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The goal of this study was the in vivo evaluation of nanoporous titanium (Ti) implants bearing a covalently linked surface hyaluronan (HA) layer. Implant surface topography and surface chemistry were previously evaluated by scanning electron microscopy and X-ray photoelectron spectroscopy. Results showed that the surface modification process did not affect surface topography, yielding a homogeneously HA-coated nanotextured implant surface. In vivo evaluation of implants in both cortical and trabecular bone of rabbit femurs showed a significant improvement of both bone-to-implant contact and bone ingrowth at HA-bearing implant interfaces at 4 weeks. The improvement in osteointegration rate was particularly evident in the marrow-rich trabecular bone (bone-to-implant contact: control 22.5%; HA-coated 69.0%, p < 0.01). Mechanical testing (push-out test) and evaluation of interfacial bone microhardness confirmed a faster bone maturation around HA-coated implants (Bone Maturation Index: control 79.1%; HA-coated 90.6%, p < 0.05). Suggestions based on the biochemical role of HA are presented to account for the observed behavior.

Ti (PG60) and Ti plus HA (HPG60) dense coatings with ultrahigh roughness (Ra: 74 +/- 8 microm and 53 +/- 18 microm, respectively) were compared to high Ti (Ti60) and Ti plus HA (HT60) high roughened porous coatings (Ra: 40 +/- 7 microm and 36 +/- 3 microm, respectively). Surfaces were implanted in cortical and trabecular bone of young adult (YOUNG), aged (AGED) and estrogen-deficient sheep (OVX) and analyzed by means of histology, histomorphometry and push-out tests 3 months after implantation. A significantly lower value in affinity index (AI) of PG60 when compared to TI60 (p < 0.01) was observed in cortical bone. In trabecular bone, lower values in AI were found in TI60 and PG60 when compared to their HA-coated surfaces (p < 0.0005). Bone ingrowth (BI) of TI60 and PG60 was significantly lower than that of the HA-coated surfaces in trabecular bone (p < 0.05). Significantly lower values in BI in OVX sheep in comparison to YOUNG sheep in both cortical and trabecular bone were observed (p < 0.05). Data showed that high roughness and Ti and HA-coated surfaces are suitable for aged and osteoporotic patients. HA coatings represent the most successful strategy in trabecular bone.

Chronic alcohol consumption is associated with pathological effects on bone, and it is correlated with the increasing risk of osteoporosis and fractures. The negative effects of alcohol intake also influence bone repair processes and the osseointegration of implants. The aim of the present in vitro study was to investigate the proliferation and synthetic activity of osteoblasts isolated from the trabecular bone of rats previously exposed to 7-week intermittent exposure to ethanol vapour (EE-OB), and sham-aged rats (SA-OB), when cultured on standard commercially pure Ti (cpTi). Osteoblast proliferation (WST-1), alkaline phosphatase (ALP), osteocalcin (OC), collagen type I (CICP), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and transforming growth factor-beta1 (TGF-beta1) were measured at 1, 7, and 14 days of culture. Our results showed a decrease in the cell viability and synthetic activity of osteoblasts exposed to ethanol when cultured on cpTi. Moreover, the release of local regulatory factors from osteoblasts was imbalanced: TGF-beta1 production was reduced and TNF-alpha and IL-6 were up-regulated. These in vitro data suggest that alcohol abuse affects bone repair and decreases the ability to form bone around standard cpTi. Innovative surfaces and adjuvant therapies could be useful when implants are required in alcoholics.

The osteointegration rate of titanium (Ti; TI01) and duplex Ti plus HA (HT01) coating systems with high surface roughness was investigated in healthy, aged, and oestrogen-deficient sheep. After having evaluated the bone quality, TI01 and HT01 rods were implanted in the tibial diaphyses (two implants for each tibia) and epiphyses (1 implant for each tibia) of five young (YOUNG), five aged (AGED), and five aged and ovariectomized (OVX) sheep. The iliac crest trabecular bone volume (BV/TV) and number (Tb.N) in OVX sheep were respectively 33.5% and 28.5% lower than in YOUNG sheep (p < 0.005) and lower than in the AGED group (BV/TV, -17%; Tb.N, -13.5%; not significant); in the OVX group the trabecular separation was 77.9% higher than in YOUNG (p < 0.05) and 30.9% higher than in AGED animals. Lumbar vertebrae L5 bone mineral density was significantly lower in AGED (8.9%, p < 0.05) and OVX sheep (19.3%, p < 0.0005) when compared with YOUNG animals. Five samples of five sheep from each group were analyzed for each observation. At 3 months, in cortical bone both affinity index and pushout test results showed no significant differences between the two materials in each group of animals. In trabecular bone, the affinity index of HT01 was significantly higher than that of TI01 in each group of animals (YOUNG, 90.7%; AGED, 76.9%; OVX, 49.9%) with no significant differences between groups. In conclusion, the performance of TI01 and HT01 surfaces was high not only in YOUNG, but also in OVX animals and, therefore, they might be useful for aged and osteoporotic patients.

