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.
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.
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.
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.
surface treatment; bone to implant contact; removal torque; dental implant
Given the popularity of cementless orthopedic implants, it is imperative for orthopedic surgeons to have a basic understanding of the process of peri-implant bone healing. Contact and distance osteogenesis have been used to explain peri-implant bone healing. In contact osteogenesis, de novo bone forms on the implant surface, while in distance osteogenesis, the bone grows from the old bone surface toward the implant surface in an appositional manner. Contact osteogenesis may lead to bone bonding if the surface of the implant displays the appropriate surface topography. The early stage of peri-implant bone healing is very important and involves the body’s initial response to a foreign material: protein adsorption, platelet activation, coagulation, and inflammation. This results in the formation of a stable fibrin clot that is a depot for growth factors and allows for osteoconduction. Osteoconduction is the migration and differentiation of osteogenic cells, such as pericytes, into osteoblasts. Osteoconduction allows for contact osteogenesis to occur at the implant surface. The late stage of healing involves the remodeling of this woven bone. In many respects, this process is similar to the bone healing occurring at a fracture site.
Bone bonding; cementless implants; peri-implant bone healing
The purpose of this study was to characterize the osseointegration of the fibronectin-coated implant surface.
Sand-blasted, large-grit, acid-etched (SLA) surface implants, with or without a thin calcium phosphate and fibronectin coating, were placed in edentulous mandibles of dogs 8 weeks after extraction. All dogs were sacrificed forhistological and histomorphometric evaluation after 4- and 8-week healing periods.
All types of implants were clinically stable without any mobility. Although the bone-to-implant contact and bone density of the SLA implants coated with calcium phosphate (CaP)/fibronectin were lower than the uncoated SLA implants, there were no significant differences between the uncoated SLA surface group and the SLA surface coated with CaP/fibronectin group.
Within the limits of this study, SLA surfaces coated with CaP/fibronectin were shown to have comparable bone-to-implant contact and bone density to uncoated SLA surfaces.
Biocompatible coated materials; Bone density; Calcium phosphate; Dental implants; Fibronectins
Statement of Problem. The chemical or topographic modification of the dental implant surface can affect bone healing, promote accelerated osteogenesis, and increase bone-implant contact and bonding strength. Objective. In this work, the effects of dental implant surface treatment and fibronectin adsorption on the adhesion of osteoblasts were analyzed. Materials and Methods. Two titanium dental implants (Porous-acid etching and PorousNano-acid etching followed by fluoride ion modification) were characterized by high-resolution scanning electron microscopy, atomic force microscopy, and X-ray diffraction before and after the incorporation of human plasma fibronectin (FN). The objective was to investigate the biofunctionalization of these surfaces and examine their effects on the interaction with osteoblastic cells. Results. The evaluation techniques used showed that the Porous and PorousNano implants have similar microstructural characteristics. Spectrophotometry demonstrated similar levels of fibronectin adsorption on both surfaces (80%). The association indexes of osteoblastic cells in FN-treated samples were significantly higher than those in samples without FN. The radioactivity values associated with the same samples, expressed as counts per minute (cpm), suggested that FN incorporation is an important determinant of the in vitro cytocompatibility of the surfaces. Conclusion. The preparation of bioactive titanium surfaces via fluoride and FN retention proved to be a useful treatment to optimize and to accelerate the osseointegration process for dental implants.
