Surface functionalization of TiO2 nanotubes with bone morphogenetic protein 2 and its synergistic effect on the differentiation of mesenchymal stem cells

To investigate the influence of surface-functionalized substrates with nanostructures on the behaviors of mesenchymal stem cells, we conjugated bone morphogenetic protein 2 (BMP2) onto TiO(2) nanotubes with different diameter sizes of 30, 60, and 100 nm for in vitro study. Polydopamine was employed as the intermediate layer for the conjugation of BMP2. The successful conjugation of BMP2 onto TiO(2) nanotubes was revealed by field-emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and contact angle measurements. Immunofluorescence staining of vinculin, osteocalcin (OCN), and osteopontin (OPN) revealed that BMP2-functionalized TiO(2) nanotubes was favorable for cell growth. More importantly, MSCs cultured onto BMP2-functionalized TiO(2) nanotubes displayed significantly higher (p < 0.05 or p < 0.01) differentiation levels of ALP and mineralization after 7 and 14 day cultures, respectively. The results suggested that surface functionalization of TiO(2) nanotubes with BMP2 was beneficial for cell proliferation and differentiation. The approach presented here has potential application for the development of titanium-based implants for enhanced bone osseointegration.     

Effect of heparin and alendronate coating on titanium surfaces on inhibition of osteoclast and enhancement of osteoblast function

The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue.In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance osteoblast differentiation and to inhibit osteoclast differentiation simultaneously.     

Increased osteoblast cell density on nanostructured PLGA-coated nanostructured titanium for orthopedic applications

There are more than 30,000 orthopedic implant revision surgeries necessary each year in part due to poor implant fixation with juxtaposed bone. A further emphasis on the current problems associated with insufficient bone implant performance is the fact that many patients are receiving hip implants earlier in life, remaining active older, and that the human lifespan is continuously increasing. Collectively, it is clear that there is a strong clinical need to improve implant performance through proper, prolonged fixation. For these reasons, the objective of the present in vitro study was to improve the performance of titanium (Ti), one of the most popular orthopedic implant materials. Accordingly, the proliferative response of osteoblasts (bone-forming cells) on novel nanostructured Ti/PLGA (poly-lactic-co-glycolic acid) composites was examined. This study showed that nano-topography can be easily applied to Ti (through anodization) and porous PLGA (through NaOH chemical etching) to enhance osteoblast cell proliferation which may lead to better orthopedic implant performance. This straight forward application of nano-topography on current bone implant materials represents a new direction in the design of enhanced biomaterials for the orthopedic industry.     

Coating with a Modular Bone Morphogenetic Peptide Promotes Healing of a Bone-Implant Gap in an Ovine Model

Despite the potential for growth factor delivery strategies to promote orthopedic implant healing, there is a need for growth factor delivery methods that are controllable and amenable to clinical translation. We have developed a modular bone growth factor, herein termed “modular bone morphogenetic peptide (mBMP)”, which was designed to efficiently bind to the surface of orthopedic implants and also stimulate new bone formation. The purpose of this study was to coat a hydroxyapatite-titanium implant with mBMP and evaluate bone healing across a bone-implant gap in the sheep femoral condyle. The mBMP molecules efficiently bound to a hydroxyapatite-titanium implant and 64% of the initially bound mBMP molecules were released in a sustained manner over 28 days. The results demonstrated that the mBMP-coated implant group had significantly more mineralized bone filling in the implant-bone gap than the control group in C-arm computed tomography (DynaCT) scanning (25% more), histological (35% more) and microradiographic images (50% more). Push-out stiffness of the mBMP group was nearly 40% greater than that of control group whereas peak force did not show a significant difference. The results of this study demonstrated that mBMP coated on a hydroxyapatite-titanium implant stimulates new bone formation and may be useful to improve implant fixation in total joint arthroplasty applications.     

Approaches to promoting bone marrow mesenchymal stem cell osteogenesis on orthopedic implant surface

Bone marrow-derived mesenchymal stem cells(BMSCs) play a critical role in the osseointegration of bone and orthopedic implant. However, osseointegration between the Ti-based implants and the surrounding bone tissue must be improved due to titanium's inherent defects. Surface modification stands out as a versatile technique to create instructive biomaterials that can actively direct stem cell fate. Here, we summarize the current approaches to promoting BMSC osteogenesis on the surface of titanium and its alloys. We will highlight the utilization of the unique properties of titanium and its alloys in promoting tissue regeneration, and discuss recent advances in understanding their role in regenerative medicine. We aim to provide a systematic and comprehensive review of approaches to promoting BMSC osteogenesis on the orthopedic implant surface.     

