Microtextured Titanium Research Articles

Evaluation of Osteoblastic Differentiation Induced by Microtextured Titanium Surface Produced by Laser Metal Fusion 3D Printing

Evaluation of Osteoblastic Differentiation Induced by Microtextured Titanium Surface Produced by Laser Metal Fusion 3D Printing

Semaphorin 3A delivered by a rapidly polymerizing click hydrogel overcomes impaired implant osseointegration in a rat type 2 diabetes model.

Semaphorin 3A (sema3A) is an osteoprotective factor that enhances bone formation while inhibiting osteoclast bone resorption. It is produced by rat calvarial osteoblasts cultured on grit-blasted/acid-etched microtextured (SLA) titanium surfaces at higher levels than on tissue culture polystyrene, suggesting that it may improve performance of titanium implants in vivo, particularly in conditions characterized by compromised bone quality. To test this, we established a clinically relevant type 2 diabetes mellitus (T2DM) rat model and used a non-toxic click hydrogel that rapidly polymerizes in situ (GEL) to provide localized controlled delivery of sema3A. In vitro studies confirmed that sema3A released from GEL was biologically active, increasing osteoblast differentiation of a pre-osteoblast cell-line. Whereas increased sema3A production was not observed in T2DM calvarial osteoblasts cultured on SLA, exogenous sema3A enhanced surface-induced osteoblast differentiation, indicating that it would be a viable candidate for in vivo use. Delivery of sema3A either by GEL or by local injection to bone defects enhanced osseointegration of SLA implants in the T2DM rats. Trabecular bone mass and bone-to-implant contact were decreased in T2DM rats compared to normal rats; sema3A delivered locally improved both parameters. These findings suggest that reduced trabecular bone contributes to poor osseointegration in T2DM patients and support GEL as a promising treatment option for sustained release of therapeutic doses of sema3A. Moreover, using this clinically translatable T2DM model and developing a biocompatible, Cu-free click chemistry hydrogel platform for the non-invasive delivery of therapeutics has major implications for regenerative medicine as a whole. STATEMENT OF SIGNIFICANCE: Osseointegration is compromised in patients with poor bone quality due to conditions like type 2 diabetes mellitus (T2DM). Previously, we showed that semaphorin 3A (sema3A) production is increased when human bone marrow stromal cells are cultured on titanium substrates that support osseointegration in vivo, suggesting it may enhance peri-implant osteogenesis in diabetes. Here we established a spontaneously developing T2DM rat model with clinical translatability and used it to assess sema3A effectiveness. Sema3A was delivered to the implant site via a novel copper-free click hydrogel, which has minimal swelling behavior and superior rheological properties. Osseointegration was successfully restored, and enhanced compared to burst release through injections. This study provides scientific evidence for using sema3A to treat impaired osseointegration in T2DM patients.

Micro/nano-textured hierarchical titanium topography promotes exosome biogenesis and secretion to improve osseointegration

BackgroundMicro/nano-textured hierarchical titanium topography is more bioactive and biomimetic than smooth, micro-textured or nano-textured titanium topographies. Bone marrow mesenchymal stem cells (BMSCs) and exosomes derived from BMSCs play important roles in the osseointegration of titanium implants, but the effects and mechanisms of titanium topography on BMSCs-derived exosome secretion are still unclear. This study determined whether the secretion behavior of exosomes derived from BMSCs is differently affected by different titanium topographies both in vitro and in vivo.ResultsWe found that both micro/nanonet-textured hierarchical titanium topography and micro/nanotube-textured hierarchical titanium topography showed favorable roughness and hydrophilicity. These two micro/nano-textured hierarchical titanium topographies enhanced the spreading areas of BMSCs on the titanium surface with stronger promotion of BMSCs proliferation in vitro. Compared to micro-textured titanium topography, micro/nano-textured hierarchical titanium topography significantly enhanced osseointegration in vivo and promoted BMSCs to synthesize and transport exosomes and then release these exosomes into the extracellular environment both in vitro and in vivo. Moreover, micro/nanonet-textured hierarchical titanium topography promoted exosome secretion by upregulating RAB27B and SMPD3 gene expression and micro/nanotube-textured hierarchical titanium topography promoted exosome secretion due to the strongest enhancement in cell proliferation.ConclusionsThese findings provide evidence that micro/nano-textured hierarchical titanium topography promotes exosome biogenesis and extracellular secretion for enhanced osseointegration. Our findings also highlight that the optimized titanium topography can increase exosome secretion from BMSCs, which may promote osseointegration of titanium implants.