Titanium (Ti) has been widely used as an implant material due to the excellent biocompatibility and corrosion resistance of its oxide surface. Biomaterials must be sterile before implantation, but the effects of sterilization on their surface properties have been less well studied. The effects of cleaning and sterilization on surface characteristics were bio-determined using contaminated and pure Ti substrata first manufactured to present two different surface structures: pretreated titanium (PT, Ra = 0.4 μm) (i.e. surfaces that were not modified by sandblasting and/or acid etching); (SLA, Ra = 3.4 μm). Previously cultured cells and associated extracellular matrix were removed from all bio-contaminated specimens by cleaning in a sonicator bath with a sequential acetone–isopropanol–ethanol–distilled water protocol. Cleaned specimens were sterilized with autoclave, gamma irradiation, oxygen plasma, or ultraviolet light. X-ray photoelectron spectroscopy (XPS), contact angle measurements, profilometry, and scanning electron microscopy were used to examine surface chemical components, hydrophilicity, roughness, and morphology, respectively. Small organic molecules present on contaminated Ti surfaces were removed with cleaning. XPS analysis confirmed that surface chemistry was altered by both cleaning and sterilization. Cleaning and sterilization affected hydrophobicity and roughness. These modified surface properties affected osteogenic differentiation of human MG63 osteoblast-like cells. Specifically, autoclaved SLA surfaces lost the characteristic increase in osteoblast differentiation seen on starting SLA surfaces, which was correlated with altered surface wettability and roughness. These data indicated that recleaned and resterilized Ti implant surfaces cannot be considered the same as the first surfaces in terms of surface properties and cell responses. Therefore, the reuse of Ti implants after resterilization may not result in the same tissue responses as found with never-before-implanted specimens.
Titanium; Sterilization; Roughness; Hydrophilicity; MG63 cells
Microtexture and chemistry of implant surfaces are important variables for modulating cellular responses. Surface chemistry and wettability are connected directly. While each of these surface properties can influence cell response, it is difficult to decouple their specific contributions. To address this problem, the aims of this study were to develop a surface wettability gradient with a specific chemistry without altering micron scale roughness and to investigate the role of surface wettability on osteoblast response. Microtextured sandblasted/acid-etched (SLA, Sa = 3.1 μm) titanium disks were treated with oxygen plasma to increase reactive oxygen density on the surface. At 0, 2, 6, 10, and 24 h after removing them from the plasma, the surfaces were coated with chitosan for 30 min, rinsed and dried. Modified SLA surfaces are denoted as SLA/h in air prior to coating. Surface characterization demonstrated that this process yielded differing wettability (SLA0 < SLA2 < SLA10 < SLA24) without modifying the micron scale features of the surface. Cell number was reduced in a wettability-dependent manner, except for the most water-wettable surface, SLA24. There was no difference in alkaline phosphatase activity with differing wettability. Increased wettability yielded increased osteocalcin and osteoprotegerin production, except on the SLA24 surfaces. mRNA for integrins α1, α2, α5, β1, and β3 was sensitive to surface wettability. However, surface wettability did not affect mRNA levels for integrin α3. Silencing β1 increased cell number with reduced osteocalcin and osteoprotegerin in a wettability-dependent manner. Surface wettability as a primary regulator enhanced osteoblast differentiation, but integrin expression and silencing β1 results indicate that surface wettability regulates osteoblast through differential integrin expression profiles than microtexture does. The results may indicate that both microtexture and wettability with a specific chemistry have important regulatory effects on osseointegration. Each property had different effects, which were mediated by different integrin receptors.
Wettability; Oxygen plasma; Chitosan; Titanium; Osteoblast; Integrin
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.
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.
phosphate; titanium; acid-etched; mineral apposition rate; BIC
The aim of the present study was to investigate bone formation to recombinant human bone morphogenetic protein-2 (rhBMP-2)-biocoated and rhBMP-2-nonbiocoated titanium implants after implantation in dogs. Implantation of sand-blasted and acid-etched (C), chromosulfuric acid surface-enhanced (CSA), and rhBMP-2-biocoated CSA [BMP-A: noncovalently immobilized rhBMP-2 (596 ng/cm2), BMP-B: covalently immobilized rhBMP-2 (819 ng/cm2)] implants was performed in both the mandible and tibia of dogs. After 4 weeks of healing, the percentage of direct bone to implant contact (BIC) and the induced bone density (BD) at a distance of less than and greater than 1 mm adjacent to each implant was assessed. Histomorphometric analysis of implants inserted in the mandible and tibia revealed that BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C. BD as measured at a distance of <1 mm revealed obvious differences between groups: BMP-B>BMP-A>CSA>C. However, no differences between groups were observed at a distance of >1 mm. Within the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependant manner.
rhBMP-2; Titanium implant; Bioactive surface; Ultrahydrophilic surface; Animal study
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.
biomimetic material; cell adhesion; collagen; osseointegration; integrin
Electron beam melting (E-beam) is a new technology to produce 3-dimensional surface topographies for cementless orthopedic implants.
