Loading deproteinized bovine bone with strontium enhances bone regeneration in rat calvarial critical size defects.

OBJECTIVE: To evaluate the effect of grafting with strontium (Sr)-loaded deproteinized bovine bone (DBB) on bone healing in calvarial critical size defects (CSD) in rats. MATERIAL AND METHODS: Two circular bone defects (5 mm in diameter) were created in the calvaria of 42 rats. One of the defects, randomly chosen, was grafted with (a) DBB, (b) DBB loaded with 19.6 µg/g of Sr (DBB/Sr1), or (c) DBB loaded with 98.1 µg/g of Sr (DBB/Sr2). The other defect was left empty as negative control. Groups of seven animals from each of the groups were euthanized 15 and 60 days post-op. Bone healing in the CSD was evaluated by micro-CT and histology/histomorphometry and immunohistochemistry. RESULTS: DBB/Sr2-grafted sites showed statistically significantly shorter radiographic residual defect length compared with DBB/Sr1- and DBB-grafted sites, and with empty controls at 60 days. Further, the amount of new bone formation in the DBB/Sr1- and DBB/Sr2-grafted sites was significantly higher compared with that in the DBB-grafted sites at 60 days. A larger number of DBB/Sr1- and DBB/Sr2-grafted sites presented with no- or only limited to mild inflammation, compared with the DBB-grafted sites, especially at 60 days. Higher expression of osteocalcin was observed in DBB/Sr1- and DBB/Sr2-grafted sites as compared to DBB-grafted sites. CONCLUSION: Grafting with Sr-loaded DBB enhanced bone formation in CSD in rats, when compared with grafting with non-loaded DBB. CLINICAL RELEVANCE: Grafting with Sr-loaded DBB may enhance bone formation in bone defects.

Biofunctional surface patterns retaining activity after exposure to whole blood.

Biofunctional surface patterns capable of resisting nonspecific bioadsorption while retaining bioactivity play crucial roles in the advancement of life science and biomedical technologies. The currently available functional surface coatings suffer from a high level of nonspecific surface adsorption of proteins under biologically challenging conditions, leading to a loss of activity in functional moieties over time. In this study, the recently discovered facile method of temperature-induced polyelectrolyte (TIP) grafting has been used to graft two biofunctional variants (biotin and nitrilotriacetic acid, NTA) of poly(l-lysine)-grafted PEG (PLL-g-PEG) onto a titanium surface. A significant increase in the polymer adsorption was observed from the TIP-grafted surfaces assembled at 80 °C, compared to the polymer surfaces assembled at ambient temperature (20 °C). These functional PLL-g-PEG surfaces were subsequently incubated in whole human blood continuously for up to 7 days, and the TIP-grafted surfaces achieved close-to-zero nonspecific protein adsorption, as confirmed by ultrasensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). To test the maintenance of the bioactivity of the biotin and NTA moieties, submicrometer-scale mono- (biotin) and bi- (biotin/NTA) functional surface chemical patterns were fabricated via two-step TIP grafting using colloidal lithography (CL), preincubated in blood for up to 7 days and sequentially exposed to streptavidin and Ni(2+)-histidine-tagged calmodulin. The fluorescence microscopy studies revealed that the PLL-g-PEG-NTA and -biotin surfaces grafted from the TIP method were still capable of recognizing the corresponding affinity proteins for up to 1 and 7 days of preincubation in blood, respectively. These results highlight the bioresistant robustness realized by the facile TIP grafting method, which in turn preserves the activities of biofunctional moieties over a prolonged period in whole blood.

Assessing the osseointegration potential of a strontium releasing nanostructured titanium oxide surface: A biomechanical study in the rabbit tibia plateau model.