Characterization of a Macro- and Micro-Textured Titanium Grade 5 Alloy Surface Obtained by Etching Only without Sandblasting.

Our purpose was to physically characterize the surface, and the subsurface, of a macro- and micro-textured titanium grade 5 dental implant surface obtained by etching only, without sandblasting. The topography, surface roughness, as well as the surface structure and subsurface distribution of elements, were determined by scanning electronic microscopy (SEM), non-contact profilometry, X-ray diffraction (XRD), and a concentration profile performed by Auger electron spectroscopy (AES). The hydrogen concentration in the implants was measured; the ability to generate nanostructures when stored in deionized water was also investigated. Under SEM, the surface resembled a sandblasted and etched titanium surface with its typical macro- and micro-texture; roughness was moderate with average roughness (Sa) 1.29 µm. No titanium hydride was found at the implant surface and no enrichment of any alloying element was identified at the surface and subsurface. Hydrogen concentration was 79 ppm, within the normative tolerance (<130 ppm). After storage in water for 6 months, densely packed finger-like nanostructures were observed. The clinical advantage of this textured titanium alloy surface is that it displays the typical macro- and micro-features of a moderately rough sandblasted and etched (SLA) titanium surface without leaving behind any foreign sandblasting material.

Oxidation facilitated antimicrobial ability of laser micro-textured titanium alloy against gram-positive Staphylococcus aureus for biomedical applications

Micro-textures were engineered on titanium samples by a rapid, highly repeatable, and cost effective nanosecond laser texturing process. The controlled and repeatable micro-texture was effective in the reduction of adhesion and proliferation of Staphylococcus aureus. However, the adhesion and proliferation of human bone marrow mesenchymal stem cells were found unaffected. The authors observed time-delayed saturation of oxidation on laser micro-textured Ti6Al4V as compared to pristine material yielding a super-hydrophobic surface (contact angle of ∼158.2°). An in-house setup was designed to expose a three-dimensionally laser micro-textured Ti6Al4V sample to S. aureus-spiked nutrient broth for 24 h in a closed loop continuous flow system. The authors observed a 50% reduction of S. aureus adhesion and proliferation over the laser micro-textured surface compared to pristine samples. In vitro tests confirmed that blood coagulation remains largely unaffected, while protein adsorption was decreased by fourfold on the micro-textured surfaces, a desirable property for surgical tools. Nanosecond laser micro-textured surfaces were effective in reducing S. aureus adhesion and its proliferation due to phase evolution, oxidative transformation of Ti6Al4V, and reduction of surface potential. In addition, laser based micro-texturing is a nontoxic, environment friendly green process that is also a cost effective alternative to conventional surface modification techniques such as mechanical micromachining and chemical treatments.

Biophysical characterization of nanostructured TiO2 as a good substrate for hBM-MSC adhesion, growth and differentiation

Mesenchymal stem cells from human bone marrow (hBM-MSC) are widely utilized for clinical applications involving bone healing. In this context, their use has been often optimized in association to variously designed titanium substrates, being this material of great use in orthopaedic implants.According to recent findings, the ability of hBM-MSC to differentiate towards a specific lineage is not only driven by biochemical signals, but physical stimuli, such as rigidity or roughness of the substrate, can also support a commitment towards osteogenic differentiation. Moreover, the presence of features with defined dimensional scales, in particular nanometer-size, also proved to elicit specific biological effects. Here we evaluated the effectiveness of a nano-patterned titanium surface in sustaining hBM-MSC adhesion, growth and differentiation by means of a panel of biophysical tools: morphometry, electrophysiology, intracellular calcium measurements and immunocytochemistry. The results substantiate the idea that this micro-textured titanium dioxide is a good surface for growth and differentiation of hBM-MSC and it exhibits a stimulating action mainly in the initial period of differentiation. Moreover, the basal concentration of free cytosolic Calcium [Ca2+]i is confirmed to be a good hallmark of the hBM-MSC maturation stage. The study could provide relevant hints to help improving the biocompatibility and osteointegration potential of clinical titanium implants.