The friction coefficients of two newly developed E-beam produced surface topographies were in vitro compared with sandblasted E-beam and titanium plasma sprayed controls. Bone ingrowth (direct bone–implant contact) was determined by implanting the samples in the femoral condyles of 6 goats for a period of 6 weeks.
Friction coefficients of the new structures were comparable to the titanium plasma sprayed control. The direct bone–implant contact was 23.9 and 24.5% for the new surface structures. Bone–implant contact of the sandblasted and titanium plasma sprayed control was 18.2 and 25.5%, respectively.
The frictional and bone ingrowth properties of the E-beam produced surface structures are similar to the plasma-sprayed control. However, since the maximal bone ingrowth had not been reached for the E-beam structures during the relatively short-term period, longer-term follow-up studies are needed to assess whether the E-beam structures lead to a better long-term performance than surfaces currently in use, such as titanium plasma spray coating.
Electron beam melting; Bone ingrowth; Friction; Surface characteristics; Prosthesis
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.
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.
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.
The results demonstrated that zirconia implants with modified surfaces result in an osseointegration which is comparable with that of titanium implants.
Early bone ongrowth secures long-term fixation of primary implants inserted without cement. Implant surfaces roughened with a texture on the micrometer scale are known to be osseoconductive. The aim of this study was to evaluate the bone formation at the surface of acid etched implants modified on the micro-scale. We compared implants with a nonparticulate texture made by chemical milling (hydrofluoric acid, nitric acid) (control) with implants that had a dual acid etched (hydrofluoric acid, hydrochloric acid) microtexture surface superimposed on the primary chemically milled texture. We used an experimental joint replacement model with cylindrical titanium implants (Ti-6Al-4V) inserted paired and press-fit in cancellous tibia metaphyseal bone of eight canines for 4 weeks and evaluated by histomorphometric quantification. A significant twofold median increase was seen for bone ongrowth on the acid etched surface [median, 36.1% (interquartile range, 24.3–44.6%)] compared to the control [18.4% (15.6–20.4%)]. The percentage of fibrous tissue at the implant surface and adjacent bone was significantly less for dual acid textured implants compared with control implants. These results show that secondary roughening of titanium alloy implant surface by dual acid etching increases bone formation at the implant bone interface.
osseointegration; surface texture; acid etching; implants; histology
Peri-implant bone formation depends on the ability of mesenchymal cells to colonize the implant surface and differentiate into osteoblasts. Human mesenchymal stem cells (HMSCs) undergo osteoblastic differentiation on microstructured titanium (Ti) surfaces in the absence of exogenous factors, but the mechanisms are unknown. Wnt proteins are associated with an osteoblast phenotype, but how Wnt signaling regulates HMSC differentiation on microstructured Ti surfaces is not known. HMSCs were cultured on tissue culture polystyrene or Ti (PT [Sa=0.33μm, θ=96°], SLA [Sa=2.5μm, θ=132°], modSLA [hydrophilic-SLA]). Expression of calcium-dependent Wnt ligand WNT5A increased and canonical Wnt pathway ligands decreased on microstructured Ti in a time-dependent manner. Treatment of HMSCs with canonical ligand Wnt3a preserved the mesenchymal phenotype on smooth surfaces. Treatment with Wnt5a increased osteoblastic differentiation. Expression of integrins ITGA1, ITGA2, and ITGAV increased over time and correlated with increased WNT5A expression. Treatment of HMSCs with Wnt5a, but not Wnt3a, increased integrin expression. Regulation of integrin expression due to surface roughness and energy was ablated in WNT5A-knockdown HMSCs. This indicates that surface properties regulate stem cell fate and induce osteoblast differentiation via the Wnt calcium-dependent pathway. Wnt5a enhances osteogenesis through a positive feedback with integrins and local factor regulation, particularly though BMP signaling.
Cell signaling; Surface roughness; Titanium; Stem cell; Growth factors
To gain basic information regarding the biologic stability of plasma ion-implanted miniscrews and their potential clinical applications.
