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1.  Bio-inspired stable antimicrobial peptide coatings for dental applications 
Acta biomaterialia  2013;9(9):8224-8231.
We developed a novel titanium coating that has applications for preventing infection-related implant failures in dentistry and orthopedics. The coating incorporates an antimicrobial peptide, GL13K, derived from parotid secretory protein, which has been previously shown to be bactericidal and bacteriostatic in solution. We characterized the resulting physicochemical properties, resistance to degradation, activity against Porphyromonas gingivalis, and in vitro cytocompatibility. P. gingivalis is a pathogen associated with dental peri-implantitis, an inflammatory response to bacteria resulting in bone loss and implant failure. Our surface modifications obtained a homogeneous, highly hydrophobic, and strongly-anchored GL13K-coating that was resistant to mechanical, thermochemical, and enzymatic degradation. The GL13K-coatings had bactericidal effect and thus, significantly reduced the number of viable bacteria compared to control surfaces. Finally, adequate proliferation of osteoblasts and human-gingival-fibroblasts demonstrated the GL13K-coating’s cytocompatibility. The robustness, antimicrobial activity, and cytocompatibility of GL13K-biofunctionalized titanium make it a promising candidate for sustained inhibition of bacterial biofilm growth. This surface chemistry provides a basis for development of multifunctional bioactive surfaces to reduce patient morbidities and improve long-term clinical efficacy of metallic dental and orthopedic implants.
doi:10.1016/j.actbio.2013.06.017
PMCID: PMC3758876  PMID: 23791670
2.  Morphology, Composition, and Bioactivity of Strontium-Doped Brushite Coatings Deposited on Titanium Implants via Electrochemical Deposition 
Surface modification techniques have been applied to generate titanium implant surfaces that promote osseointegration for use in dental applications. In this study, strontium-doped brushite coatings were deposited on titanium by electrochemical deposition. The phase composition of the coating was investigated by energy dispersive X-ray spectroscopy and X-ray diffraction. The surface morphologies of the coatings were studied through scanning electron microscopy, and the cytocompatibility and bioactivity of the strontium-doped brushite coatings were evaluated using cultured osteoblasts. Osteoblast proliferation was enhanced by the addition of strontium, suggesting a possible mechanism by which strontium incorporation in brushite coatings increased bone formation surrounding the implants. Cell growth was also strongly influenced by the composition of the deposited coatings, with a 10% Sr-doped brushite coating inducing the greatest amount of bone formation among the tested materials.
doi:10.3390/ijms15069952
PMCID: PMC4100132  PMID: 24901526
strontium; brushite; coating; implant; osteoblast; biomedical materials
3.  The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration 
Biomaterials  2008;29(19):2849-2857.
Implant osseointegration, defined as bone apposition and functional fixation, is a requisite for clinical success in orthopaedic and dental applications, many of which are restricted by implant loosening. Modification of implants to present bioactive motifs such as the RGD cell-adhesive sequence from fibronectin (FN) represents a promising approach in regenerative medicine. However, these biomimetic strategies have yielded only marginal enhancements in tissue healing in vivo. In this study, clinical-grade titanium implants were grafted with a non-fouling oligo(ethylene glycol)-substituted polymer coating functionalized with controlled densities of ligands of varying specificity for target integrin receptors. Biomaterials presenting the α5β1-integrin-specific FN fragment FNIII7–10 enhanced osteoblastic differentiation in bone marrow stromal cells compared to unmodified titanium and RGD-presenting surfaces. Importantly, FNIII7–10-functionalized titanium significantly improved functional implant osseointegration compared to RGD-functionalized and unmodified titanium in vivo. This study demonstrates that bioactive coatings that promote integrin binding specificity regulate marrow-derived progenitor osteoblastic differentiation and enhance healing responses and functional integration of biomedical implants. This work identifies an innovative strategy for the rational design of biomaterials for regenerative medicine.
doi:10.1016/j.biomaterials.2008.03.036
PMCID: PMC2397448  PMID: 18406458
4.  Techniques for dental implant nanosurface modifications 
PURPOSE
Dental implant has gained clinical success over last decade with the major drawback related to osseointegration as properties of metal (Titanium) are different from human bone. Currently implant procedures include endosseous type of dental implants with nanoscale surface characteristics. The objective of this review article is to summarize the role of nanotopography on titanium dental implant surfaces in order to improve osseointegration and various techniques that can generate nanoscale topographic features to titanium implants.
MATERIALS AND METHODS
A systematic electronic search of English language peer reviewed dental literature was performed for articles published between December 1987 to January 2012. Search was conducted in Medline, PubMed and Google scholar supplemented by hand searching of selected journals. 101 articles were assigned to full text analysis. Articles were selected according to inclusion and exclusion criterion. All articles were screened according to inclusion standard. 39 articles were included in the analysis.