OBJECTIVES: To investigate the impact of a Ti-Sr-O technology, applied to either a turned surface or an SLA surface, on the mechanical robustness of osseointegration, benchmarked against the SLActive surface. MATERIAL AND METHODS: Ti discs (6.25-mm-diameter and 2-mm-thick) with three different surfaces were inserted on the proximal-anterior part of the tibial plateau of adult Swedish loop rabbits: (I) turned surface modified with Ti-Sr-O (turned + Ti-Sr-O), (II) SLA surface modified with Ti-Sr-O (SLA + Ti-Sr-O), and (III) SLActive surface (SLActive). Following a healing period of 2 weeks and 4 weeks, the pull-out (PO) force needed to detach the discs from the bone was assessed, as a surrogate of osseointegration. RESULTS: The SLActive surface exhibited statistically significant higher median PO forces, compared with the SLA + Ti-Sr-O surfaces at both 2- and 4 weeks post-op (p > .05). In this study, no single turned + Ti-Sr-O surface disk was integrated. CONCLUSIONS: The tested Ti-Sr-O technology failed to enhance osseointegration; however, this finding may be related to the inappropriateness of the rabbit tibia plateau model for assessing third-generation implant surface technologies, due to the limited diffusion and clearance at the disk-bone interface.

Surfaces Coated with Polymer Brushes Work as Carriers for Histidine Ammonia Lyase.

The immobilization of enzymes on solid supports is an important challenge in biotechnology and biomedicine. In contrast to other methods, enzyme deposition in polymer brushes offers the benefit of high protein loading that preserves enzymatic activity in part due to the hydrated 3D environment that is available within the brush structure. The authors equipped planar and colloidal silica surfaces with poly(2-(diethylamino)ethyl methacrylate)-based brushes to immobilize Thermoplasma acidophilum histidine ammonia lyase, and analyzed the amount and activity of the immobilized enzyme. The poly(2-(diethylamino)ethyl methacrylate) brushes are attached to the solid silica supports either via a "grafting-to" or a "grafting-from" method. It is found that the grafting-from method results in higher amounts of deposited polymer and, consequently, higher amounts of Thermoplasma acidophilum histidine ammonia lyase. All polymer brush-modified surfaces show preserved catalytic activity of the deposited Thermoplasma acidophilum histidine ammonia lyase. However, immobilizing the enzyme in polymer brushes using the grafting-from method resulted in twice the enzymatic activity from the grafting-to approach, illustrating a successful enzyme deposition on a solid support.

Dopamine-assisted rapid fabrication of nanoscale protein arrays by colloidal lithography.

The development of cost-effective methodologies for the precise nanometer-scale positioning of biomolecules permits the low-cost production of various biofunctional devices for a range of biomedical and nanotechnological applications. By combining colloidal lithography and the mussel-inspired multifunctional polydopamine coating, we present a novel parallel benchtop method that allows rapid nanoscale patterning of proteins without the need for electrically powered equipment in the fabrication process. The PDA-immobilized binary nanopattern consisting of BSA surrounded by PLL-g-PEG is fabricated over a large area, and the integrity of the pattern is confirmed using AFM and FM.

Temperature-induced ultradense PEG polyelectrolyte surface grafting provides effective long-term bioresistance against mammalian cells, serum, and whole blood.

We report a facile method of generating ultradense poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) surface by using high temperature alone, which in turn provides dramatic improvement in resisting nonspecific bioadsorption. X-ray photoelectron spectroscopy (XPS) revealed that the surface graft density increased ~4 times higher on the surface prepared at 80 °C compared to 20 °C. The studies from small-angle X-ray scattering (SAXS) and the effect of varying ionic strength during/post assemblies at 20 and 80 °C indicated that the "cloud point grafting effect" is not the cause for obtaining high density grafting. Stringent long-term bioresistance tests have been conducted and the temperature-induced PLL-g-PEG surfaces have achieved (1) zero mammalian cell adsorption/migration for up to 36 days and (2) extremely close-to-zero protein adsorptions have been observed even after 36 days in 10% serum media and 24 h in whole blood within the ultrasensitive detection limit of time-of-flight secondary ion mass spectrometry (ToF-SIMS).