Bone Morphogenetic Protein 2 Alters Osteogenesis and Anti-Inflammatory Profiles of Mesenchymal Stem Cells Induced by Microtextured Titanium In Vitro.

Microtextured titanium (Ti) induces osteoblast differentiation of mesenchymal stem cells (MSCs) in the absence of exogenous osteogenic factors; and high-energy surface modifications speed healing of microrough Ti implants. Bone morphogenetic protein 2 (BMP2) is used clinically to improve peri-implant bone formation and osseointegration but can cause inflammation and bone-related complications. In this study, we determined whether BMP2 alters human MSC differentiation, apoptosis, and inflammatory factor production when grown on Ti implants with different surface properties. Human MSCs were cultured on Ti substrates (smooth [PT], sandblasted acid-etched [SLA], hydrophilic-SLA [modSLA]), or tissue culture polystyrene (TCPS). After 7 days, inflammatory mRNAs were measured by polymerase chain reaction array. In addition, 7-day cultures were treated with exogenous BMP2 and osteogenic differentiation and production of local factors, proinflammatory interleukins, and anti-inflammatory interleukins assessed. Finally, osteogenic markers and interleukins were measured in MSCs cultured for 48 h on BMP2 dip-coated SLA and modSLA surfaces. Expression of interleukins, chemokines, cytokines, and growth factors was affected by surface properties, particularly on modSLA. MSCs on Ti produced fewer resorptive and more osteogenic/anti-inflammatory factors than cells on TCPS. Addition of 100 ng/mL BMP2 not only increased differentiation but also increased proinflammatory and decreased anti-inflammatory/antiresorptive factors. Two hundred nanograms per milliliter BMP2 abolished osteogenesis and dramatically increased pro-osteoclastogenic factors. MSCs cultured on BMP2-dip-coated disks produced similar proinflammatory profiles with inhibited osteogenic differentiation and had increased apoptotic markers at the highest doses. MSCs underwent osteogenesis and regulated inflammatory cytokines on microtextured Ti. Exogenous BMP2 inhibited MSC differentiation and stimulated a dose-dependent proinflammatory and apoptotic response. Use of BMP2 with microtextured metal implants may increase inflammation and possibly delay bone formation dependent on dose, suggesting that application of BMP2 clinically during implant insertion may need to be reevaluated.

Deposition of Ultrathin Nano-Hydroxyapatite Films on Laser Micro-Textured Titanium Surfaces to Prepare a Multiscale Surface Topography for Improved Surface Wettability/Energy.

The primary aim of this study was to analyse the correlation between topographical features and chemical composition with the changes in wettability and the surface free energy of microstructured titanium (Ti) surfaces. Periodic microscale structures on the surface of Ti substrates were fabricated via direct laser interference patterning (DLIP). Radio-frequency magnetron sputter deposition of ultrathin nanostructured hydroxyapatite (HA) films was used to form an additional nanoscale grain morphology on the microscale-structured Ti surfaces to generate multiscale surface structures. The surface characteristics were evaluated using atomic force microscopy and contact angle and surface free energy measurements. The structure and phase composition of the HA films were investigated using X-ray diffraction. The HA-coated periodic microscale structured Ti substrates exhibited a significantly lower water contact angle and a larger surface free energy compared with the uncoated Ti substrates. Control over the wettability and surface free energy was achieved using Ti substrates structured via the DLIP technique followed by the deposition of a nanostructured HA coating, which resulted in the changes in surface chemistry and the formation of multiscale surface topography on the nano- and microscale.