RESULTS
Out of 39 studies, seven studies demonstrated that bone implant contact increases with increase in surface roughness. Five studies showed comparative evaluation of techniques producing microtopography and nanotopography. Eight studies concluded that osteoblasts preferably adhere to nano structure as compared to smooth surface. Six studies illustrated that nanotopography modify implant surface and their properties. Thirteen studies described techniques to produce nano roughness.
CONCLUSION
Modification of dental osseous implants at nanoscale level produced by various techniques can alter biological responses that may improve osseointegration and dental implant procedures.
doi:10.4047/jap.2014.6.6.498
PMCID: PMC4279049  PMID: 25558347
Intelligent surfaces; Sputtering; Superhydrophillic; Chemical vapor deposition; Osseointegration; Engineered surface
5.  Evaluation of Biological Properties of Electron Beam Melted Ti6Al4V Implant with Biomimetic Coating In Vitro and In Vivo 
PLoS ONE  2012;7(12):e52049.
Background
High strength porous titanium implants are widely used for the reconstruction of craniofacial defects because of their similar mechanical properties to those of bone. The recent introduction of electron beam melting (EBM) technique allows a direct digitally enabled fabrication of patient specific porous titanium implants, whereas both their in vitro and in vivo biological performance need further investigation.
Methods
In the present study, we fabricated porous Ti6Al4V implants with controlled porous structure by EBM process, analyzed their mechanical properties, and conducted the surface modification with biomimetic approach. The bioactivities of EBM porous titanium in vitro and in vivo were evaluated between implants with and without biomimetic apatite coating.
Results
The physical property of the porous implants, containing the compressive strength being 163 - 286 MPa and the Young’s modulus being 14.5–38.5 GPa, is similar to cortical bone. The in vitro culture of osteoblasts on the porous Ti6Al4V implants has shown a favorable circumstance for cell attachment and proliferation as well as cell morphology and spreading, which were comparable with the implants coating with bone-like apatite. In vivo, histological analysis has obtained a rapid ingrowth of bone tissue from calvarial margins toward the center of bone defect in 12 weeks. We observed similar increasing rate of bone ingrowth and percentage of bone formation within coated and uncoated implants, all of which achieved a successful bridging of the defect in 12 weeks after the implantation.
Conclusions
This study demonstrated that the EBM porous Ti6Al4V implant not only reduced the stress-shielding but also exerted appropriate osteoconductive properties, as well as the apatite coated group. The results opened up the possibility of using purely porous titanium alloy scaffolds to reconstruct specific bone defects in the maxillofacial and orthopedic fields.
doi:10.1371/journal.pone.0052049
PMCID: PMC3525565  PMID: 23272208
6.  Reality of Dental Implant Surface Modification: A Short Literature Review 
Screw-shaped endosseous implants that have a turned surface of commercially pure titanium have a disadvantage of requiring a long time for osseointegration while those implants have shown long-term clinical success in single and multiple restorations. Titanium implant surfaces have been modified in various ways to improve biocompatibility and accelerate osseointegration, which results in a shorter edentulous period for a patient. This article reviewed some important modified titanium surfaces, exploring the in vitro, in vivo and clinical results that numerous comparison studies reported. Several methods are widely used to modify the topography or chemistry of titanium surface, including blasting, acid etching, anodic oxidation, fluoride treatment, and calcium phosphate coating. Such modified surfaces demonstrate faster and stronger osseointegration than the turned commercially pure titanium surface. However, there have been many studies finding no significant differences in in vivo bone responses among the modified surfaces. Considering those in vivo results, physical properties like roughening by sandblasting and acid etching may be major contributors to favorable bone response in biological environments over chemical properties obtained from various modifications including fluoride treatment and calcium phosphate application. Recently, hydrophilic properties added to the roughened surfaces or some osteogenic peptides coated on the surfaces have shown higher biocompatibility and have induced faster osseointegration, compared to the existing modified surfaces. However, the long-term clinical studies about those innovative surfaces are still lacking.
doi:10.2174/1874120701408010114
PMCID: PMC4231373  PMID: 25400716
Anodic oxidation; BMP; fluoride; functional peptide; hydrophilicity; implant surface; SLA; surface modification.