Roughness of glancing angle deposited titanium thin films: an experimental and computational study.

The characterization of roughness at the nanoscale by the means of atomic force microscopy (AFM) was performed on high aspect ratio glancing angle deposited titanium thin films. With the use of scanning electron microscopy as well as x-ray photoelectron spectroscopy, it was shown that the AFM measurements gave rise to incorrect roughness values for the films consisting of the highest aspect ratio structures. By correcting for this experimental artefact, the difference between the saturated roughness value of a film grown with conventional physical vapour deposition and films grown with a glancing angle of deposition was shown to behave as a power law function of the deposition angle, with a saturated roughness exponent of κ = 7.1 ± 0.2. This power law scaling was confirmed by three-dimensional molecular dynamics simulations of glancing angle deposition, where the saturated roughness exponent was calculated to κ = 6.7 ± 0.4.

Local Release of Strontium from Sputter-Deposited Coatings at Implants Increases the Strontium-to-Calcium Ratio in Peri-implant Bone.

It is well known that strontium (Sr) has a significant effect on peri-implant bone healing when administered systemically. Due to the risk of adverse effects of such treatments, new routes focusing on the local, sustained release of Sr from bone-implant contact surfaces have been explored, with success in in vivo experiments. However, the increase of Sr concentrations in the peri-implant bone has not been described in depth yet. Here, we show that a local, sustained Sr release from Ti-Sr-O physical vapor deposition (PVD) coatings by magnetron sputter coating increases the Sr/Ca ratio close to the implant in a rabbit model and that the Sr/Ca background level is reached approximately 500 µm from the implant.

Post-treatments of polydopamine coatings influence cellular response.

Polydopamine (PDA) is the final oxidation product of dopamine or other catecholamines. Since the first reports of PDA coatings starting around 2007, these coatings have been widely studied as a versatile and inexpensive one-step coating option for biomaterial functionalization. The coating attach to a wide range of materials and can subsequently be modified with biomolecules or nanoparticles. However, as a strong candidate for biomaterial research and even clinical use, it is important to unravel the changes in physico-chemical properties and the cell-PDA interaction as a function of heat sterilization procedures and shelf storage periods. Four groups were examined in this study: titanium (Ti), PDA-coated Ti samples and PDA-coated Ti samples either stored for up to two weeks at room temperature or heated at 121 °C for 24 h, respectively. We used X-ray Photoelectron Spectroscopy (XPS), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Water contact angle (WCA) to characterize chemical composition and surface properties of the groups. Cell adhesion and proliferation was examined by three different cell types: human primary dermal fibroblasts (hDF), human epidermal keratinocytes (HaCaTs) and a murine preosteoblastic cell line (MC3T3-E1), respectively. Cells were cultured on PDA coated samples for 4 h, 3 days and 5 days. Both thermal treatment of PDA at 121℃ for 24 h and storage of the samples for 2 weeks increased the amount of quinone groups at the surface and decreased the amount of primary amine groups as detected by XPS and ToF-SIMS. Even though these surface reactions increased the WCA of the PDA coating, we found that the post-treatments increased cell proliferation for both hDFs, HaCaTs and MC3T3-E1 s as compared to pristine PDA. This emphasizes the importance of post-treatment and shelf-time for PDA coatings.

Strontium enhances proliferation and osteogenic behavior of bone marrow stromal cells of mesenchymal and ectomesenchymal origins in vitro.

