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The aim of the study was to investigate cell adhesion to micro-structured titanium. Osteoblastic MC3T3 cells were cultured on smooth (P) or sand-blasted/acid-etched (SLA) titanium discs and were observed at scanning electron microscope/focused ion beam (SEM/FIB). Myosin II and actin microfilaments were labelled for epifluorescence microscopy. FIB revealed that cell adhesion initiated centrally and expanded to the cell periphery and that cells attached on the substrate by bridging over the titanium irregularities and adhering mostly on surface peaks. Gaps were visible between concave areas and cytoplasm and areas around ridges represented preferred attachment points for cells. A different myosin distribution was observed between samples and myosin inhibition affected cell responses. Taken together our data indicate that cells attach on micro-rough titanium by bridging over its irregularities. This is likely mediated by myosin II, whose distribution is altered in cells on SLA discs.

The aim of the present study was to investigate how the enrichment of chitosan films with anti-fibronectin aptamers could enhance scaffold colonization by osteoblasts, by improving their adhesion and accelerating their proliferation. Chitosan discs were enriched with excess of anti-fibronectin aptamer. Aptamer adsorption on chitosan was monitored by measuring aptamer concentration in the supernatant by spectrophotometry, as well as its release, while functionalization was confirmed by labelling aptamers with a DNA intercalating dye. Chitosan samples were then characterized morphologically with atomic force microscopy and physically with contact angle measurement. Chitosan enrichment with fibronectin was then investigated by immunofluorescence and Bradford assay. 2% chitosan discs were then enriched with increasing doses of aptamers and used as culture substrates for MC3T3-E1 cells. Cell growth was monitored by optical microscopy, while cell viability and metabolic activity were assessed by chemiluminescence and by Resazurin Sodium Salt assay. Cell morphology was investigated by cytofluorescence and by scanning electron microscopy. Chitosan films efficiently bound and retained aptamers. Aptamers did not affect the amount of adsorbed fibronectin, but affected osteoblasts behavior. Cell growth was proportional to the amount of aptamer used for the functionalization, as well as aptamers influenced cell morphology and their adhesion to the substrate. Our results demonstrate that the enrichment of chitosan films with aptamers could selectively improve osteoblasts behavior. Furthermore, our results support further investigation of this type of functionalization as a suitable modification to ameliorate the biocompatibility of biomaterial for hard tissue engineering applications.

Rough titanium surfaces enhance cell response to activation of Wnt canonical signalling, a pathway required for osteoblast differentiation. The present study investigated the effects of GSK3β-inhibitors SB216763 and SB415286 on osteoblastic differentiation on titanium surfaces with different topography and wettability. Osteoblastic MC3T3 cells were plated on smooth (Pickled), sand-blasted/acid-etched (SLA) or hyper hydrophilic SLA (modSLA) titanium discs and transfected with a reporter vector sys-tem for Wnt canonical signalling. Cells were also seeded in the presence or in the absence of GSK3b-inhibitors SB216763 or SB415286 and their viability, morphology and the expression of Wnt target and osteoblast specific genes was assessed by Real Time PCR. Inhibitors altered cell morphology and mostly reduced cell viability at high concentration. SB415286 markedly increased the expression of ALP in MC3T3 cells on rough surfaces at the concentration of 100 nM before decreasing its expression at higher concentrations. OCN expression was unaffected. Increasing concentrations of SB216763 increased the expression of ALP in MC3T3 cells on rough surfaces but OCN expression was not changed at any con-centration. SB216763 and SB415286 inhibitors should be further investigated as potential tools to improve cell differentiation on titanium surfaces for endosseous implants.

Background: Rough surface topography enhances the activation of Wnt canonical signaling, a pathway required for osteoblast differentiation. The present study investigated the effects of the modulation of prostaglandin E2 (PGE2) signaling on osteoblastic differentiation on titanium surfaces for endosseous implants with different topographies., Methods: C2C12 cells were plated on polished or acid-etched/sand-blasted (SLA) titanium discs and stimulated with 1 μM PGE2 or 100 nM cyclooxygenase inhibitor indomethacin. Activation of Wnt canonical signaling was measured with a reporter system. Gene expression was measured in the same cell system by real-time polymerase chain reaction (RT-PCR). Osteoblastic MC3T3 cells were then plated on polished or SLA titanium discs with or without indomethacin, and their proliferation and the expression of osteoblast-specific genes was assessed by RT-PCR. Cell morphology was furthermore studied on SEM, and cell adhesion was assessed by fluorescent labeling of focal adhesion., Results: PGE2 decreased Wnt signaling stimulation in cells growing on polished or SLA surfaces, while indomethacin increased the expression of Wnt target genes in C2C12 and MC3T3 cells, by reporter assay. Moreover, indomethacin increased the expression of early differentiation marker alkaline phosphatase in MC3T3 cells on polished discs and of late marker osteocalcin in cells on SLA titanium., Conclusions: Prostaglandin signaling affects the activation of Wnt canonical pathway in osteoblastic and mesenchymal cells on microstructured surfaces.