Background: Recently, the use of titanium micromesh for alveolar bone augmentation has drawn interest; however, only limited histologic data are available on the quality of the bone regenerated. Therefore, this study compared the use of 100% intraoral autogenous bone to a combination of intraoral autogenous bone (70%) and bovine porous bone mineral (BPBM) (30%) for alveolar ridge augmentation with titanium micromesh histologically and histomorphometrically., Methods: Twelve partially edentulous patients required alveolar bone augmentation before implant insertion because of ridge resorption. The defect sites, six in the maxilla and six in the mandible, were reconstructed with particulate autologous bone (control group, N = 6) or a mixture of autologous bone and BPBM (test group, N = 6) in combination with titanium micromesh. Core biopsies were taken from the defect sites 8 to 9 months after grafting at the time of implant insertion., Results: Newly formed compact bone with a well-organized lamellar pattern was identified in all specimens. In the samples taken from the test group, the BPBM particles were surrounded completely by newly formed bone with no signs of resorption. The mean total bone volume was 62.38% +/- 13.02% in the control group and 52.88% +/- 11.47% in the test group. The soft tissue volume was 37.61% +/- 13.02% and 29.96% +/- 12.58%, respectively, and the residual BPBM volume was 17.15% +/- 2.72% in the test group. No statistical difference was observed in the histologic parameters evaluated, irrespective of graft type and site (P >0.05)., Conclusion: Within the limits of this study, BPBM (30%) in combination with autogenous bone (70%) did not yield a lower percentage of new bone formed compared to autogenous bone alone in ridge augmentation with titanium micromesh.

In this study we coated a new biocompatible, nanostructured titanium alloy, Ti13Nb13Zr, with a thin layer of hydroxyapatite nanocrystals and we investigated the response of human bone-marrow-derived mesenchymal cells. The coating was realized using a slightly supersaturated CaP solution, which provokes a fast deposition of nanocrystalline hydroxyapatite. A thin layer of deposition is appreciable on the etched Ti13Nb13Zr substrates after just 1.5 h soaking in the CaP solution, and it reaches a thickness of 1-2 mum after 3 h soaking. The coating seems thinner than that deposited on Ti6Al4V, which was examined for comparison, likely because of the different roughness profiles of the two etched alloys, and it is constituted of elongated HA nanocrystals, with a mean length of about 100 nm. Mesenchymal stem cells were seeded onto coated and uncoated Ti alloys and cultured for up to 35 days. Cell morphology, proliferation and differentiation were evaluated. The cells display good adhesion and proliferation on the uncoated substrates, whereas the presence of hydroxyapatite coating slightly reduces cell proliferation and induces differentiation of MSCs towards a phenotypic osteoblastic lineage, in agreement with the increase of the expression of osteopontin, osteonectin and collagen type I, evaluated by means of rt-PCR. Type I collagen expression is higher in Ti13Nb13Zr MSC culture compared to Ti6Al4V, standing for a more efficient extracellular matrix deposition., (Copyright 2007 Wiley Periodicals, Inc.)

To evaluate how aging and estrogen deficiency influence the success rate of Sandblasted Titanium (Ti/SA) implants, the osteointegration of Ti/SA rods was studied in the cortical and trabecular bone of 5 young, 5 aged and 5 ovariectomized (OVX) sheep. The characterization of the host bone by transiliac biopsies of the iliac crest showed a progressive rarefaction of trabecular bone in aged and OVX animals when compared to young ones. A significant reduction, both in cortical and trabecular bone, of the osteointegration rate of Ti/SA rods in the presence of estrogen deficiency compared to young animals was observed, while only a minor reduction was observed in aged animals. These results were confirmed by the pushout test in cortical bone. Bone quality affected the biological response of bone to Ti/SA implants in both trabecular and cortical bone; consequently, strategies to maximize the bone osteogenic properties of osteoporotic patients should be adopted.

Small titanium particles may detach from titanium plasma sprayed (TPS) implants during implant insertion, when no preliminary tapping is used, probably for the frictional force between titanium coating and host bone. Aim of this study was to investigate the destination of these titanium particles observed in the peri-implant environment. Twenty-four TPS screws were implanted in tibiae of two sheep. Fourteen and 90 days after implantation the implants with the surrounding bone were removed and processed to be analyzed by light microscope and scanning electron microscope (secondary electron and back-scattered electron probes). Small titanium particles detached from the unloaded TPS implants were observed both in the newly-formed bone matrix and in marrow tissue. Histomorphometric analysis showed that both at 14 and 90 days after implantation the titanium particles appeared more concentrated in marrow tissue than in calcified bone matrix, decreasing by 66.4% over time. In particular, smaller particles (<250 microm(2)) decreased by 81.5%, whereas the larger ones (250-2000 microm(2)) did not show any significant variations over time, suggesting that most of the smaller particles may undergo to ionic dissolution, probably migrating into the peri-implant marrow lacunae. A slight migration of titanium particles from the implant surface towards the more distant peri-implant tissues was also demonstrated over time.