7.  Ag-plasma modification enhances bone apposition around titanium dental implants: an animal study in Labrador dogs 
Dental implants with proper antibacterial ability as well as ideal osseointegration are being actively pursued. The antimicrobial ability of titanium implants can be significantly enhanced via modification with silver nanoparticles (Ag NPs). However, the high mobility of Ag NPs results in their potential cytotoxicity. The silver plasma immersion ion-implantation (Ag-PIII) technique may remedy the defect. Accordingly, Ag-PIII technique was employed in this study in an attempt to reduce the mobility of Ag NPs and enhance osseointegration of sandblasted and acid-etched (SLA) dental implants. Briefly, 48 dental implants, divided equally into one control and three test groups (further treated by Ag-PIII technique with three different implantation parameters), were inserted in the mandibles of six Labrador dogs. Scanning electron microscopy, X-ray photoelectron spectroscopy, and inductively coupled plasma optical emission spectrometry were used to investigate the surface topography, chemical states, and silver release of SLA- and Ag-PIII-treated titanium dental implants. The implant stability quotient examination, Microcomputed tomography evaluation, histological observations, and histomorphometric analysis were performed to assess the osseointegration effect in vivo. The results demonstrated that normal soft tissue healing around dental implants was observed in all the groups, whereas the implant stability quotient values in Ag-PIII groups were higher than that in the SLA group. In addition, all the Ag-PIII groups, compared to the SLA-group, exhibited enhanced new bone formation, bone mineral density, and trabecular pattern. With regard to osteogenic indicators, the implants treated with Ag-PIII for 30 minutes and 60 minutes, with the diameter of the Ag NPs ranging from 5–25 nm, were better than those treated with Ag-PIII for 90 minutes, with the Ag NPs diameter out of that range. These results suggest that Ag-PIII technique can reduce the mobility of Ag NPs and enhance the osseointegration of SLA surfaces and have the potential for future use.
doi:10.2147/IJN.S73467
PMCID: PMC4298332  PMID: 25609967
surface modification; micro/nanostructure; silver; ion implantation; osseointegration
8.  Biomechanical evaluation of dental implants with different surfaces: Removal torque and resonance frequency analysis in rabbits 
STATEMENT OF PROBLEM
Macroscopic and especially microscopic properties of implant surfaces play a major role in the osseous healing of dental implants. Dental implants with modified surfaces have shown stronger osseointegration than implants which are only turned (machined). Advanced surface modification techniques such as anodic oxidation and Ca-P application have been developed to achieve faster and stronger bonding between the host bone and the implant.
PURPOSE
The purpose of this study was to investigate the effect of surface treatment of titanium dental implant on implant stability after insertion using the rabbit tibia model.
MATERIAL AND METHODS
Three test groups were prepared: sandblasted, large-grit and acid-etched (SLA) implants, anodic oxidized implants, and anodized implants with Ca-P immersion. The turned implants served as control. Twenty rabbits received 80 implants in the tibia. Resonance frequencies were measured at the time of implant insertion, 2 weeks and 4 weeks of healing. Removal torque values (RTV) were measured 2 and 4 weeks after insertion.
RESULTS
The implant stability quotient (ISQ) values of implants for resonance frequency analysis (RFA) increased significantly (P < .05) during 2 weeks of healing period although there were no significant differences among the test and control groups (P > .05). The test and control implants also showed significantly higher ISQ values during 4 weeks of healing period (P < .05). No significant differences, however, were found among all the groups. All the groups showed no significant differences in ISQ values between 2 and 4 weeks after implant insertion (P > .05). The SLA, anodized and Ca-P immersed implants showed higher RTVs at 2 and 4 weeks of healing than the machined one (P < .05). However, there was no significant difference among the experimental groups.
CONCLUSION
The surface-modified implants appear to provide superior implant stability to the turned one. Under the limitation of this study, however, we suggest that neither anodic oxidation nor Ca-P immersion techniques have any advantage over the conventional SLA technique with respect to implant stability.
doi:10.4047/jap.2009.1.2.107
PMCID: PMC2994679  PMID: 21165264
surface treatment; bone to implant contact; removal torque; dental implant
9.  The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and differentiation 
Biomaterials  2011;32(13):3395-3403.
Titanium (Ti) osseointegration is critical for the success of dental and orthopaedic implants. Previous studies have shown that surface roughness at the micro- and submicro-scales promotes osseointegration by enhancing osteoblast differentiation and local factor production. Only relatively recently have the effects of nanoscale roughness on cell response been considered. The aim of the present study was to develop a simple and scalable surface modification treatment that introduces nanoscale features to the surfaces of Ti substrates without greatly affecting other surface features, and to determine the effects of such superimposed nano-features on the differentiation and local factor production of osteoblasts. A simple oxidation treatment was developed for generating controlled nanoscale topographies on Ti surfaces, while retaining the starting micro-/submicro-scale roughness. Such nano-modified surfaces also possessed similar elemental compositions, and exhibited similar contact angles, as the original surfaces, but possessed a different surface crystal structure. MG63 cells were seeded on machined (PT), nano-modified PT (NMPT), sandblasted/acid-etched (SLA), and nano-modified SLA (NMSLA) Ti disks. The results suggested that the introduction of such nanoscale structures in combination with micro-/submicro-scale roughness improves osteoblast differentiation and local factor production, which, in turn, indicates the potential for improved implant osseointegration in vivo.