Enhanced osteoblast adhesion on nanostructured selenium compacts for anti-cancer orthopedic applications

Metallic bone implants possess numerous problems limiting their long-term efficacy, such as poor prolonged osseointegration, stress shielding, and corrosion under in vivo environments. Such problems are compounded for bone cancer patients since numerous patients receive orthopedic implants after cancerous bone resection. Unfortunately, current orthopedic materials were not originally developed to simultaneously increase healthy bone growth (as in traditional orthopedic implant applications) while inhibiting cancerous bone growth. The long-term objective of the present research is to investigate the use of nano-rough selenium to prevent bone cancer from re-occurring while promoting healthy bone growth for this select group of cancer patients. Selenium is a well known anti-cancer chemical. However, what is not known is how healthy bone cells interact with selenium. To determine this, selenium, spherical or semispherical shots, were pressed into cylindrical compacts and these compacts were then etched using 1N NaOH to obtain various surface structures ranging from the micron, submicron to nano scales. Changes in surface chemistry were also analyzed. Through these etching techniques, results of this study showed that biologically inspired surface roughness values were created on selenium compacts to match that of natural bone roughness. Moreover, results showed that healthy bone cell adhesion increased with greater nanometer selenium roughness (more closely matching that of titanium). In this manner, this study suggests that nano-rough selenium should be further tested for orthopedic applications involving bone cancer treatment.     

Micro-plasma textured Ti-implant surfaces

The surface state of titanium implants modulates bone response and implant anchorage. This evidence brought implant manufacturers to switch from the standard surface refinements and implement new surface treatments for more bone apposition and enhanced interfacial strength measured by removal torque or push-out tests. Anodic plasma-chemical treatment of implant surfaces is a cost-effective process to modify surface topography and chemistry. This technique is used for structuring connected with a coating of implant surfaces. The aim of our investigations, here, is to texture the implant surface in the nanoscale without coating. Ti disks with different mechanical pre-treatment (grinded, glass blasted) were used as substrate. Micro-plasma texturing was carried out in an aqueous electrolyte. By applying a pulsed DC voltage to the specimen, micro-plasma discharge was generated in the thin steam film between immersed specimen and electrolyte. The electrical process parameter current density was varied. The micro-plasma textured Ti surfaces were characterised optically by SEM and electrochemically by CV- (for testing the corrosion parameters), CA- (to give the enlargement of the real surface) and EIS-measurement in range of 100 kHz-100 microHz. We found that the initial structure of the material surface has small or no influence on the results of the micro-plasma treatment. The properties of the thick oxide layer resulting from the plasma process are influenced by electrical process parameters. After removal of the thick oxide layer a fine, micro- and nanoscaled surface structure of the titanium remains.     

Functionalized titanium oxide surfaces with phosphated carboxymethyl cellulose: characterization and bonelike cell behavior

The performance of dental or orthopedic implants is closely dependent on surface properties in terms of topography and chemistry. A phosphated carboxymethylcellulose containing one phosphate group for each disaccharide unit was synthesized and used to functionalize titanium oxide surfaces with the aim to improve osseointegration with the host tissue. The modified surfaces were chemically characterized by means of X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The investigation of the surface topography was performed by atomic force microscopy measurements before and after the polysaccharide coating. In vitro biological tests using osteoblastlike cells demonstrated that functionalized TiO(2) surfaces modulated cell response, in terms of adhesion, proliferation,and morphology. Phosphated carboxymethylcellulose promoted better cell adhesion and significantly enhanced their proliferation. The morphology of cells was polygonal and more spread on this type of modified surface.These findings suggest that the presence of a phosphate polysaccharide coating promotes osteoblast growth on the surface potentially improving biomaterial osseointegration.     