The electrochemical impedance spectroscopy (EIS) technique was used for the study of the electrochemical behavior of an equiatomic NiTi alloy and an implant quality AISI 316 stainless steel type ASTM F138. Experiments were carried out using four different different test solutions: phosphate buffered saline (PBS), Dulbecco minimum essential medium (MEM), MEM + fetal calf serum (FCS), and MEM + fetal calf serum + fibroblast cell (CELL). Specimens were finished to 600-grit SiC paper and were tested in conditions that did not provoke abrupt mechanical damage of the passive film. Bode-phase spectra showed the presence of two maxima and were fitted with an equivalent circuit characterized by two parallel combinations (R, resistance; CPE, constant phase element). The R(1) and CPE(1) branch was assigned to the inner compact passive film and the R(2) and CPE(2) branch to the external porous film. The resistance of the inner film R(1), roughly corresponding to the polarization resistance (R(p)), which is inversely proportional to the material's corrosion rate, increased with the immersion time and was generally greater in PBS than in other media. With the exception of FCS solution, R(1) for NiTi alloy is better or similar to that of ASTM F138., ((c) 2006 Wiley Periodicals, Inc.)

The goal of the study was the evaluation of the effect of modification of titanium implants by acrylic acid surface grafting-collagen I coupling. Tests were performed on titanium samples treated by galvanostatic anodization to create a porous surface topography. Surface characterization by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) confirms the biochemical modification of the surface and shows a surface topography characterized by pores mostly below 1 mum diameter. In vitro evaluation involving human mesenchymal cells shows enhanced cell growth on collagen coated surfaces as compared to titanium ones. Four weeks in vivo evaluation of implants in rabbit femur trabecular bone shows improvements of bone-to-implant contact, while improvement of bone ingrowth is slightly not significant (p = 0.056), when compared to the control. Overall, these data indicate that integration in trabecular, or cancellous, bone can be enhanced by the surface collagen layer, confirming previous findings obtained by modification of machined surfaces by the same approach in cortical bone implants.

This paper describes the effect of surface functionalization on surface composition and cell adhesion to titanium samples by high and low molecular weight Hyaluronan (HA). HA was covalently linked to aminated Ti surfaces obtained by two different surface functionalization techniques, that is polyethyleneimine (PEI) adsorption and deposition from allylamine plasma. The two approaches yield very different surface densities of available amino groups, affecting this way the number and frequency of surface-HA bonds and the configurational freedom of the latter. Results of cell adhesion test are dependent on the surface functionalization approach adopted, low molecular weight HA coupled to PEI functionalized Ti does not yield the same degree of resistance to cell adhesion found on other samples. These results indicate that the details of the surface functionalization step are crucial for surface engineering of implant devices by biological molecules.

The development of novel mechanical and chemical surface modification treatments to improve the osteointegration properties of osseointegrated dental implants is nowadays a topic of great applicative interest. The aim of the present study was to analyse the role of surface topography and chemistry of four different surface treatments on titanium by an in vitro human osteosarcoma immortalised cell line model (MG63). The surface treatments considered were (a) machined titanium, (b) chemical etched on machined titanium, (c) sandblasted titanium and (d) chemical etching on sandblasted titanium. Chemical and physical surface properties were investigated by Scanning Electron Microscopy, Thin Film-X ray Diffraction and by Laser Profilometry. The in vitro biological response was characterised using the MG63 cell line by elution cytotoxicity tests, cell morphology, adhesion, proliferation activity, alkaline phosphatase activity and total DNA content in order to show a relationship between osteoblast response and surface features. Chemical and physical characterisation showed that the considered treatments differently modify the surface morphology in the micro and sub-micrometric scale. Although some differences in alkaline phosphatase activity were observed in the biological characterisation, depending on the specific material's surface finishing, the results showed that cells were well responsive on all the tested materials and grew and differentiated with similar proliferation rate.