doi:10.1016/j.biomaterials.2011.01.029
PMCID: PMC3350795  PMID: 21310480
(4 to 6) nanotopography; titanium oxide; surface roughness; titanium; bone; implant; osteoblasts
10.  Simple application of fibronectin-mimetic coating enhances osseointegration of titanium implants 
Integrin-mediated cell adhesion to biomolecules adsorbed onto biomedical devices regulates device integration and performance. Because of the central role of integrin-fibronectin (FN) interactions in osteoblastic function and bone formation, we evaluated the ability of fibronectin-inspired biomolecular coatings to promote osteoblastic differentiation and implant osseointegration. Notably, these biomolecular coatings relied on physical adsorption of FN-based ligands onto biomedical-grade titanium as a simple, clinically-translatable strategy to functionalize medical implants. Surfaces coated with a recombinant fragment of FN spanning the central cell binding domain enhanced osteoblastic differentiation and mineralization in bone marrow stromal cell cultures and increased implant osseointegration in a rat cortical bone model compared to passively adsorbed RGD peptides, serum proteins, and full-length FN. Differences in biological responses correlated with integrin binding specificity and signaling among surface coatings. This work validates a simple, clinically-translatable, surface biofunctionalization strategy to enhance biomedical device integration.
doi:10.1111/j.1582-4934.2008.00476.x
PMCID: PMC2819599  PMID: 18752639
fibronectin; osseointegration; coating; integrins; biomimetic; implant
11.  Porous Tantalum Coatings Prepared by Vacuum Plasma Spraying Enhance BMSCs Osteogenic Differentiation and Bone Regeneration In Vitro and In Vivo 
PLoS ONE  2013;8(6):e66263.
Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration.
doi:10.1371/journal.pone.0066263
PMCID: PMC3679049  PMID: 23776648
12.  Bioactive glass enhances bone ingrowth into the porous titanium coating on orthopaedic implants 
International Orthopaedics  2012;36(8):1739-1745.
Purpose
The aim of the study was to verify the ability of nanoparticulate bioactive glass (BAG) to infiltrate into the porous titanium (Ti) layer on Ti-based implants to promote osseointegration.
Methods
The porous titanium layer on Ti-based implants was impregnated with nanoparticulate BAG. The implants without or with BAG were implanted bilaterally in tibial holes of ten New Zealand white rabbits. The rabbits were sacrificed after ten weeks for examinations. Beside histological examination, EDXS analysis of polished cross-sections of explanted implants was also performed with the aim to quantitatively evaluate the bone-to-pore contact and bone-in-pore ratio.
Results
After ten weeks, EDXS analyses of cross-sections of the explanted implants confirmed that bioactive glass was fully resorbed and that the pores throughout the thickness of the porous titanium layer were to a large extent filled with a new bone. In the absence of bioactive glass, only the outer part of the porous layer was filled with bone. The implants without BAG in the porous Ti-layer exhibited similar bone-to-pore contact, while significant improvement of bone ingrowth into the pores was observed for the implants with BAG (38%), as opposed to those without it (22%).
Conclusion
This study confirmed that the nanoparticulate bioactive glass within the porous titanium surface layer on implants promotes osseointegration and stimulates the formation of bone within the pores.
doi:10.1007/s00264-012-1520-y
PMCID: PMC3535029  PMID: 22422142
13.  Biomolecular surface coating to enhance orthopaedic tissue healing and integration 
Biomaterials  2007;28(21):3228-3235.
Implant osseointegration is a prerequisite for clinical success in orthopaedic and dental applications, many of which are restricted by loosening. Biomaterial surface modification approaches, including calcium-phosphate ceramic coatings and macro/microporosity, have had limited success in promoting integration. To improve osseointegration, titanium surfaces were coated with the GFOGER collagen-mimetic peptide, selectively promoting α2β1 integrin binding, a crucial event for osteoblastic differentiation. Titanium surfaces presenting GFOGER triggered osteoblastic differentiation and mineral deposition in bone marrow stromal cells, leading to enhanced osteoblastic function compared to unmodified titanium. Furthermore, this integrin-targeted coating significantly improved in vivo peri-implant bone regeneration and osseointegration, as characterized by bone-implant contact and mechanical fixation, compared to untreated titanium in a rat cortical bone-implant model. GFOGER-modified implants also significantly enhanced osseointegration compared to surfaces modified with full-length type I collagen, highlighting the importance of presenting specific biofunctional domains within the native ligand. In addition, this biomimetic implant coating is generated using a simple, single-step procedure that readily translates to a clinical environment with minimal processing and cytotoxicity concerns. Therefore, this study establishes a biologically active and clinically relevant implant coating strategy that enhances bone repair and orthopaedic implant integration.
doi:10.1016/j.biomaterials.2007.04.003
PMCID: PMC2034748  PMID: 17448533
biomimetic material; cell adhesion; collagen; osseointegration; integrin
14.  Nano-TiO2/PEEK bioactive composite as a bone substitute material: in vitro and in vivo studies 
Video abstract
Video
Background
Compared with titanium (Ti) and other metal implant materials, poly(ether-ether ketone) (PEEK) shows outstanding biomechanical properties. A number of studies have also reported attractive bioactivity for nano-TiO2 (n-TiO2).