Testing biomaterials by the in-situ evaluation of cell response

In this work, we describe a technique for the in-situ observation of cells adhered on opaque biomaterial surfaces. The visualisation of the morphology of cells adhered onto a surface allows to derive nuclear apoptotic signs or even the existence of organisation between groups of these cells. The technique is based on the use of an auto-immune reaction combined with a fluorescent agent that allows a direct inspection of the cell behaviour. The versatility of the technique is demonstrated by presenting several examples with different cultured cells (human chondrosarcome cells (CSRCs) and pluripotential mesenchymal stem cells (MSCs) from bone marrow) seeded over two different Ti-based surfaces (TiO(2) and TiN, respectively). These in-vitro observations are compared with the behaviour of the same cells on bare TiAlV alloy. From our results it is concluded that both TiO(2) and TiN surfaces show enhanced biological responses.     

Artificial composite bone as a model of human trabecular bone: the implant-bone interface

The use of artificial bones in implant testing has become popular due to their low variability and ready availability. However, friction coefficients, which are critical to load transfer in uncemented implants, have rarely been compared between human and artificial bone, particularly for wet and dry conditions. In this study, the static and dynamic friction coefficients for four commercially used titanium surfaces (polished, Al(2)O(3) blasted, plasma sprayed, beaded) acting on the trabecular component of artificial bones (Sawbones) were compared to those for human trabecular bone. Artificial bones were tested in dry and wet conditions and normal interface stress was varied (0.25, 0.5, 1.0MPa). Friction coefficients were mostly lower for artificial bones than real bone. In particular, static friction coefficients for the dry polished surface were 20% of those for real bone and 42-61% for the dry beaded surface, with statistical significance (alpha<0.05). Less marked differences were observed for dynamic friction coefficients. Significant but non-systematic effects of normal stress or wet/dry condition on friction coefficients were observed within each surface type. These results indicate that the use of artificial bone models for pre-clinical implant testing that rely on interface load transfer with trabecular bone for mechanical integrity can be particularly sensitive to surface finish and lubrication conditions.     

Staphylococcus aureus adhesion to bone matrix and bone-associated biomaterials

Staphylococcus aureus is a frequent cause of orthopedic infections in humans. The bacterium expresses several adhesins that facilitate bacterial binding to the bone matrix and to bone implant biomaterials coated with host plasma constituents. The relevant S. aureus adhesins are termed microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and specific MSCRAMMs are involved in bone and joint infections.     

The influence of bone damage on press-fit mechanics

Press-fitting is used to anchor uncemented implants in bone. It relies in part on friction resistance to relative motion at the implant–bone interface to allow bone ingrowth and long-term stability. Frictional shear capacity is related to the interference fit of the implant and the roughness of its surface. It was hypothesised here that a rough implant could generate trabecular bone damage during implantation, which would reduce its stability. A device was constructed to simulate implantation by displacement of angled platens with varying surface finishes (polished, beaded and flaked) onto the surface of an embedded trabecular bone cube, to different nominal interferences. Push-in (implantation) and Pull-out forces were measured and micro-CT scans were made before and after testing to assess permanent bone deformation. Depth of permanent trabecular bone deformation (‘damage’), Pull-out force and Radial force all increased with implantation displacement and with implantation force, for all surface roughnesses. The proposed hypothesis was rejected, since primary stability did not decrease with trabecular bone damage. In fact, Pull-out force linearly increased with push-in force, independently of trabecular bone damage or implant surface. This similar behaviour for the different surfaces might be explained by the compaction of bone into the surfaces during push-in so that Pull-out resistance is governed by bone-on-bone, rather than implant surface-on-bone friction. The data suggest that maximum stability is achieved for the maximum implantation force possible (regardless of trabecular bone damage or surface roughness), but this must be limited to prevent periprosthetic cortical bone fracture, patient damage and component malpositioning.     

Nano rough micron patterned titanium for directing osteoblast morphology and adhesion

Previous studies have demonstrated greater functions ofosteoblasts (bone-forming cells) on nanophase compared with conventional metals. Nanophase metals possess a biologically inspired nanostructured surface that mimics the dimensions of constituent components in bone, including collagen and hydroxyapatite. Not only do these components possess dimensions on the nanoscale, they are aligned in a parallel manner creating a defined orientation in bone. To date, research has yet to evaluate the effect that organized nanosurface features can have on the interaction of osteoblasts with material surfaces. Therefore, to determine if surface orientation of features can mediate osteoblast adhesion and morphology, this study investigated osteoblast function on patterned titanium substrates containing alternating regions of micron rough and nano rough surfaces prepared by novel electron beam evaporation techniques. This study was also interested in determining whether or not the size of the patterned regions had an effect on osteoblast behavior and alignment. Results indicated early controlled osteoblast alignment on these patterned materials as well as greater osteoblast adhesion on the nano rough regions of these patterned substrates. Interestingly, decreasing the width of the nano rough regions (from 80 microm to 22 microm) on these patterned substrates resulted in a decreased number of osteoblasts adhering to these areas. Changes in the width of the nano rough regions also resulted in changes in osteoblast morphology, thus, suggesting there is an optimal pattern dimension that osteoblasts prefer. In summary, results of this study provided evidence that aligned nanophase metal features on the surface of titanium improved early osteoblast functions (morphology and adhesion) promising for their long term functions, criteria necessary to improve orthopedic implant efficacy.     