The aim of this study was to characterize and compare various titanium (Ti) and hydroxyapatite (HA) coatings on Ti6Al4V, in view of their application on noncemented orthopedic implants. Two innovative vacuum plasma sprayed (VPS) coatings, the first of ultrahigh rough and dense Ti (PG60, Ra=74 microm) and the second of ultrahigh rough and dense Ti coated with HA (HPG60, Ra=52 microm), have been developed, and the response of osteoblast-like cells (MG-63) seeded on these new coatings was evaluated in comparison to: a low roughness and sandblasted (Ti/SA, Ra=4 microm) Ti6Al4V surface; Ti medium (TI01, Ra=18 microm), and high (TI60, Ra=40 microm) roughness VPS coatings; and the relative Ti plus HA duplex coatings (HT01, Ra=12 microm and HT60, Ra=36 microm respectively), also obtained by VPS. PG60 coating presented no open porosity, making it dense and potentially intrinsically stronger. Cell adhesion and proliferation on PG60 was similar to those of the smoothest one (Ti/SA) and adhesion on ultrahigh roughness was lower than the medium- and high-roughness coatings, whereas cell proliferation on PG60 was lower than TI60. The HA coating determined significant increases in cell proliferation at medium and high roughness levels when compared to the relative Ti coating, but not compared to the ultrahigh one; all HA-coated surfaces showed a decrease in alkaline phosphatase activity and collagen I production. Surface morphology and the HA coating strongly affected cell behavior. However, ultrahigh values of roughness are not correctly seen by cells, and the presence of HA has no improving effects., (Copyright (c) 2005 Wiley Periodicals, Inc.)

A new implant surface has been developed with the purpose of avoiding as much stress shielding as possible, and thus prolong the prosthesis lifespan. The aim of this study was to investigate the in vitro effect of this new ultra-high roughness and dense Titanium (Ti) surface (PG60, Ra = 74 microm) in comparison with medium (TI01, Ra = 18 microm) and high (TI60, Ra = 40 microm) roughness and open porous coatings; all the coatings were obtained by vacuum plasma spraying. MG63 osteoblast-like cells were seeded on the tested materials and polystyrene, as control, for 3 and 7 days. Cells proliferated on the material surfaces similarly to the control. Alkaline phosphatase activity had lower values for TI60 than TI01 (p < 0.0005) and PG60 (p < 0.005). Osteocalcin levels measured on TI60 were significantly (p < 0.0005) lower in comparison with TI01 and PG60 at 7 days. Procollagen-I synthesis reduced with increasing roughness and the lowest data was found for PG60. While at 3 days Transforming Growth Factor beta1 levels augmented with increasing roughness, at 7 days TI60, the high roughness surface, was significantly lower than PG60 (p < 0.005) and TI01 (p < 0.001). All tested materials showed significantly higher Interleukin-6 levels than those of polystyrene at both experimental times. Nitric Oxide activity on TI01 was significantly (p < 0.05) higher than on TI60 and polystyrene. In conclusion, the new ultra-high roughness and dense coating PG60 provided a good biological response, even though, at least in vitro, it behaved similarly to the coatings already used in orthopaedics.

Over the past decade the increase of elderly population has determined a rise in the incidence of bone fractures, and the improvement of the implant-bone interface remains an open problem. Metal-organic chemical vapour deposition (MOCVD) has recently been proposed as a technique to coat orthopaedic and dental prostheses with metal nanostructured oxide films either through the decomposition of oxygenated compounds (single-source precursors) or the reaction of oxygen-free metal compounds with oxygenating agents. The present study was performed to assess the in vivo biocompatibility of commercially pure Ti (control material: TI/MA) implants ( psi 2 mm x 5 mm length) coated with nanostructured TiO2 films by MOCVD (Ti/MOCVD) and then inserted into rabbit femoral cortical (middhiaphysis) and cancellous (distal epiphysis) bone. Histomorphometric, ultrastructural and microhardness investigations were carried out. Four and 12 weeks after surgery, significant (p<0.0005) increases in AI of Ti/MOCVD implants were observed as compared to Ti/MA implants (distal femoral epiphysis: 4 weeks=8.2%, ns; 12 weeks=52.3%, p <0.005; femoral diaphysis: 4 weeks=20.2%, p <0.0005; 12 weeks=10.7%, p <0.005). Bone microhardness results showed significant increases for the Ti/MOCVD versus Ti/MA implants at 200 microm in the femoral diaphysis (4 weeks=14.2, p <0.005) and distal femoral epiphysis (12 weeks=14.5, p <0.01) at 4 and 12 weeks, respectively. In conclusion, the current findings demonstrate that the nanostructured TiO2 coating positively affects the osseointegration rate of commercially pure Ti implants and the bone mineralization at the bone-biomaterial interface in both cortical and cancellous bone.

Two different cationic antimicrobial molecules, chlorhexidine (CH) and poly(hexamethylenebiguanide) (PH), were adsorbed from aqueous solution to titanium implant devices surface-modified by the covalent coupling of collagen on a polyanionic acrylic acid overlayer. Results show that more antimicrobial was adsorbed on surface modified implants as compared to control titanium devices. Moreover, the kinetic of release was affected by the interaction between the polyanionic overlayer and the cationic antimicrobial, leading to slower kinetic of release in the case of CH and stable adsorption in the case of polycationic PH . These data indicate that biochemically modified collagen coated surfaces could be endowed also by antimicrobial properties, in the spirit of present researches on multifunctional implant surfaces.