Obejective: To investigate the effect of increasing Strontium (Sr) concentrations on the growth and osteogenic behavior of human bone marrow stromal cells (BMSCs) from mesenchymal (i.e., fibula) and ectomesenchymal (i.e., mandible) embryonic origins. Materials and methods: Fibula and mandible BMSCs were cultured in media without (Ctrl) or with Sr in four diverse concentrations: Sr1, 11.3 × 10-3 mg/L, human seric physiological level; Sr2, 13 mg/L, human seric level after strontium ranelate treatment; Sr3, 130 mg/L, and Sr4, 360 mg/L. Proliferation rate (1, 3, and 7 days), osteogenic behavior (alkaline phosphatase [ALP] activity, 7 and 14 days; expression of osteogenic genes (ALP, osteopontin, and osteocalcin at 7, 14, and 21 days), and formation of mineralized nodules (14 and 21 days) of the BMSCs were assessed. Data was compared group- and period-wise using analysis of variance tests. Results: Fibula and mandible BMSCs cultured with Sr4 showed increased proliferation rate, and osteocalcin and osteopontin gene expression together with more evident formation of mineralized nodules, compared all other Sr concentrations. For both cell populations, Sr4 led to lower ALP activity, and ALP gene expression, compared with the other Sr concentrations. Conclusion: BMSCs from mesenchymal (i.e., fibula) and ectomesenchymal (i.e., mandible) embryonic origins showed increased cellular proliferation and osteogenic behavior when cultured with Sr4, in vitro.

Extracellular matrix remodelling during cell adhesion monitored by the quartz crystal microbalance.

A cell's ability to remodel adsorbed protein layers on surfaces is influenced by the nature of the protein layer itself. Remodelling is often required to accomplish cellular adhesion and extracellular matrix formation which forms the basis for cell spreading, increased adhesion and expression of different phenotypes. The adhesion of NIH3T3 (EGFP) fibroblasts to serum protein (albumin or fibronectin) precoated tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces was examined using the quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy. The cells were either untreated or treated with cycloheximide to examine the contribution of endogenous protein production during cell adhesion to the QCM-D response over a period of 2h. Following adsorption of albumin onto Ta and PS(ox) there was no difference detected between the response to seeding untreated and cycloheximide treated cells. The QCM-D was able to detect differences in the untreated cellular responses to fibronectin versus serum precoated Ta and PS(ox) substrates, while cycloheximide treatment of the cells produced the same QCM-D response for fibronectin and serum precoatings on each of the materials. This confirmed that the process of matrix remodelling by the cells is dependent on the underlying substrate and the preadsorbed proteins and that the QCM-D response is dominated by changes in the underlying protein layer. Changes in dissipation correspond to the development of the actin cytoskeleton as visualised by actin staining.

Surface chemistry, substrate, and topography guide the behavior of human articular chondrocytes cultured in vitro.

Understanding the behavior of chondrocytes in contact with artificial culture surfaces is becoming increasingly important in attaining appropriate ex vivo culture conditions of chondrocytes in cartilage regeneration. Chondrocyte transplantation-based cartilage repair requires efficiently expanded chondrocytes, and the culture surface plays an important role in guiding the behavior of the cell. Micro- and nano-engineered surfaces make it possible to modulate cell behavior. We hypothesized that the combined influence of topography, substrate, and surface chemistry may affect the chondrocyte culturing in terms of proliferation and phenotypic means. Human chondrocytes were cultured on polystyrene fabricated microstructures, flat polydimethylsiloxane (PDMS), or polystyrene treated with fibronectin or oxygen plasma and cultured for 1, 4, 7, and 10 days. The behavior of chondrocytes was evaluated by proliferation, viability, chondrogenic gene expression, and cell morphology. Contrary to our hypothesis, microstructures in polystyrene did not significantly influence the behavior of chondrocytes neither under normoxic- nor hypoxic conditions. However, changes in the substrate stiffness and surface chemistry were found to influence cell viability, gene expression, and morphology of human chondrocytes. Oxygen plasma treatment was the most important parameter followed by the softer substrate type PDMS. The findings indicate the culture of human chondrocytes on softer substratum and surface activation by oxygen plasma may prevent dedifferentiation and may improve chondrocyte transplantation-based cartilage repair. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2805-2816, 2018.

Comparing the effect of strontium-functionalized and fluoride-modified surfaces on early osseointegration.