Methods
In this study, n-TiO2/PEEK nanocomposites were prepared, taking advantage of the unique properties of both PEEK polymer and n-TiO2. The in vitro and in vivo bioactivity of these nanocomposites was assessed against a PEEK polymer control. The effect of surface morphology or roughness on the bioactivity of the n-TiO2/PEEK nanocomposites was also studied. n-TiO2/PEEK was successfully fabricated and cut into disks for physical and chemical characterization and in vitro studies, and prepared as cylindrical implants for in vivo studies. Their presence on the surface and dispersion in the composites was observed and analyzed by scanning and transmission electron microscopy and X-ray photoelectron spectroscopy.
Results
Bioactivity evaluation of the nanocomposites revealed that pseudopods of osteoblasts preferred to anchor at areas where n-TiO2 was present on the surface. In a cell attachment test, smooth PEEK showed the lowest optical density value (0.56 ± 0.07) while rough n-TiO2/PEEK exhibited the highest optical density value (1.21 ± 0.34, P < 0.05). In in vivo studies, the percent bone volume value of n-TiO2/PEEK was approximately twice as large as that of PEEK (P < 0.05). Vivid three-dimensional and histologic images of the newly generated bone on the implants further supported our test results.
Conclusion
Our study demonstrates that n-TiO2 significantly improves the bioactivity of PEEK, especially if it has a rough composite surface. A n-TiO2/PEEK composite with a rough surface could be a novel alternative implant material for orthopedic and dental applications.
doi:10.2147/IJN.S28101
PMCID: PMC3298387  PMID: 22419869
polyether-ether-ketone; bioactivity; TiO2; nanocomposite; polymer; implant
15.  Behavior of osteoblastic cells cultured on titanium and structured zirconia surfaces 
Head & Face Medicine  2008;4:29.
Background
Osseointegration is crucial for the long-term success of dental implants and depends on the tissue reaction at the tissue-implant interface. Mechanical properties and biocompatibility make zirconia a suitable material for dental implants, although surface processings are still problematic. The aim of the present study was to compare osteoblast behavior on structured zirconia and titanium surfaces under standardized conditions.
Methods
The surface characteristics were determined by scanning electron microscopy (SEM). In primary bovine osteoblasts attachment kinetics, proliferation rate and synthesis of bone-associated proteins were tested on different surfaces.
Results
The results demonstrated that the proliferation rate of cells was significantly higher on zirconia surfaces than on titanium surfaces (p < 0.05; Student's t-test). In contrast, attachment and adhesion strength of the primary cells was significant higher on titanium surfaces (p < 0.05; U test). No significant differences were found in the synthesis of bone-specific proteins. Ultrastructural analysis revealed phenotypic features of osteoblast-like cells on both zirconia and titanium surfaces.
Conclusion
The study demonstrates distinct effects of the surface composition on osteoblasts in culture. Zirconia improves cell proliferation significantly during the first days of culture, but it does not improve attachment and adhesion strength. Both materials do not differ with respect to protein synthesis or ultrastructural appearance of osteoblasts. Zirconium oxide may therefore be a suitable material for dental implants.
doi:10.1186/1746-160X-4-29
PMCID: PMC2614982  PMID: 19063728
16.  In vivo and in vitro investigations of a nanostructured coating material – a preclinical study 
Immediate loading of dental implants is only possible if a firm bone-implant anchorage at early stages is developed. This implies early and high bone apposition onto the implant surface. A nanostructured coating material based on an osseoinductive bone grafting is investigated in relation to the osseointegration at early stages. The goal is to transmit the structure (silica matrix with embedded hydroxyapatite) and the properties of the bone grafting into a coating material. The bone grafting substitute offers an osseoinductive potential caused by an exchange of the silica matrix in vivo accompanied by vascularization. X-ray diffraction and transmission electron microscopy analysis show that the coating material consists of a high porous silica matrix with embedded nanocrystalline hydroxyapatite with the same morphology as human hydroxyapatite. An in vitro investigation shows the early interaction between coating and human blood. Energy-dispersive X-ray analysis showed that the silica matrix was replaced by an organic matrix within a few minutes. Uncoated and coated titanium implants were inserted into the femora of New Zealand White rabbits. The bone-to-implant contact (BIC) was measured after 2, 4, and 6 weeks. The BIC of the coated implants was increased significantly at 2 and 4 weeks. After 6 weeks, the BIC was decreased to the level of the control group. A histological analysis revealed high bone apposition on the coated implant surface after 2 and 4 weeks. Osteoblastic and osteoclastic activities on the coating material indicated that the coating participates in the bone-remodeling process. The nanostructure of the coating material led to an exchange of the silica matrix by an autologous, organic matrix without delamination of the coating. This is the key issue in understanding initial bone formation on a coated surface.
