribution profiles at the surface and subsurface regions of human dentine samples irradiated with a Nd:YAG laser were simulated using Crank-Nicolson's finite difference method for different laser energies and pulse durations. Heat distribution throughout the dentin layer, from the external dentin surface to the pulp chamber wall, were calculated in each case, to investigate the details of pulsed laser-hard dental tissue interactions. The results showed that the final temperature at the pulp chamber wall and at the dentin surface are strongly dependent on the pulse duration, exposure time, and the energy contained in each pulse.     

Differential adhesion of Streptococcus gordonii to anatase and rutile titanium dioxide surfaces with and without functionalization with chlorhexidine

The majority of dental implants are composed primarily of titanium and have an outer layer of titanium dioxide. Crystalline titanium dioxide most commonly exists in one of the two structures, anatase and rutile, and both of these have been observed on commercially available dental implants. Early implant failure can be associated with postoperative infection due to implant contamination during or immediately after surgery. The impetus of this study was to investigate whether functionalization of anatase and rutile titanium dioxide surfaces with chlorhexidine-reduced subsequent colonization of the surface by Streptococcus gordonii. Exposure to 100 mg x L(-1) chlorhexidine for 60 s resulted in a fivefold reduction in S. gordonii coverage on anatase and a twofold reduction on rutile. This may be related to a preferential adsorption of chlorhexidine to anatase compared with rutile. The reduction in bacterial coverage was not due to desorption of chlorhexidine into solution. More bacteria were observed on anatase than rutile surfaces without chlorhexidine functionalization, indicating that crystal structure may have a significant effect on bacterial colonization. In conclusion, functionalization with chlorhexidine reduced bacterial coverage on titanium dioxide surfaces, and anatase surfaces may be more amenable to such treatment than rutile.     

Characterization and biocompatibility of a titanium dental implant with a laser irradiated and dual-acid etched surface

The biological properties of commercial pure titanium (cp-Ti) dental implants can be improved by surface treatment. In this study, the cp-Ti surfaces were prepared to enable machined surfaces (TM) to be compared to the machined, sandblasted, laser irradiated and dual-acid etched surfaces (TA). The surface elements and roughness were characterized. The biocompatibility was evaluated by cell and organ culture in vitro. The removal torque was measured in rabbit implantation. Surface characterization revealed that TA surface was more oxidized than TM surface. The TA surface had micrometric, beehive-like coarse concaves. The average roughness (2.28 mum) was larger than that typical of acid-etched surfaces. Extracts of both materials were not cytotoxic to bone cells. The morphology of cells attached on the TA surface was superior to that on the TM surface. TA promoted cell migration and repaired damaged bones more effectively in organ culture. The formation of bone-like nodules on TA disk exceeded that on TM disk. Rabbit tibia implantation also proved that TA implant had greater removal torque value. These results suggested that TA had good osteoconductivity and was a potential material for dental implantation.     

Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces

The demand for orthopedic and dental implants will continue to grow, and for these applications, titanium and its alloys have been used extensively. While these implants have achieved high success rates, two major complications may be encountered: the lack of bone tissue integration and implant-centered infection. The surface of the implant, through its interactions with proteins, bacteria and tissue cells, plays a determining role in the success or failure of the implant. Ideally, to enhance the success of implants, their surfaces should inhibit bacterial colonization and concomitantly promote osteoblast functions. In this article, we discuss strategies for tailoring implant surfaces by exploiting the differences in the response of bacteria and osteoblasts to proteins and surface structures. Nevertheless, limitations still exist in the quest for an ideal implant surface. Further advances in this field will require concurrent development in surface modification techniques and a better understanding of the complex and highly inter-related events occurring at the implant surface after implantation.     

Influence of a zirconia sandblasting treated surface on peri-implant bone healing: An experimental study in sheep

A sandblasting process with round zirconia (ZrO₂) particles might be an alternative surface treatment to enhance the osseointegration of titanium dental implants. Our previous study on sheep compared smooth surface titanium implants (control) with implant surfaces sandblasted with two different granulations of ZrO₂. As the sandblasted surfaces proved superior, the present study further compared the ZrO₂ surface implant with other surface treatments currently employed: machined titanium (control), titanium oxide plasma sprayed (TPS) and alumina sandblasted (Al-SL) at different times after insertion (2, 4 and 12 weeks). Twelve sheep were divided into three groups of four animals each and underwent implant insertion in tibia cortical bone under general anaesthesia. The implants with surrounding tissues were subjected to histology, histomorphometry, scanning electron microscopy and microhardness tests. The experimentation indicated that at 2 weeks Zr-SL implants had the highest significant bone ingrowth (p < 0.05) compared to the other implant surfaces, and a microhardness of newly formed bone inside the threads significantly higher than that of Ti. The present work shows that the ZrO₂ treatment produces better results in peri-implant newly formed bone than Ti and TPS processing, whereas its performance is similar to the Al-SL surface treatment.     

The bisphosphonate pamidronate on the surface of titanium stimulates bone formation around tibial implants in rats

Many materials with differing surfaces have been developed for clinical implant therapy in dentistry and orthopedics. We analyzed the quantity of new bone formed in vivo around calcium-immobilized titanium implants with surfaces modified using pamidronate (PAM), a nitrogen-containing bisphosphonate (N-BP), implants of pure titanium, and titanium implants immobilized with calcium ions. New bone formation was visualized using fluorescent labeling (calcein blue and alizarin complexone) with intravenous injection at 1 and 3 weeks after implantation. After 4 weeks, undecalcified sections were prepared, and new bone formation around the implants was examined by morphometry using confocal laser scanning microscopy images. After 1 week, more new bone formed around the PAM-immobilized implant than around the calcium-immobilized and pure titanium implants. This was also seen with the new bone formation after 3 weeks. After 4 weeks, significantly more new bones were formed around the BP-immobilized implant than around the calcium ion-implanted and pure titanium implants. The new N-BP-modified titanium surface stimulates new bone formation around the implant, which might contribute to the success of implant therapy.     

强脉冲光联合Nd:YAG激光治疗黄褐斑疗效观察

目的:探讨强脉冲光联合Nd:YAG激光治疗黄褐斑的疗效。方法:60例患者按治疗方法不同分成3组:强脉冲光组、Nd:YAG激光组、联合组（强脉冲光联合Nd:YAG激光组）,每组20例患者,观察3组患者疗效和副作用。结果:3组之间疗效两两比较采用Mann-Whitney秩和检验,强脉冲光组和激光组疗效无显著差异,而联合组疗效显著好于强脉冲光组（Z=7.123, P=0.024）和激光组（Z=6.024, P=0.030）。3组患者治疗后均无出现色素沉着加重、色素脱失病等不良反应。强脉冲光组和激光组的满意度无显著差异,而联合组的满意度显著好于强脉冲光组（Z=7.361, P=0.029）和激光组（Z=6.831, P=0.027）。结论:强脉冲光联合Nd:YAG激光治疗黄褐斑是一种安全有效的治疗方法,值得临床推广。