BACKGROUND: Studies have shown that medical devices comprising strontium contribute to bone healing and osseointegration. The aim of this study was to evaluate the in vivo performance of surface-functionalized implants (Ti-Sr-O) showing predictable release characteristics of strontium and compare it to performance a commercially available fluoride-modified surface. METHODS: Ti-Sr-O functionalized, fluoride-modified,  Grade 4 titanium implants were inserted in the femoral condyle of adult male New Zealand white rabbits. Atomic absorption spectrometry (AAS) was utilized to monitor strontium blood serum levels. Two weeks after insertion, histomorphometric evaluation was performed with respect to bone-to-implant contact (BIC%) and bone formation (BF%) using defined regions of interest. RESULTS: Mean values for BIC% showed a comparable degree of osseointegration for Ti-Sr-O and the fluoride-modified surface, while BF% revealed a significant difference in increased BF with Ti-Sr-O. AAS measurements did not indicate any influence of the Ti-Sr-O modified implants on the strontium blood serum concentrations. CONCLUSIONS: Within the limitations of this study, it was shown that the Ti-Sr-O coating, with sustained release characteristics of strontium, enhanced bone apposition and, thus, could find practical applications, e.g., within the field of medical implantology.

Effect of strontium surface-functionalized implants on early and late osseointegration: A histological, spectrometric and tomographic evaluation.

Numerous in vivo, in vitro and clinical studies report on beneficial effects of strontium with respect to increased bone growth. Based on this knowledge the aim of this study was to evaluate early and late osseointegration stages of functionalized titanium implants showing sustained release of strontium (Sr) and further investigate its potential systemic effect. Strontium functionalized (Ti-Sr-O) and Grade 4 (Control) titanium implants were inserted in the femoral condyle of New Zealand White rabbits. The Ti-Sr-O coating was characterized using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectrometry (EDX) for structure, coating thickness and chemical composition. Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) was used to evaluate released strontium in vitro while Atomic Absorption Spectrometry (AAS) was utilized to monitor serum levels of strontium and calcium. Additionally, histological and tomographic analysis of bone-to-implant contact (BIC%) and bone formation (BF%) was performed, following implantation periods of two or twelve weeks, respectively. Median values for BIC% for Ti-Sr-O revealed significant differences within the two- and twelve-week observation periods, while exceeding BF% was discovered especially after twelve weeks when performing the histological evaluation. The results from the micro-computed tomography (µ-CT) showed no significant differences, when comparing the experimental groups. AAS measurements did not indicate a systemic effect by the local strontium release. Within the limitations of the study, it was shown that a Ti-Sr-O coating with sustained release characteristics of strontium, accelerates bone apposition and represents a potential potent surface modification for endosseous medical implant devices. STATEMENT OF SIGNIFICANCE: This study presents first data with respect to early and late in vivo response on a strontium functionalized titanium surface comprising a nanotopography manufactured by a magnetron sputtering process. We investigated different osseointegration stages of screw-shaped implants with dental implant geometries in a rabbit femur model observing beneficial effects of the functionalized surface on bone-to-implant contact and bone formation caused by tailored release of the bone anabolic strontium. Histomorphometrical data revealed that a functionalized titanium surface with controlled liberation of strontium accelerates osseointegration while spectrometry measurements did not indicate a potential systemic effect of this osteoinductive agent and could thus have impact on modifications of medical implant devices.

Monitoring cell adhesion on tantalum and oxidised polystyrene using a quartz crystal microbalance with dissipation.