doi:10.2147/IJN.S48416
PMCID: PMC3931640  PMID: 24627631
silica; hydroxyapatite; dental implants; matrix change; osseointegration; in vivo
17.  Nanotechnology and Dental Implants 
The long-term clinical success of dental implants is related to their early osseointegration. This paper reviews the different steps of the interactions between biological fluids, cells, tissues, and surfaces of implants. Immediately following implantation, implants are in contact with proteins and platelets from blood. The differentiation of mesenchymal stem cells will then condition the peri-implant tissue healing. Direct bone-to-implant contact is desired for a biomechanical anchoring of implants to bone rather than fibrous tissue encapsulation. Surfaces properties such as chemistry and roughness play a determinant role in these biological interactions. Physicochemical features in the nanometer range may ultimately control the adsorption of proteins as well as the adhesion and differentiation of cells. Nanotechnologies are increasingly used for surface modifications of dental implants. Another approach to enhance osseointegration is the application of thin calcium phosphate (CaP) coatings. Bioactive CaP nanocrystals deposited on titanium implants are resorbable and stimulate bone apposition and healing. Future nanometer-controlled surfaces may ultimately direct the nature of peri-implant tissues and improve their clinical success rate.
doi:10.1155/2010/915327
PMCID: PMC3021857  PMID: 21253543
18.  Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces 
BMC Oral Health  2014;14:75.
Background
A key element for long-term success of dental implants is integration of the implant surface with the surrounding host tissues. Modification of titanium implant surfaces can enhance osteoblast activity but their effects on soft-tissue cells are unclear. Adherence of human keratinocytes and gingival fibroblasts to control commercially pure titanium (CpTi) and two surfaces prepared by anodic oxidation was therefore investigated. Since implant abutments are exposed to a bacteria-rich environment in vivo, the effect of oral bacteria on keratinocyte adhesion was also evaluated.
Methods
The surfaces were characterized using scanning electron microscopy (SEM). The number of adhered cells and binding strength, as well as vitality of fibroblasts and keratinocytes were evaluated using confocal scanning laser microscopy after staining with Live/Dead Baclight. To evaluate the effect of bacteria on adherence and vitality, keratinocytes were co-cultured with a four-species streptococcal consortium.
Results
SEM analysis showed the two anodically oxidized surfaces to be nano-structured with differing degrees of pore-density. Over 24 hours, both fibroblasts and keratinocytes adhered well to the nano-structured surfaces, although to a somewhat lesser degree than to CpTi (range 42-89% of the levels on CpTi). The strength of keratinocyte adhesion was greater than that of the fibroblasts but no differences in adhesion strength could be observed between the two nano-structured surfaces and the CpTi. The consortium of commensal streptococci markedly reduced keratinocyte adherence on all the surfaces as well as compromising membrane integrity of the adhered cells.
Conclusion
Both the vitality and level of adherence of soft-tissue cells to the nano-structured surfaces was similar to that on CpTi. Co-culture with streptococci reduced the number of keratinocytes on all the surfaces to approximately the same level and caused cell damage, suggesting that commensal bacteria could affect adherence of soft-tissue cells to abutment surfaces in vivo.
doi:10.1186/1472-6831-14-75
PMCID: PMC4083866  PMID: 24952379
Oral keratinocytes; Gingival fibroblasts; Cell attachment; Dental implant; Surface modification; Oral bacteria
19.  Early loading of hydrophilic titanium implants inserted in low-mineralized (D3 and D4) bone: one year results of a prospective clinical trial 
Head & Face Medicine  2013;9:37.
Introduction
Pure titanium is the material of choice for contemporary dental implants. However, superficial reaction of the moderately rough titanium surface with atmospheric components decreases its hydrophilicity. INICELL® represents a chemical alteration and hydrophilization of a moderately rough i. e. sand-blasted and acid-etched titanium surface. The hydrophilicity leads to a more homogenous adsorption of proteins on the implant surface in-vitro, supporting the activation of a higher number of platelets and the generation of a homogenous, complete fibrin matrix in the early phases of osseointegration. This in turn helps to reduce the healing time and enhances the predictability of osseointegration in compromised bony situations.
The objective of this case series trial was therefore to investigate if early loading (after 8 weeks) of hydrophilic INICELL implants is feasible in patients with reduced bone quality.
Methods
In 10 patients, 35 hydrophilic implants were placed in sites revealing bone quality class 3 and 4, and uncovered after 4 weeks. Eight weeks later implants were released for loading if the tactile resistance was ≥35 Ncm. Lower resistances resulted in 12 weeks initial healing period. Insertion torque, ISQ, tactile resistance and vertical bone level were evaluated at implant installation, after 4 weeks (uncovering), 8 or 12 weeks (loading), and 12 weeks and one year after loading.
Results
Mean implant insertion torque was 21 Ncm. 31 (88.6%) showed a tactile resistance of >35 Ncm after eight weeks and were released for prosthetic loading. Eight weeks after insertion, one implant (2.9%) had to be removed following a soft tissue complication. One implant had to be removed after 4 weeks due to a technical complication (fractured Osstell-abutment), it was therefore excluded from the analysis.
33 of 34 implants (97%) were loaded to occlusion and were in situ/functional one year after implantation. ISQs increased from 43 at baseline to 63 at eight weeks, and 72 at three months after loading. Then, ISQ remained constant until one year after loading.