Sixteen plasma ion-implanted and 16 sandblasted and acid-etched (SLA) miniscrews were bilaterally inserted in the mandibles of 4 beagles (2 miniscrews of each type per quadrant). Then, 250 - 300 gm of force from Ni-Ti coil springs was applied for 2 different periods: 12 weeks on one side and 3 weeks contralaterally. Thereafter, the animals were sacrificed and mandibular specimens including the miniscrews were collected. The insertion torque and mobility were compared between the groups. The bone-implant contact and bone volume ratio were calculated within 800 µm of the miniscrews and compared between the loading periods. The number of osteoblasts was also quantified. The measurements were expressed as percentages and analyzed by independent t-tests (p < 0.05).
No significant differences in any of the analyzed parameters were noted between the groups.
The preliminary findings indicate that plasma ion-implanted miniscrews have similar biologic characteristics to SLA miniscrews in terms of insertion torque, mobility, bone-implant contact rate, and bone volume rate.
Orthodontic mini-implant; Surface treatment; Histology; Stability
Background and purpose
Intermittent administration of parathyroid hormone (PTH) has an anabolic effect on bone, as confirmed in human osteoporosis studies, distraction osteogenesis, and fracture healing. PTH in rat models leads to improved fixation of implants in low-density bone or screw insertion transcortically.
Material and methods
We examined the effect of human PTH (1–34) on the cancellous osseointegration of unloaded implants inserted press-fit in intact bone of higher animal species. 20 dogs were randomized to treatment with human PTH (1–34), 5 μg/kg/day subcutaneously, or placebo for 4 weeks starting on the day after insertion of a cylindrical porous coated plasma-sprayed titanium alloy implant in the proximal metaphyseal cancellous bone of tibia. Osseointegration was evaluated by histomorphometry and fixation by push-out test to failure.
Surface fraction of woven bone at the implant interface was statistically significantly higher in the PTH group by 1.4 fold with (median (interquartile range) 15% (13–18)) in the PTH group and 11% (7–13) in control. The fraction of lamellar bone was unaltered. No significant difference in bone or fibrous tissue was observed in the circumferential regions of 0–500, 500–1,000, and 1,000–2,000 μm around the implant. Mechanically, the implants treated with PTH showed no significant differences in total energy absorption, maximum shear stiffness, or maximum shear strength.
Intermittent treatment with PTH (1–34) improved xhistological osseointegration of a prosthesis inserted press-fit at surgery in cancellous bone, with no additional improvement of the initial mechanical fixation at this time point.
Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopaedic implants after sterilization procedures and affect osseointegration. The goals of this study were to develop a murine model of osseointegration in order to determine whether removing surface contaminants enhances osseointegration. To develop the murine model, titanium alloy implants were implanted into a unicortical pilot hole in the mid-diaphysis of the femur and osseointegration was measured over a five week time course. Histology, backscatter scanning electron microscopy and x-ray energy dispersive spectroscopy showed areas of bone in intimate physical contact with the implant, confirming osseointegration. Histomorphometric quantification of bone-to-implant contact and peri-implant bone and biomechanical pullout quantification of ultimate force, stiffness and work to failure increased significantly over time, also demonstrating successful osseointegration. We also found that a rigorous cleaning procedure significantly enhances bone-to-implant contact and biomechanical pullout measures by two-fold compared with implants that were autoclaved, as recommended by the manufacturer. The most likely interpretation of these results is that surface contaminants inhibit osseointegration. The results of this study justify the need for the development of better detection and removal techniques for contaminants on orthopaedic implants and other medical devices.
contaminants; osseointegration; murine; histomorphometry; biomechanical testing
Although the bone's capability of dental implant osseointegration has clinically been utilised as early as in the Gallo-Roman population, the specific mechanisms for the emergence and maintenance of peri-implant bone under functional load have not been identified. Here we show that under immediate loading of specially designed dental implants with masticatory loads, osseointegration is rapidly achieved.