The quartz crystal microbalance with dissipation (QCM-D) (Q-Sense AB, Sweden) has been established as a useful tool for evaluating interactions between various biological and non-biological systems, and there has been increasing interest in using the QCM-D technique for cell monitoring applications. This study investigated the potential of the QCM-D to characterise the initial adhesion and spreading of cells in contact with protein precoated biocompatible surfaces. The QCM-D technique is attractive for monitoring cell adhesion and spreading as it allows in situ real-time measurements. The adhesion of NIH3T3 (EGFP) fibroblasts to tantalum (Ta) and oxidised polystyrene (PS(ox)) surfaces precoated with serum proteins was examined using the QCM-D for a period of either 2 or 4 h. Time-lapse photography was performed at 30 min intervals to visually examine cell adhesion and spreading in order to relate cell morphology to the QCM-D response. Following adsorption of albumin, fibronectin or newborn calf serum onto the surfaces, QCM-D measurements showed that cells adhered and spread on the fibronectin and serum coated surfaces, while few cells adhered to the albumin coated surfaces. Cells adhered to albumin coated surfaces had a rounded morphology. The responses to fibronectin and serum precoated surfaces were quite different for each of the underlying substrates indicating that the process of cell adhesion and spreading elicits different responses depending on both the protein coating composition and the influence of the underlying substrate. The different response may be due to extracellular matrix remodelling as well as cytoskeletal changes. Frequency (f) and dissipation (D) changes associated with cell adhesion were less than would be expected from the Sauerbrey relation due to the viscoelastic properties of the cells.

QCM-D studies of attachment and differential spreading of pre-osteoblastic cells on Ta and Cr surfaces.

The quartz crystal microbalance with dissipation (QCM-D) technique was employed to characterize initial cell adhesion in terms of attachment and spreading of pre-osteoblastic MC3T3-E1 cells on Ta and Cr surfaces. Evaluation of initial cell adhesion established a correlation between input cell number and the shifts in frequency (f) and dissipation (D). The f-shift was found to be much larger in serum-free medium as compared to a medium including serum; hence, initial cell adhesion was subsequently evaluated in serum-free medium. During the first hour of adhesion, we found a positive correlation between the QCM-D f-shift and the average area of the spread cells, as measured by cryo-scanning electron microscopy (cryo-SEM). Finally, the QCM-D technique was used to study cell adhesion on different metal oxide surfaces. Initial cell adhesion on Ta was found to induce a larger f-shift as compared to Cr, indicating larger spreading of cells on Ta. Cryo-SEM data confirmed that spreading of cells on Cr was on average only two-thirds the spreading on Ta. Our results demonstrate that the QCM-D technique is a versatile technique to quickly distinguish initial cell-surface interactions on different biomaterials.

Bone regenerating effect of surface-functionalized titanium implants with sustained-release characteristics of strontium in ovariectomized rats.

Since strontium (Sr) is known for its anabolic and anticatabolic effect on bone, research has been focused on its potential impact on osseointegration. The objective of this study was to investigate the performance of nanotopographic implants with a Sr-functionalized titanium (Ti) coating (Ti-Sr-O) with respect to osseointegration in osteoporotic bone. The trial was designed to examine the effect of sustained-release characteristics of Sr in poor-quality bone. Three Ti-Sr-O groups, which differed from each other in coating thickness, Sr contents, and Sr release, were examined. These were prepared by a magnetron sputtering process and compared to uncoated grade 4 Ti. Composition, morphology, and mechanical stability of the coatings were analyzed, and Sr release data were gained from in vitro washout experiments. In vivo investigation was carried out in an osteoporotic rat model and analyzed histologically, 6 weeks and 12 weeks after implantation. Median values of bone-to-implant contact and new bone formation after 6 weeks were found to be 84.7% and 54.9% (best performing Sr group) as compared to 65.2% and 23.8% (grade 4 Ti reference), respectively. The 12-week observation period revealed 84.3% and 56.5% (best performing Sr group) and 81.3% and 39.4% (grade 4 Ti reference), respectively, for the same measurements. The increase in new bone formation was found to correlate with the amount of Sr released in vitro. The results indicate that sputtered nanostructured Ti-Sr-O coatings showed sustained release of Sr and accelerate osseointegration even in poor-quality bone, and thus, may have impact on practical applications for medical implants.

Combinatorial Biomolecular Nanopatterning for High-Throughput Screening of Stem-Cell Behavior.