Conclusions
Within the limitations of this prospective case series, hydrophilic implants may allow for shortening of the initial healing period even in bone with compromised density.
doi:10.1186/1746-160X-9-37
PMCID: PMC3866303  PMID: 24321192
Titanium implants; Hydrophilic surface; Healing time; Bone quality; Weak bone
20.  Effects of Coating a Titanium Alloy with Fibronectin on the Expression of Osteoblast Gene Markers in the MC3T3 Osteoprogenitor Cell Line 
Purpose
A number of environmental and patient-related factors contribute to implant failure. A significant fraction of these failures can be attributed to limited osseointegration resulting from poor bone healing responses. The overall goal of this study was to determine whether surface treatment of a titanium-aluminum-vanadium alloy (Ti-6Al-4V) implant material with a biomimetic protein coating could promote the differentiation of attached osteoblastic cells. The specific aims of the study were to investigate whether osteoprogenitor cells cultured on a rigorously cleaned implant specimen showed a normal pattern of differentiation and whether preadsorbed fibronectin accelerated or enhanced osteoblast differentiation.
Materials and Methods
Ti-6Al-4V disks were rigorously cleaned, passivated in nitric acid, and dry heat–sterilized; some of the disks were then coated with 1 nmol/L fibronectin. MC3T3 osteoprogenitor cells were then cultured on the pretreated disks for several weeks. Quantitative real-time polymerase chain reaction was performed to measure changes over time in the mRNA levels of osteoblast genes.
Results
Fibronectin increased the peak expression of all analyzed osteoblast gene markers. “Early” genes that normally mark the proliferative phase (0 to 10 days) of osteoblastic development showed peak expression within the first 10 days after cell attachment to the titanium alloy. In contrast, “late” genes that normally mark the differentiation (10 to 20 days) and mineralization (20 to 36 days) phases of osteoblastogenesis achieved peak expression only after approximately 3 to 4 weeks of culture.
Conclusions
Osteoprogenitors cultured on a rigorously cleaned Ti-6Al-4V alloy were found to demonstrate a normal pattern of osteoblast differentiation. Preadsorbed fibronectin was observed to stimulate osteoblast differentiation during the mineralization phase of osteoblastogenesis.
PMCID: PMC3810407  PMID: 23057020
coatings; differentiation; fibronectin; metal oxides; osteoblast; real-time polymerase chain reaction
21.  Nanometer Scale Titanium Surface Texturing Are Detected by Signaling Pathways Involving Transient FAK and Src Activations 
PLoS ONE  2014;9(7):e95662.
Background
It is known that physico/chemical alterations on biomaterial surfaces have the capability to modulate cellular behavior, affecting early tissue repair. Such surface modifications are aimed to improve early healing response and, clinically, offer the possibility to shorten the time from implant placement to functional loading. Since FAK and Src are intracellular proteins able to predict the quality of osteoblast adhesion, this study evaluated the osteoblast behavior in response to nanometer scale titanium surface texturing by monitoring FAK and Src phosphorylations.
Methodology
Four engineered titanium surfaces were used for the study: machined (M), dual acid-etched (DAA), resorbable media microblasted and acid-etched (MBAA), and acid-etch microblasted (AAMB). Surfaces were characterized by scanning electron microscopy, interferometry, atomic force microscopy, x-ray photoelectron spectroscopy and energy dispersive X-ray spectroscopy. Thereafter, those 4 samples were used to evaluate their cytotoxicity and interference on FAK and Src phosphorylations. Both Src and FAK were investigated by using specific antibody against specific phosphorylation sites.
Principal Findings
The results showed that both FAK and Src activations were differently modulated as a function of titanium surfaces physico/chemical configuration and protein adsorption.
Conclusions
It can be suggested that signaling pathways involving both FAK and Src could provide biomarkers to predict osteoblast adhesion onto different surfaces.
doi:10.1371/journal.pone.0095662
PMCID: PMC4085036  PMID: 24999733
22.  PHOSPHATED, ACID-ETCHED IMPLANTS DECREASE MINERAL APPOSITION RATES NEAR IMPLANTS IN CANINES 
Purpose:
This study evaluated the effects of phosphate-coated titanium on mineral apposition rate (MAR) and new bone-to-implant contact (BIC) in canines.
Materials and Methods:
2.2 mm × 4 mm electrolytically phosphated or non-phosphated titanium implants with acid-etched surfaces were placed in 48 mandibular sites in 6 foxhounds. Tetracycline and calcein dyes were administered 1 week after implant placement and 1 week before sacrifice. At twelve weeks following implant healing, animals were sacrificed. MAR and BIC were evaluated using fluorescence microscopy. Light microscopic and histological evaluation was performed on undecalcified sections.
Results:
Microscopic evaluation showed the presence of healthy osteoblasts lining bone surfaces near implants. Similar bone-to-implant contact was observed in phosphated and non-phosphated titanium implant sites. MAR was significantly higher near non-phosphated titanium implant surfaces than the phosphated titanium samples. No significant differences were found between dogs or implant sites.