Titanium (Ti) endosseous dental screws with different surfaces (smooth titanium--STi, titanium plasma-sprayed-TPS, alumina oxide sandblasted and acid-etched--Al-SLA, zirconium oxide sandblasted and acid etched--Zr-SLA) were implanted in femura and tibiae of sheep to investigate the biological evolution of the peri-implant tissues and detachment of Ti debris from the implant surfaces in early healing. Implants were not loaded. Sections of the screws and the peri-implant tissues obtained by sawing and grinding were analysed by light microscopy immediately after implantation (time 0) and after 14 days. All samples showed new bone trabeculae and vascularised medullary spaces in those areas where gaps between the implants and host bone were visible. In contrast, no osteogenesis was induced in the areas where the implants were initially positioned in close contact with the host bone. Chips of the pre-existing bone inducing new peri-implant neo-osteogenesis were surrounded by new bone trabeculae. The threads of some screws appeared to be deformed where the host bone showed fractures. Ti granules of 3-60 microm were detectable only in the peri-implant tissues of TPS implants both immediately after surgery and after 14 days, thus suggesting that this phenomenon may be related to the friction of the TPS coating during surgical insertion.

Collagen was covalently linked to the surface of Titanium (Ti) by a surface modification process involving deposition of a thin film from hydrocarbon plasma followed by acrylic acid grafting. The composition and properties of surface-modified Ti were investigated by a number of surface sensitive techniques: XPS, ATR-IR, atomic force microscopy and AFM force-separation curves. In vitro tests were performed to check samples cytotoxicity and the behavior of osteoblast-like SaOS-2 cells. In vivo experiments involved 12 weeks implants in rabbit muscle as general biocompatibility assessment and 1-month implants in rabbit bone to evaluate the effect of surface modification on osteointegration rate. Results of XPS measurements show how surface chemistry is affected throughout each step of the surface modification process, finally leading to a complete and homogeneous collagen overlayer on top of the Ti samples. AFM data clearly display the modification of the surface topography and of the surface area of the samples as a consequence of the grafting and coupling process. AFM force-distance curves show that the interfacial structure responds by shrinking or swelling to variations of ionic force of the surrounding aqueous environment, suggesting that the aqueous interface of the biochemically modified Ti samples has enhanced degrees of freedom as compared to the inorganic surface of plain Ti. As to biological evaluations, the biochemically modified Ti samples are safe in terms of cytotoxicity and in vivo biocompatibility assessment. SaOS-2 cells growth rate is lower on collagen modified surfaces, and no significant difference is detected in terms of alkaline phosphatase production as compared to control Ti. Importantly, implants in rabbit femur show a significant increase of bone growth and bone-to-implant contact in the case of the collagen modified samples, confirming that biochemical modifications of Ti surface can enhance the rate of bone healing as compared to plain Ti.

Several factors influence the healing process and the long-term mechanical stability of cementless fixed implants, such as bone remodeling and mineralization processes. Histomorphometric and bone hardness measurements were taken in implants inserted in sheep femoral cortical bone at different times to compare the in vivo osseointegration of titanium screws (diam.; 3.5 x 7 mm length) with the following surface treatments: machined (Ti-MA); acid-etched (Ti-HF); HA vacuum plasma spray (Ti-HA); and Ca-P anodization followed by a hydrothermal treatment (Ti-AM/HA). Ti-MA and Ti-AM/HA implants presented the lowest (Ra = 0.20 +/- 0.01 microm) and highest (Ra = 1.97 +/- 0.64 microm) significant (p < 0.0005) roughness value, respectively. Bone-to-implant contact of Ti-HF was lower than that of the other surface treatments at both experimental times (8 weeks: -20%, ns; 12 weeks: -30%, p < 0.01). Significant differences in MAR (mineral apposition rate) were also found between the different experimental times for Ti-MA (115%, p < 0.01) and Ti-HF (57%, p < 0.01), demonstrating that bone growth had slowed inside the screw threads of Ti-HA and Ti-AM/HA after 12 weeks. No bone microhardness changes in preexisting host bone were found, while Ti-MA showed the lowest value for the inner thread area at 8 weeks (HV(200 microm)= 49.8 +/- 3.8 HV). These findings confirm that osseointegration may be accelerated by adequate surface roughness and bioactive ceramic coating such as Ca-P anodization followed by a hydrothermal treatment, which enhance bone interlocking and mineralization., (Copyright 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 112-120, 2003)