We examined the bone reaction around non- and immediately loaded dental implants inserted in the mandible of mature minipigs during the presently assumed time for osseointegration. We used threaded conical titanium implants containing a titanium2+ oxide surface, allowing direct bone contact after insertion. The external geometry was designed according to finite element analysis: the calculation showed that physiological amplitudes of strain (500–3,000 ustrain) generated through mastication were homogenously distributed in peri-implant bone. The strain-energy density (SED) rate under assessment of a 1 Hz loading cycle was 150 Jm-3 s-1, peak dislocations were lower then nm.
Bone was in direct contact to the implant surface (bone/implant contact rate 90%) from day one of implant insertion, as quantified by undecalcified histological sections. This effect was substantiated by ultrastructural analysis of intimate osteoblast attachment and mature collagen mineralisation at the titanium surface. We detected no loss in the intimate bone/implant bond during the experimental period of either control or experimental animals, indicating that immediate load had no adverse effect on bone structure in peri-implant bone.
In terms of clinical relevance, the load related bone reaction at the implant interface may in combination with substrate effects be responsible for an immediate osseointegration state.
Interaction between implant surface and surrounding bone influences implant fixation. We attempted to improve the bone-implant interaction by 1) adding surface micro scale topography by acid etching, and 2) removing surface-adherent pro-inflammatory agents by plasma cleaning. Implant fixation was evaluated by implant osseointegration and biomechanical fixation.
The study consisted of two paired animal sub-studies where 10 skeletally mature Labrador dogs were used. Grit blasted titanium alloy implants were inserted press fit in each proximal tibia. In the first study grit blasted implants were compared with acid etched grit blasted implants. In the second study grit blasted implants were compared with acid etched grit blasted implants that were further treated with plasma sterilization. Implant performance was evaluated by histomorphometrical investigation (tissue-to-implant contact, peri-implant tissue density) and mechanical push-out testing after four weeks observation time.
Neither acid etching nor plasma sterilization of the grit blasted implants enhanced osseointegration or mechanical fixation in this press-fit canine implant model in a statistically significant manner.
Acid etching; biocompatibility; endotoxin; implant surgery; grit blasting; plasma sterilization; titanium.
The mechanism by which hydroxyapatite (HA)-coated titanium promotes bone–implant integration is largely unknown. Furthermore, refining the fabrication of nano-structured HA to the level applicable to the mass production process for titanium implants is challenging. This study reports successful creation of nanopolymorphic crystalline HA on microroughened titanium surfaces using a combination of flame spray and low-temperature calcination and tests its biological capability to enhance bone–implant integration. Sandblasted microroughened titanium implants and sandblasted + HA-coated titanium implants were subjected to biomechanical and histomorphometric analyses in a rat model. The HA was 55% crystallized and consisted of nanoscale needle-like architectures developed in various diameters, lengths, and orientations, which resulted in a 70% increase in surface area compared to noncoated microroughened surfaces. The HA was free from impurity contaminants, with a calcium/phosphorus ratio of 1.66 being equivalent to that of stoichiometric HA. As compared to microroughened implants, HA-coated implants increased the strength of bone–implant integration consistently at both early and late stages of healing. HA-coated implants showed an increased percentage of bone–implant contact and bone volume within 50 μm proximity of the implant surface, as well as a remarkably reduced percentage of soft tissue intervention between bone and the implant surface. In contrast, bone volume outside the 50 μm border was lower around HA-coated implants. Thus, this study demonstrated that the addition of pure nanopolymorphic crystalline HA to microroughened titanium not only accelerates but also enhances the level of bone–implant integration and identified the specific tissue morphogenesis parameters modulated by HA coating. In particular, the nanocrystalline HA was proven to be drastic in increasing osteoconductivity and inhibiting soft tissue infiltration, but the effect was limited to the immediate microenvironment surrounding the implant.
osseointegration; dental and orthopedic implant; nanotechnology; bone–implant integration; HA; calcium phosphate
Background: The goal of this study was to evaluate the behavior of neonatal rat calvarial osteoblast-like cells cultured on different implant surfaces and exposed once or three times to a 660-nm light-emitting diode (LED).