A novel combinatorial biomolecular nanopatterning method is reported, in which multiple biomolecular ligands can be patterned in multiple nanoscale dimensions on a single surface. The applicability of the combinatorial platform toward cell-biology applications is demonstrated by screening the adhesion behavior of a population of human dental pulp stem cell (hDPSC) on 64 combinations of nanopatterned extracellular matrix (ECM) proteins in parallel.

Modulation of human mesenchymal stem cell behavior on ordered tantalum nanotopographies fabricated using colloidal lithography and glancing angle deposition.

Ordered surface nanostructures have attracted much attention in biotechnology and biomedical engineering because of their potential to modulate cell-surface interactions in a controllable manner. However, the ability to fabricate large area ordered nanostructures is limited because of high costs and low speed of fabrication. Here, we have fabricated ordered nanostructures with large surface areas (1.5 × 1.5 cm(2)) using a combination of facile techniques including colloidal self-assembly, colloidal lithography and glancing angle deposition (GLAD). Polystyrene (722 nm) colloids were self-assembled into a hexagonally close-packed (hcp) crystal array at the water-air interface, transferred on a biocompatible tantalum (Ta) surface and used as a mask to generate an ordered Ta pattern. The Ta was deposited by sputter coating through the crystal mask creating approximately 60-nm-high feature sizes. The feature size was further increased by approximately 200-nm-height respectively using GLAD, resulting in the fabrication of four different surfaces (FLAT, Ta60, GLAD100, and GLAD200). Cell adhesion, proliferation, and osteogenic differentiation of primary human adipose-derived stem cells (hADSCs) were studied on these ordered nanostructures for up to 2 weeks. Our results suggested that cell spreading, focal adhesion formation, and filopodia extension of hADSCs were inhibited on the GLAD surfaces, while the growth rate was similar between each surface. Immunostaining for type I collagen (COL1) and osteocalcin (OC) showed that there was higher osteogenic components deposited on the GLAD surfaces compared to the Ta60 and FLAT surfaces after 1 week of osteogenic culture. After 2 weeks of osteogenic culture, alkaline phosphatase (ALP) activity and the amount of calcium was higher on the GLAD surfaces. In addition, osteoblast-like cells were confluent on Ta60 and FLAT surfaces, whereas the GLAD surfaces were not fully covered suggesting that the cell-cell interactions are stronger than cell-substrate interactions on GLAD surfaces. Visible extracellular matrix deposits decorated the porous surface can be found on the GLAD surfaces. Depth profiling of surface components using a new Ar cluster source and X-ray photoelectron spectroscopy (XPS) showed that deposited extracellular matrix on GLAD surfaces is rich in nitrogen. The fabricated ordered surface nanotopographies have potential to be applied in diverse fields, and demonstrate that the behavior of human stem cells can be directed on these ordered nanotopographies, providing new knowledge for applications in biomaterials and tissue engineering.

Low-aspect ratio nanopatterns on bioinert alumina influence the response and morphology of osteoblast-like cells.

Topographical features on the nanometer scale are known to influence cellular behavior. The response of specific cell types to various types of surface structures is currently still being investigated. Alumina ceramics play an important role as biomaterials, e.g., in medical and dental applications. In this study, we investigated the influence of nanoscale surface features with low aspect ratio (< 0.1) on the response of osteoblast-like MG-63 cells. To this end, low-energy ion irradiation was employed to produce shallow nanoscale ripple patterns on Al2O3(0001) surfaces with lateral periodicities of 24 nm and 179 nm and heights of only 0.7 and 11.5 nm, respectively. The nanopatterning was found to increase the proliferation of MG-63 cells and may lead to pseudopodia alignment along the ripples. Furthermore, focal adhesion behavior and cell morphology were analyzed. We found that MG-63 cells are able to recognize surface nanopatterns with extremely low vertical variations of less than 1 nm. In conclusion, it is shown that surface topography in the sub-nm range significantly influences the response of osteoblast-like cells.