A new austenitic stainless steel compound, P558, has been widely recognized to have good mechanical properties, excellent potential for corrosion resistance and negligible nickel ion release, making it a promising substitute for more expensive metallic prostheses with limited machinable features. The effect of P558 was studied in vitro and human osteoblast- like cells (MG63) were cultured directly on P558, Ti6Al4V alloy (Ti), and polystyrene (Control) for 72 hours. Osteoblast functions were evaluated by assaying cell proliferation and synthetic activity after 1.25(OH)2D3 stimulation. Results demonstrated that growth of MG63 on P558 was not negatively affected when compared to the Ti and Control groups and showed no alteration in the production of ALP, NO and PICP. Moreover, IL-6 was lower, whereas OC and TGFbeta1 were significantly higher. SEM images revealed that cells proliferated and differentiated on P558 without any alteration in their morphology. The current findings have demonstrated that P558 promotes osteoblast proliferation, activation and differentiation without negative effects and, thus, its good biocompatibility when used for orthopedic application.

The influence of fluorohydroxyapatite (FHA) coating and surface roughness of Ti6Al4V implants on bone response was investigated. Uncoated and FHA-coated screws with lower (LR and LR+FHA; Ra: 5.7+/-0.2 microm) and higher (HR and HR+FHA; Ra: 21.8+/-0.9 microm) surface roughness, were inserted into the diaphyses of 8 sheep tibiae. Twelve weeks after implantation, extraction torque and bone-to-implant contact were evaluated. The smoothest surfaces showed an improved extraction torque and significant differences were observed between LR and HR (-24.6%, p<0.0005), LR and HR+FHA (-30.7%, p<0.0005), LR+FHA and HR (-17.4%, p<0.005), and LR+FHA and HR+FHA (-24.0%, p<0.005). The bone-to-implant contact data paralleled the biomechanical data: the smoother the surface, the greater the bone-to-implant contact. Significant (p<0.0005) decreases in bone-to-implant contact were observed between LR+FHA and HR (-24.2%), and between LR+FHA and HR+FHA (-29.2%). The current findings suggest that LR surfaces significantly improve the osteointegration rate of implanted cortical screws independently of the FHA coating.

Metal-Organic Chemical Vapor Deposition (MOCVD) has recently been proposed to coat orthopedic and dental prostheses with metal nanostructured oxide films through the decomposition of oxygenated compounds (single-source precursors) or the reaction of oxygen-free metal compounds with oxygenating agents. The present study was carried out to assess the in vitro biocompatibility in terms of cell proliferation and activation, of commercially pure Ti (control material: TI/MA) coated with nanostructured TiO2 film by MOCVD (Ti/MOCVD) using osteoblast-like cell cultures (MG-63). Evaluations were performed at 3, 7 and 14 days. Cell proliferation showed a similar trend for Ti/MA and TilMOCVD compared to polystyrene; cell number increased with time from seeding to day 7 (p < 0.005), and then decreased progressively until day 14 (ranging from -14% to -47%). The ALP level and OC production showed no significant differences between Ti/MOCVD and Ti/MA at each experimental time. Significantly higher ALP levels were found in Ti/MA at 3 days and in Ti/MOCVD at 7 and 14 days when compared to the polystyrene group. OC production decreased over time and the highest values were observed at 3 days, when it was significantly higher in the Ti/MA than in the polystyrene group (50%, p < 0.05). CICP synthesis was positively affected by the presence of Ti/MOCVD and was higher in Ti/MOCVD than in the polystyrene group. No significant differences were found between Ti/MOCVD and Ti/MA in terms of IL-6 and TGF-beta1 synthesis at any experimental time. In conclusion, the current findings demonstrate that the nanostructured TiO2 coating positively affects the osteoblast-like cell behavior in terms of cell proliferation and activity, thus confirming its high level of in vitro biocompatibility in accordance with expectations.

Improvement of the implant-bone interface is still an open problem and the interest in chemical modification of implant surfaces for cementless fixation has grown steadily over the past decade. Mechanical and histomorphometric investigations were performed at different times on implants inserted into sheep femoral cortical bone to compare the in vivo osseointegration of titanium screws ( X 3.5 x 7 mm length) with different surface treatments. After 8 weeks of implantation, the push-out force of anodized and hydrothermally treated implants (ANODIC) was significantly higher than that of machined implants (MACH) (36%, p<0.0005), whereas a decrease of 39% was observed for acid-etched implants (HF) when compared to other surface treatments. After 12 weeks of implantation, the push-out force values of HF implants were still significantly lower than those observed for MACH (-19%, p<0.01) and hydroxyapatite vacuum plasma-sprayed implants (HAVPS, -25%, p<0.0005), and the highest push-out force was found in HAVPS (p<0.001) implants. After 8 and 12 weeks of implantation, the AI of HF implants was significantly (p<0.05) lower ( approximately -25%) than that of MACH, HAVPS and ANODIC implants. In conclusion, results appear to confirm that there are no specific differences between ANODIC and HAVPS implants in terms of behavior. Moreover, although MACH implants show some surface contaminating agents, they appear to ensure good osseointegration within 12 weeks both mechanically and histomorphometrically, as do ANODIC and HAVPS implants. However, further studies are required to investigate bone hardness and mineralization around implants.