Modification of the Micro Arc-oxidized Ti Surface for Implant Applications

Surface treatments applied to titanium and its alloys for implant applications are important for the development of bio properties.In this study,first an oxide layer was formed on the surface of the titanium plate by micro arc oxidation,and then both calcium phosphate and calcium phosphate/chitosan accumulation were performed for different samples by the sol-gel method.FE-SEM/EDS examinations,XRD,FTIR and thermal analysis were performed for these micro arc-oxidized,cal-cium phosphate-coated and calcium phosphate/chitosan-coated surfaces.The surface roughnesses for these surfaces were measured between 10 μm and 100 μm,suitable for bone development on the surface.The effect of chitosan addition on the calcium phosphate-coated surface on apatite formation ability and antibacterial properties was investigated.Although the addition of chitosan slows down the formation of apatite,it ensured that the coating had antibacterial properties.The calcium phosphate/chitosan biocomposite obtained can be recommended for dental and orthopedic implants.     

Different titanium surfaces modulate the bone phenotype of SaOS-2 osteoblast-like cells

Commercially pure titanium implants presenting a relatively smooth, machined surface or a roughened endosseous surface show a large percentage of clinical success. Surface properties of dental implants seem to affect bone cells response. Implant topography appears to modulate cell growth and differentiation of osteoblasts affecting the bone healing around the titanium implant. The aim of the present study was to examine the effects of 1cm diameter and 1mm thick titanium disks on cellular morphology, adhesion and bone phenotypic expression of human osteoblast-like cells, SaOS-2. SaOS-2 cells were cultured on commercially 1 cm pure titanium disks with three different surface roughness: smooth (S), sandblasted (SB) and titanium plasma sprayed (TPS). Differences in the cellular morphology were found when they were grown on the three different surfaces. An uniform monolayer of cells recovered the S surface, while clusters of multilayered irregularly shaped cells were distributed on the rough SB and TPS surfaces. The adhesion of SaOS-2 cells, as measured after 3h of culture, was not affected by surface roughness. ECM components such as Collagen I (CoI), Fibronectin (FN), Vitronectin (VN) and Tenascin (TN) were secreted and organized only on the SB and TPS surfaces while they remained into the cytoplasm on the S surfaces. Osteopontin and BSP-II were largely detected on the SB and TPS surfaces, while only minimal production was observed on the S ones. These data show that titanium surface roughness affects bone differentiation of osteoblast like-cells, SaOS-2, indicating that surface properties may be able to modulate the osteoblast phenotype. These observations also suggest that the bone healing response around dental implants can be affected by surface topography.     

Early adhesion of human mesenchymal stem cells on TiO(2) surfaces studied by single-cell force spectroscopy measurements

Understanding the interactions involved in the adhesion of living cells on surfaces is essential in the field of tissue engineering and biomaterials. In this study, we investigate the early adhesion of living human mesenchymal stem cells (hMSCs) on flat titanium dioxide (TiO(2) ) and on nanoporous crystallized TiO(2) surfaces with the use of atomic force microscopy-based single-cell force spectroscopy measurements. The choice of the substrate surfaces was motivated by the fact that implants widely used in orthopaedic and dental surgery are made in Ti and its alloys. Nanoporous TiO(2) surfaces were produced by anodization of Ti surfaces. In a typical force spectroscopy experiment, one living hMSC, immobilized onto a fibronectine-functionalized tipless lever is brought in contact with the surface of interest for 30 s before being detached while recording force-distance curves. Adhesion of hMSCs on nanoporous TiO(2) substrates having inner pore diameter of 45 nm was lower by approximately 25% than on TiO(2) flat surfaces. Force-distance curves exhibited also force steps that can be related to the pulling of membrane tethers from the cell membrane. The mean force step was equal to 35 pN for a given speed independently of the substrate surface probed. The number of tethers observed was substrate dependent. Our results suggest that the strength of the initial adhesion between hMSCs and flat or nanoporous TiO(2) surfaces is driven by the adsorption of proteins deposited from serum in the culture media.     