Methods: An LED with a 660-nm wavelength was applied once or three times to cultured cells on standard and modified sandblasted acid-etched surfaces (SLA and SLActive; Straumann, Basel, Switzerland). To analyze the effect of the LED on cell proliferation, numbers, and viability, cells were cultured on titanium discs, and measurements were taken after 72 h. Cell proliferation rates were assessed using a bromodeoxyuridine immunohistochemical technique. Cell morphologies were evaluated by scanning electron microscopy (SEM).
Results: Osteoblast-like cells proliferated on all tested surfaces, with differences among groups in cell counts and DNA synthesis values. The application of one LED treatment caused a significant increase in cell count in the SLActive group in comparison with the SLA group (p = 0.001), whereas the application of three LED treatments caused a significant decrease in cell count in the SLA group compared with the SLActive group (p < 0.001). After 72 h, the number of cells was highest in the SLActive group exposed once to the LED.
Conclusions: One LED application in the SLActive group resulted in significantly increased cell numbers. However, these findings were not exactly compatible with the SEM findings, which demonstrated fewer cells and weak attachments between cells and to the surface. Thus, further studies using different LED application times are needed to clarify the reason for the increased number of cells that are apparently incapable of attaching to the titanium surfaces after 72 h.
Light-emitting diode; SLA and SLActive surface; primary osteoblast cell culture
An implantable model system was developed to investigate the effects of nanoscale surface properties on the osseointegration of titanium implants in rat tibia. Topographical nanostructures with a well-defined shape (semispherical protrusions) and variable size (60 nm, 120 nm and 220 nm) were produced by colloidal lithography on the machined implants. Furthermore, the implants were sputter-coated with titanium to ensure a uniform surface chemical composition. The histological evaluation of bone around the implants at 7 days and 28 days after implantation was performed on the ground sections using optical and scanning electron microscopy. Differences between groups were found mainly in the new bone formation process in the endosteal and marrow bone compartments after 28 days of implantation. Implant surfaces with 60 nm features demonstrated significantly higher bone-implant contact (BIC, 76%) compared with the 120 nm (45%) and control (57%) surfaces. This effect was correlated to the higher density and curvature of the 60 nm protrusions. Within the developed model system, nanoscale protrusions could be applied and systematically varied in size in the presence of microscale background roughness on complex screw-shaped implants. Moreover, the model can be adapted for the systematic variation of surface nanofeature density and chemistry, which opens up new possibilities for in vivo studies of various nanoscale surface-bone interactions.
in vivo; nanotopography; osseointegration; titanium implant; colloidal lithography
Rough titanium (Ti) surface microarchitecture and high surface energy have been shown to increase osteoblast differentiation, and this response occurs through signaling via the α2β1 integrin. However, clinical success of implanted materials is dependent not only upon osseointegration but also on neovascularization in the peri-implant bone. Here we tested the hypothesis that Ti surface microtopography and energy interact via α2β1 signaling to regulate the expression of angiogenic growth factors. Primary human osteoblasts (HOB), MG63 cells and MG63 cells silenced for α2 integrin were cultured on Ti disks with different surface microtopographies and energies. Secreted levels of vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), and angiopoietin-1 (Ang-1) were measured. VEGF-A increased 170% and 250% in MG63 cultures, and 178% and 435% in HOB cultures on SLA and modSLA substrates, respectively. In MG63 cultures, FGF-2 levels increased 20 and 40-fold while EGF increased 4 and 6-fold on SLA and modSLA surfaces. These factors were undetectable in HOB cultures. Ang-1 levels were unchanged on all surfaces. Media from modSLA MG63 cultures induced more rapid differentiation of endothelial cells and this effect was inhibited by anti-VEGF-A antibodies. Treatment of MG63 cells with 1α,25(OH)2D3 enhanced levels of VEGF-A on SLA and modSLA. Silencing the α2 integrin subunit increased VEGF-A levels and decreased FGF-2 levels. These results show that Ti surface microtopography and energy modulate secretion of angiogenic growth factors by osteoblasts and that this regulation is mediated at least partially via α2β1 integrin signaling.