Discussion and Conclusion:
Acid-etched only implants showed significantly higher mineral apposition rates compared to acid-etched, phosphate-coated implants.
PMCID: PMC2946350  PMID: 20369085
phosphate; titanium; acid-etched; mineral apposition rate; BIC
23.  Heat and Radiofrequency Plasma Glow Discharge Pretreatment of a Titanium Alloy Promote Bone Formation and Osseointegration 
Journal of cellular biochemistry  2013;114(10):10.1002/jcb.24585.
Orthopedic and dental implants manifest increased failure rates when inserted into low density bone. We determined whether chemical pretreatments of a titanium alloy implant material stimulated new bone formation to increase osseointegration in vivo in trabecular bone using a rat model. Titanium alloy rods were untreated or pretreated with heat (600°C) or radiofrequency plasma glow discharge (RFGD). The rods were then coated with the extracellular matrix protein fibronectin (1 nM) or left uncoated and surgically implanted into the rat femoral medullary cavity. Animals were euthanized 3 or 6 weeks later, and femurs were removed for analysis. The number of trabeculae in contact with the implant surface, surface contact between trabeculae and the implant, and the length and area of bone attached to the implant were measured by histomorphometry. Implant shear strength was measured by a pull-out test. Both pretreatments and fibronectin enhanced the number of trabeculae bonding with the implant and trabeculae-to-implant surface contact, with greater effects of fibronectin observed with pretreated compared to untreated implants. RFGD pretreatment modestly increased implant shear strength, which was highly correlated (r2 = 0.87 – 0.99) with measures of trabecular bonding for untreated and RFGD-pretreated implants. In contrast, heat pretreatment increased shear strength 3 to 5-fold for both uncoated and fibronectin-coated implants at 3 and 6 weeks, suggesting a more rapid increase in implant-femur bonding compared to the other groups. In summary, our findings suggest that the heat and RFGD pretreatments can promote the osseointegration of a titanium alloy implant material.
doi:10.1002/jcb.24585
PMCID: PMC3786157  PMID: 23649564
Dental implant; fibronectin; osteoblast; cell differentiation; bone mineralization; osseointegration
24.  In vitro preliminary study of osteoblast response to surface roughness of titanium discs and topical application of melatonin 
Objectives: To observe human osteoblast behavior cultured in vitro on titanium discs (Ti) in relation to surface roughness and melatonin application. Study Design: Human osteoblasts (MG-63) were cultured on 60 Ti6Al4V discs divided into three groups: Group I: discs treated with dual acid etching; Group II dual acid etching and blasting with calcium phosphate particles; Group III (control) machined discs. Surface roughness and topography of the discs were examined with scanning electron microscope (SEM) and confocal laser scanning electron microscope( CLSM). Osteoblast adhesion, proliferation and cell morphology were determined by means of fluorescence microscopy with Image-Pro Plus software and SEM. Results: Group II presented the roughest discs, while the least rough were Group III. Cell adhesion was greatest in Group II. The addition of melatonin improved cell proliferation. Conclusions: 1. Surface treatments (dual acid etching, calcium phosphate impaction) increase surface roughness in comparison with machined titanium. 2. Greater surface roughness tends to favor cell adhesion after 24-hour cell culture. 3. The addition of melatonin tends to favor osteoblast proliferation.
Key words:Osteoblasts, titanium, roughness, melatonin, dental implants, osseointegration.
doi:10.4317/medoral.19953
PMCID: PMC4320427  PMID: 25129252
25.  Osseointegration of zirconia implants compared with titanium: an in vivo study 
Head & Face Medicine  2008;4:30.
Background
Titanium and titanium alloys are widely used for fabrication of dental implants. Since the material composition and the surface topography of a biomaterial play a fundamental role in osseointegration, various chemical and physical surface modifications have been developed to improve osseous healing. Zirconia-based implants were introduced into dental implantology as an altenative to titanium implants. Zirconia seems to be a suitable implant material because of its tooth-like colour, its mechanical properties and its biocompatibility. As the osseointegration of zirconia implants has not been extensively investigated, the aim of this study was to compare the osseous healing of zirconia implants with titanium implants which have a roughened surface but otherwise similar implant geometries.
Methods
Forty-eight zirconia and titanium implants were introduced into the tibia of 12 minipigs. After 1, 4 or 12 weeks, animals were sacrificed and specimens containing the implants were examined in terms of histological and ultrastructural techniques.
Results
Histological results showed direct bone contact on the zirconia and titanium surfaces. Bone implant contact as measured by histomorphometry was slightly better on titanium than on zirconia surfaces. However, a statistically significant difference between the two groups was not observed.
Conclusion
The results demonstrated that zirconia implants with modified surfaces result in an osseointegration which is comparable with that of titanium implants.
doi:10.1186/1746-160X-4-30
PMCID: PMC2614983  PMID: 19077228

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