Peri-implant bone remodeling after total hip replacement combined with systemic alendronate treatment: a finite element analysis

In order to decrease the peri-implant bone loss during the life-time of the implant, oral use of anti-osteoporosis drugs (like bisphosphonates) has been suggested. In this study, bone remodeling parameters identified from clinical trials of alendronate were used to simulate the effect of those drugs used after total hip arthroplasty on the peri-implant bone density. Results of the simulation show that the oral administrated drugs increase bone density around the implant and decreases, at the same time, the micromovements between the implant and the surrounding bone tissue. Incorporation of drug effect in numerical studies of bone remodeling is a promising tool especially to predetermine safe bisphosphonate doses that could be used with orthopedic implants.     

Enhanced osteoblast adhesion to drug-coated anodized nanotubular titanium surfaces

Current orthopedic implants have functional lifetimes of only 10-15 years due to a variety of reasons including infection, extensive inflammation, and overall poor osseointegration (or a lack of prolonged bonding of the implant to juxtaposed bone). To improve properties of titanium for orthopedic applications, this study anodized and subsequently coated titanium with drugs known to reduce infection (penicillin/streptomycin) and inflammation (dexamethasone) using simple physical adsorption and the deposition of such drugs from simulated body fluid (SBF). Results showed improved drug elution from anodized nanotubular titanium when drugs were coated in the presence of SBF for up to 3 days. For the first time, results also showed that the simple physical adsorption of both penicillin/streptomycin and dexamethasone on anodized nanotubular titanium improved osteoblast numbers after 2 days of culture compared to uncoated unanodized titanium. In addition, results showed that depositing such drugs in SBF on anodized titanium was a more efficient method to promote osteoblast numbers compared to physical adsorption for up to 2 days of culture. In addition, osteoblast numbers increased on anodized titanium coated with drugs in SBF for up to 2 days of culture compared to unanodized titanium. In summary, compared to unanodized titanium, this preliminary study provided unexpected evidence of greater osteoblast numbers on anodized titanium coated with either penicillin/streptomycin or dexamethasone using simple physical adsorption or when coated with SBF; results which suggest the need for further research on anodized titanium orthopedic implants possessing drug-eluting nanotubes.     

Peri-implant Bone Remodeling after Total Hip Replacement Combined with Systemic Alendronate Treatment: A Finite Element Analysis

In order to decrease the peri-implant bone loss during the life-time of the implant, oral use of anti-osteoporosis drugs (like bisphosphonates) has been suggested.

In this study, bone remodeling parameters identified from clinical trials of alendronate were used to simulate the effect of those drugs used after total hip arthroplasty on the peri-implant bone density. Results of the simulation show that the oral administrated drugs increase bone density around the implant and decreases, at the same time, the micromovements between the implant and the surrounding bone tissue.

Incorporation of drug effect in numerical studies of bone remodeling is a promising tool especially to predetermine safe bisphosphonate doses that could be used with orthopedic implants.     

Effect of surface roughness of Ti, Zr, and TiZr on apatite precipitation from simulated body fluid

Some of the critical properties for a successful orthopedic or dental implant material are its biocompatibility and bioactivity. Pure titanium (Ti) and zirconium (Zr) are widely accepted as biocompatible metals, due to their non-toxicity. While the bioactivity of Ti and some Ti alloys has been extensively investigated, there is still insufficient data for Zr and titanium-zirconium (TiZr) alloys. In the present study, the bioactivity, that is, the apatite forming ability on the alkali and heat treated surfaces of Ti, Zr, and TiZr alloy in simulated body fluid (SBF), was studied. In particular, the effect of the surface roughness characteristics on the bioactivity was evaluated for the first time. The results indicate that the pretreated Ti, Zr and TiZr alloy could form apatite coating on their surfaces. It should be noted that the surface roughness also critically affected the bioactivity of these pretreated metallic samples. A surface morphology with an average roughness of approximately 0.6 microm led to the fastest apatite formation on the metal surfaces. This apatite layer on the metal surface is expected to bond to the surrounding bones directly after implantation.