Titanium; microstructure; surface energy; osteoblast; angiogenesis; VEGF
Osteoblasts grown on microstructured Ti surfaces enhance osteointegration by producing local factors that regulate bone formation as well as bone remodeling, including the RANK ligand decoy receptor osteoprotegerin (OPG). The objective of this study was to explore the mechanism by which surface microstructure and surface energy mediate their stimulatory effects on OPG expression. Titanium disks were manufactured to present different surface morphologies: a smooth pretreatment surface (PT, Ra<0.2μm), microstructured sandblasted/acid etched surface (SLA, Ra=3-4μm), and a microstructured Ti plasma-sprayed surface (TPS, Ra=4μm). Human osteoblast-like MG63 cells were cultured on these substrates and the regulation of OPG production by TGF-β1, PKC, and α2β1 integrin signaling determined. Osteoblasts produced increased amounts of OPG as well as active and latent TGF-β1 and had increased PKC activity when grown on SLA and TPS. Exogenous TGF-β1 increased OPG production in a dose-dependent manner on all surfaces, and this was prevented by adding blocking antibody to the TGF-β type II receptor or by reducing TGF-β1 binding to the receptor by adding exogenous soluble type II receptor. The PKC inhibitor chelerythrine inhibited the production of OPG in a dose-dependent manner, but only in cultures on SLA and TPS. shRNA knockdown of α2 or a double knockdown of α2β1 also reduced OPG, as well as production of TGF-β1. These results indicate that substrate dependent OPG production is regulated by TGF-β1, PKC, and α2β1 and suggest a mechanism by which α2β1-signaling increases PKC, resulting in TGF-β1 production and TGF-β1 then acts on its receptor to increase transcription of OPG.
Osteoblast; TGF-β1; Osteoprotegerin; Titanium; Microtopography
Implant surface topography influences osteoblastic proliferation, differentiation and extracellular matrix protein expressions. Previous researches proved that chemical surface modification of titanium implants could be used to improve Bone-to-implant contact. In this study, the surface topography, chemistry and biocompatibility of polished titanium surfaces treated with mixed solution of three acids containing HCl, HF and H3PO4 with different etched conditions for example concentration, time and addition of calcium chloride were studied. Osteoblast cells (MG-63) were cultured on different groups of titanium surfaces. In order to investigate titanium surfaces, SEM, AFM and EDS analyses were carried out. The results showed that surfaces treated with HCl–HF–H3PO4 had higher roughness, lower cytotoxicity level and better biocompatibility than controls. Moreover, addition of calcium chloride into mixed solution of three acids containing HCl, HF and H3PO4 is an important, predominant and new technique for obtaining biofunction in metals for biomedical use including dentistry.
Osseointegration depends on the implant surface, bone quality and the local and systemic host environment, which can differ in male and female patients. This study was undertaken in order to determine if male and female cells respond differently to titanium surfaces that have micron-scale roughness and if interactions of calciotropic hormones [1α,25(OH)2D3 and 17β-oestradiol (E2)] and microstructured surfaces on osteoblasts are sex dependent.
Osteoblasts from 6-week old Sprague-Dawley rats were cultured on tissue culture polystyrene (TCPS) or on titanium (Ti) disks with two different surface topographies, a smooth pretreated (PT) surface and a coarse grit-blasted/acid-etched (SLA) surface, and treated with 1α,25(OH)2D3, E2, or E2 conjugated to bovine serum albumin (E2-BSA).
Male and female cells responded similarly to Ti microstructure with respect to cell number and levels of osteocalcin, transforming growth factor-β1, osteoprotegerin and prostaglandin E2 in their conditioned media, exhibiting a more differentiated phenotype on SLA than on PT or TCPS. E2 and E2-BSA increased differentiation and local factor production, an effect that was microstructure dependent and found only in female osteoblasts. 1α,25(OH)2D3 increased osteoblast differentiation and local factor production in female and male cells, but the effect was more robust in male cells.
Male and female rat osteoblasts respond similarly to surface microstructure but exhibit sexual dimorphism in substrate-dependent responses to systemic hormones. Oestrogen affected only female cells while 1α,25(OH)2D3 had a greater effect on male cells. These results suggest that successful osseointegration in males and females may depend on the implant surface design and correct levels of calciotropic hormones.