Purpose: To analyze the surface composition of 34 different commercially available titanium dental implants., Materials and Methods: Surface composition was evaluated by x-ray photoelectron spectroscopy (XPS). Samples were divided into 4 groups, depending on their surface topography (machined, sandblasted, acid etched, or plasma sprayed)., Results: Statistical analysis of the data showed a clear relationship between surface composition and topography, which can be easily accounted for by the chemical effects of the surface treatment performed. On average, acid-etched and plasma-sprayed surfaces had higher titanium and lower carbon concentration than machined surfaces., Discussion and Conclusion: Current studies aimed at the evaluation of implants with different topography should not implicitly assume that topography is the only variable controlling the biologic response. Rather, when comparing different topographies, it should be taken into account that surface chemistry may be a variable as well.

Purpose: To determine, in vitro, cytotoxicity and cell adhesion on 3 different implant surfaces., Materials and Methods: All samples had machined surfaces, but they were subjected to different cleaning procedures, which produced 3 different surface chemistries. One of the samples was "as-produced" from the machining tools. The other samples were subjected to partial and total cleaning routines. Cytotoxicity was evaluated using mouse fibroblast cultures, and cell adhesion was evaluated with osteoblast-like SaOS-2 cells., Results: The "as-produced" sample showed a pronounced surface contamination by lubricating oils. For partially and totally cleaned samples, an increasing amount of titanium and a decreasing carbon/titanium ratio was observed as cleaning became more complete., Discussion: Differences in surface chemistry such as those normally found on titanium implant surfaces (see part 1 of this series) can lead to those same effects which, in in vitro experiments, are normally accounted for in terms of surface topography alone., Conclusion: Effects related to surface chemistry can operate over and above surface topography, making it impossible, without proper characterization, to make definite statements about the role of topography alone.

Purpose: Histomorphometry and microhardness measurements were performed after Ti6AI4V implantation in cancellous and cortical bone of healthy and ovariectomized animals to determine characterization of the bone-biomaterial interface in osteopenic bone., Materials and Methods: Nine ovariectomized and 9 sham-aged rats were used. Four months later, nails were implanted in the distal femurs, and the animals were sacrificed after 8 weeks. Moreover, 3 ovariectomized and 3 sham-aged sheep were used. Twenty-four months later, screws were implanted in the tibial diaphyses and the animals were sacrificed after 12 weeks., Results: Histomorphometry showed the development of osteopenia in both trabecular and cortical bone, and revealed a significant decrease in the osseointegration rate in osteopenic versus sham-aged animals for both trabecular (Affinity Index: -18.6%, P < .001) and cortical bone (Affinity Index: -23.5%, P < .005; Bone Ingrowth: -9%, P <.05). At the interfaces of the sham-aged animals in both trabecular and cortical bone, a decrease of bone microhardness was observed in comparison with preexisting bone (trabecular: -9.8%, P < .0005; cortical: -19.3%, P < .0005). In case of osteopenia, this decrease was even more extensive (trabecular: -15.5%, P < .0005; cortical: -24.7%, P < .0005)., Discussion: The present data suggest that bone formation around Ti6AI4V was not associated with complete bone maturation, even in healthy animals. In case of osteopenia, both bone formation and maturation were delayed., Conclusion: These results apparently demonstrate the utility of investigating biomaterials in osteopenic bone and the importance of careful evaluation of the healing rate and bone maturation degree around implanted biomaterials.

It is known that titanium alloys cause more extensive local metallosis due to fretting corrosion than stainless steel implants. The aim of the present study was to investigate possible systemic metal releases (Ti, Al, V, Cr, Ni) in sheep where L4-L5 were implanted with titanium alloy (Ti6Al4V, ASTM F 136) and stainless steel (AISI 316 L). 16 sheep were used: 8 were implanted with Ti6Al4V and 8 with stainless steel. At 6, 12, 24 and 36 months, the following examinations were performed: histology, atomic absorption spectrophotometry (AAS) and scanning electron microscopy (SEM), on liver, lung, kidney, brain, spleen and lumbo-aortic lymph nodes. Hair, urine and arteria blood samples were also analysed by AAS before implantation and at sacrifices. A histologic and ultrastructural study was performed on peri-implant tissues, too. Particular attention was paid to avoid contamination from dissection instruments or use of containers. In basal and in samples at 6 and 12 months, no metals were found in blood, urine, hair or other target tissues of the animals implanted with either Ti6Al4V or stainless steel. Regarding Al, V, Co and Ni, negative results in all tissues and body fluids were obtained also at 24 and 36 months. On the contrary, Ti traces were found in lumbo-aortic lymph nodes and lungs of one sheep only (10 and 30 ng/g, respectively) at 24 months. At 36 months, a systemic diffusion of Ti was observed in all tissues of both sheep instrumented with Ti6Al4V (2-16.5 ng/g), except for body fluids and hair. Metal research in target tissues by light and SEM micro-probe analysis provided negative results. Current data suggest that the amount of Ti found in organs after stable pedicular fixation is extremely low and not biologically available. This observation would lead us to exclude the hypothesis of any toxic reaction and such a release seems to be due to the passive diffusion through lymphatic fluids. Additional studies are needed to confirm if this long-term release of Ti particles might cause tissue damage.