Enhanced photocatalytic activity of micro/nano textured TiO2 surfaces prepared by sandblasting/acid-etching/anodizing process

In this study, micro-textured titanium surfaces were created by sandblasting and acid-etching methods. Nanostructured titanium dioxide (TiO2) films were synthesized on the micro-textured titanium surfaces by anodizing method. Subsequently, anodized (A), acid-etched/anodized (EA), sandblasted/anodized (SA) and sandblasted/acid-etched/anodized (SEA) samples were fabricated. Structural and morphological properties of the prepared samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and atomic force microscopy (AFM). Furthermore, surface roughness and wettability of the samples were evaluated by means of profilometry and contact angle measurement systems, respectively. Finally, the photocatalytic activities of the samples were determined by using aqueous methylene blue (MB) solutions under a specific UV light illumination for different periods of time. The obtained results pointed out that sandblasting and acid-etching treatment improved the surface roughness of the samples significantly. The photocatalytic experimental results showed that micro-textured surface accelerated the degradation of MB, if compared to samples which are anodized without any of the regarding surface treatments. To sum up, sandblasted/acid-etched/anodized titanium surfaces exhibited the highest wettability, surface roughness and photocatalytic activity among the samples. The present study may shed new light on the modification of titanium surfaces.

Human Mesenchymal Stem Cell Morphology and Migration on Microtextured Titanium

The implant used in spinal fusion procedures is an essential component to achieving successful arthrodesis. At the cellular level, the implant impacts healing and fusion through a series of steps: first, mesenchymal stem cells (MSCs) need to adhere and proliferate to cover the implant; second, the MSCs must differentiate into osteoblasts; third, the osteoid matrix produced by the osteoblasts needs to generate new bone tissue, thoroughly integrating the implant with the vertebrate above and below. Previous research has demonstrated that microtextured titanium is advantageous over smooth titanium and PEEK implants for both promoting osteogenic differentiation and integrating with host bone tissue; however, no investigation to date has examined the early morphology and migration of MSCs on these surfaces. This study details cell spreading and morphology changes over 24 h, rate and directionality of migration 6–18 h post-seeding, differentiation markers at 10 days, and the long-term morphology of MSCs at 7 days, on microtextured, acid-etched titanium (endoskeleton), smooth titanium, and smooth PEEK surfaces. The results demonstrate that in all metrics, the two titanium surfaces outperformed the PEEK surface. Furthermore, the rough acid-etched titanium surface presented the most favorable overall results, demonstrating the random migration needed to efficiently cover a surface in addition to morphologies consistent with osteoblasts and preosteoblasts.

Ti Nano-nodular Structuring for Bone Integration and Regeneration

Nanostructuring technology has been proven to create unique biological properties in various biomaterials. Here we present a discovered phenomenon of titanium nano-nodular self-assembly that occurs during physical vapor depositions of titanium (Ti) onto specifically conditioned micro-textured titanium surfaces, and test a hypothesis that the Ti nanostructure has the potential to enhance bone-titanium integration. The nanostructure creation effectively provided geometrical undercut and increased the surface area by up to 40% compared with the acid-etched surface with microtopography. Depending on the size control, the nano-nodules can be added without smearing the existing micro-texture, creating a nano-micro-hybrid architecture. Titanium implants with 560-nm nano-nodules produced 3.1 times greater strength of osseointegration than those with an acid-etched surface in a rat femur model. The discovered titanium nano-nodular self-structuring has been proven feasible on biocompatible materials other than titanium, offering new avenues for the development of implant surfaces and other implantable materials for better bone-generative and -regenerative potential.

Differential regulation of osteoblasts by substrate microstructural features

Microtextured titanium implant surfaces enhance bone formation in vivo and osteoblast phenotypic expression in vitro, but the mechanisms are not understood. To determine the roles of specific microarchitectural features in modulating osteoblast behavior, we used Ti surfaces prepared by electrochemical micromachining as substrates for MG63 osteoblast-like cell culture. Cell response was compared to tissue culture plastic, a sand-blasted with large grit and acid-etched surface with defined mixed microtopography (SLA), polished Ti surfaces, and polished surfaces electrochemically machined through a photoresist pattern to produce cavities with 100, 30 and 10μm diameters arranged so that the ratio of the microscopic-scale area of the cavities versus the microscopic-scale area of the flat region between the cavities was equal to 1 or 6. Microstructured disks were acid-etched, producing overall sub-micron-scale roughness (Ra=0.7μm). Cell number, differentiation (alkaline phosphatase; osteocalcin) and local factor levels (TGF-β1; PGE2) varied with microarchitecture. 100μm cavities favored osteoblast attachment and growth, the sub-micron-scale etch enhanced differentiation and TGF-β1 production, whereas PGE2 depended on cavity dimensions but not the sub-micron-scale roughness.