The influence of different surface preparations on titanium biocompatibility and bone integration was evaluated. Commercially grade 2 titanium rods (diameter 2 mm, length: 3 mm), vacuum annealed and hydrofluoric acid etched was selected for its promising surface characteristics to achieve good direct osseointegration. Some rods were surface modified by Anodic Spark Discharge anodization and a thin layer (approximately 5 microm) of amorphous TiO2 containing Ca and P (Ti/AM) was obtained. Some of the Ti/AM specimens underwent a further hydrothermal treatment to produce a thin outermost layer (approximately 1 microm) of hydroxyapatite (Ti/AM/HA). Cytotoxicity tests (direct contact: ISO 10993-5) showed good cytocompatibility for all tested samples. Ti and tissue culture substrate + DMEM control, respectively, were associated with a significant higher proportion of attached cells than Ti/AM and Ti/AM/HA (P < 0.0005), but this was in the normal range of 10-20% of unattached cells for cytocompatible materials. Histomorphometric analysis conducted on samples inserted in the cancellous bone of distal femoral epiphysis of Sprague-Dawley rats gave the following results at 4 and 8 weeks: Affinity index (AI%) data proving the surface osteconductive properties of non-anodized acid etched Ti (AI-4 weeks: 67.1 +/- 17.0%; AI-8 weeks: 74.8 +/- 11.5%). Ti/AM samples showed the lowest values (AI-4 weeks: 45.8 +/- 15.9%; AI-8 weeks: 68.5 +/- 13.6%) while the best performances of the Ti/AM/HA samples (AI-4 weeks: 60.4 +/- 21.8%; AI-8 weeks: 79.5 + 9.37%) indicated that hydroxyapatite allowed a higher bone to implant contact respect to Ti only. Further investigations should be performed in order to better understand the mechanism of observed in vitro behaviour and to achieve information on long-term osseointegration process.

The article presents a study that examines the effect of different ions incorporated into bioactive apatite during the biomimetic coating of titanium substrates on in vitro osteoblast cultures. The study shows that strontium (TiSr10) maintains a significant high proliferation rate, while alkaline phosphatase (ALP) activity shows no difference among groups. It suggests that different effects of the studied ions seem to promote cell proliferation and differentiation of osteoblast.

New nickel (Ni)-reduced stainless-steel metals have recently been developed to avoid sensitivity to Ni. In the present study, an austenitic Ni-reduced SSt named P558 (P558, Bo¨hler, Milan, Italy) was studied in vitro on primary osteoblasts and in vivo after bone implantation in the sheep tibia, and was compared to ISO 5832-9 SSt (SSt) and Ti6Al4V. Cells were cultured directly on P558 and Ti6Al4V. Cells cultured on polystyrene were used as controls. Osteoblast proliferation, viability and synthetic activity were evaluated at 72 h by assaying WST1, alkaline phosphatase activity (ALP), nitric oxide, pro-collagen I (PICP), osteocalcin (OC), transforming growth factor-β1 (TGFβ-1) and interleukin-6 (IL-6) after 1.25(OH)2D3 stimulation. Under general anaesthesia, four sheep were submitted for bilateral tibial implantation of P558, SSt and Ti6Al4V rods. In vitro results demonstrated that the effect of P558 on osteoblast viability, PICP, TGF β-1, tumor necrosis factor-α production did not significantly differ from that exerted by Ti6Al4V and controls. Furthermore, P558 enhanced osteoblast differentiation, as confirmed by ALP and OC levels, and reduced IL-6 production. At 26 weeks, the bone-to-implant contact was higher in P558 than in SSt (28%, p<0.005) and Ti6Al4V (4%, p<0.05), and was higher in Ti6Al4V than in SSt (22%, p<0.005). The tested materials did not affect bone microhardness in pre-existing host bone as evidenced by the measurements taken at 1000 μm from the bone–biomaterial interface (F=1.89, ns). At the bone–biomaterial interface the lowest HV value was found for SSt, whereas no differences in HV were observed between materials (F=1.55, ns). The current findings demonstrate P558 biocompatibility both in vitro and in vivo, and osteointegration processes are shown to be significantly improved by P558 as compared to the other materials tested. [Copyright &y& Elsevier]