Aim: The aim of the present study was to investigate the efficacy of environmental scanning electron microscopy (ESEM), in low vacuum mode (LV-ESEM) and in wet mode (wet-ESEM) in the assessment of cell-material interactions., Methods: Mouse calvaria MC3T3 cells (ATCC) were seeded on commercially pure machined titanium discs of 10 mm diameter in Dulbecco modified MEM, 10% Fetal Bovine Serum, 1% Penicillin and Streptomycin and 1% Glutamine. Samples were then processed for microscope observation by rinse in Phosphate Buffer saline and fixation in 4.5% Glutaraldehyde. Samples were then rinsed in Sodium Cacodylate buffer and observed or dehydrated in alcohol prior to LV-ESEM observation. Fresh samples in 0.9% NaCl solution were observed in wet- ESEM., Results: No significant loss of detail was observed when dehydrated or non dehydrated samples were analysed at LV-ESEM.The observation of fresh samples in wet-ESEM however proved difficult for the need to eliminate water which forms a layer covering the sample, thus hiding cell surface details. When reducing the vapor pressure in the chamber, the layer evaporated and NaCl immediately started to precipitate and cells collapsed, thus no further investigation was possible., Conclusions: The use of low vacuum-ESEM after cell fixation, but without dehydration or gold sputter coating proved a viable alternative to traditional high vacuum SEM observation.

The aim of the present work was to investigate the morphology and activity of the murine osteoblastic cell line MC3T3 on control smooth (Machined), commercially available rough (ZT) titanium discs, and on titanium samples obtained by modifying the ZT treatment protocol, and herein labelled as ZTF, ZTM and ZTFM. Cells were evaluated at SEM and immunofluorescence for morphology and cell-to-cell interactions and by MTT assay and real time PCR for cell growth and function. Microscopy showed that ZT modified protocols could differently affect cell shape and distribution. All the tested surfaces showed good biocompatibility by viability assay. However, cells on smoother surfaces appeared to express higher levels of transcript for Collagen 1a1, the main component of extracellular matrix, by real time PCR. Expression of the early differentiation marker Alkaline Phosphatase was higher on ZTF surfaces and ZTM enhanced the expression of later osteoblastic markers Osteoprotegerin and Osteocalcin. Noteworthy, the expression of Connexin 43, a component of cell-to-cell contacts and hemichannels, followed a similar pattern to differentiation marker genes and was higher in cells on ZTM surfaces, consistently with the microscopic observation of cell clusters. Taken together, this data showed that ZTF and ZTM treatment protocols appeared to improve the basal sand-blasting/acid-etching ZT procedure with ZTM surfaces promoting the most mature stage of differentiation.

The aim of the present study was to investigate whether chitosan-based scaffolds modified with D-(+) raffinose and enriched with thiol-modified gelatin could selectively improve osteoblast adhesion and proliferation. 2, 3 and 4.5% chitosan films were prepared. Chitosan suitability for tissue engineering was confirmed by protein adsorption assay. Scaffolds were incubated with a 2.5 mg ml(-1) BSA solution and the decrease of protein content in the supernatants was measured by spectrophotometry. Chitosan films were then enriched with thiol-modified gelatin and their ability to bind BSA was also measured. Then, 2% chitosan discs with or without thiol-modified gelatin were used as culture substrates for MC3T3-E1 cells. After 72 h cells were stained with trypan blue or with calcein AM and propidium iodide for morphology, viability and proliferation assays. Moreover, cell viability was measured at 48, 72, 96 and 168 h to obtain a growth curve. Chitosan films efficiently bound and retained BSA proportionally to the concentration of chitosan discs. The amount of protein retained was higher on chitosan enriched with thiol-modified gelatin. Moreover, chitosan discs allowed the adhesion and the viability of cells, but inhibited their proliferation. The functionalization of chitosan with thiol-modified gelatin enhanced cell spreading and proliferation. Our data confirm that chitosan is a suitable material for tissue engineering. Moreover, our data show that the enrichment of chitosan with thiol-modified gelatin enhances its biological properties.

Periostin is a matricellular protein highly expressed in periodontal ligament and periostium and has been shown to be required for tissue development and maintenance. We showed that the adhesion of murine osteoblastic MC3T3 cells to thiolated hyaluronic acid/polyethyleneglycol hydrogels was greatly improved by enrichment with periostin. Polished or sand-blasted/acid-etched (SLA) commercially pure titanium surfaces were also coated with this protein and periostin ameliorated cell adhesion and dramatically affected cell morphology on both surfaces, as assessed at fluorescence microscopy, scanning electron microscopy, and chemiluminescence-based viability assay. Moreover, periostin increased the expression of alkaline phosphatase, osteoprotegerin, connective tissue growth factor, collagen 1a1, osteocalcin, Runx2, and osterix transcription factors on smooth surfaces. However, it did not affect, or even decreased, the expression of these genes on SLA discs. Transcript levels for connexin 43 were greatly increased on both surfaces in the presence of periostin. Taken together, these results show that periostin coatings can be a viable approach to improve cell adhesion and differentiation on implantable biomaterials., (© 2013 Wiley Periodicals, Inc.)

Rough titanium surfaces enhance the activation of Wnt canonical signaling, a pathway required for osteoblast differentiation. The present study investigated the effects of GSK3b-inhibitor (2'Z,3'E)- 6-Bromoindirubin-3'-oxime (BIO) on osteoblastic differentiation on titanium surfaces with different topography and wettability. C2C12 cells were plated on pickled, acid-etched/sand-blasted (SLA), modified hydrophilic SLA titanium discs (modSLA) and stimulated with increasing doses of BIO. Activation of Wnt canonical signaling was measured with a reporter system. Gene expression was measured in the same cell system by Real Time PCR. Osteoblastic MC3T3 cells were then plated on discs with or without BIO and the expression of osteoblast specific genes was assessed by Real Time PCR. One mM BIO activated Wnt canonical signaling in C2C12 cells on all surfaces, and the highest effect was on rough surfaces. BIO markedly increased the expression of Osteoprotegerin and Osteocalcin in MC3T3 cells on rough surfaces at the concentration of 100 nM, and on all surfaces at the concentration of 1 mM. BIO enhances Wnt signaling activation and the expression of osteoblastic genes on rough surfaces and could be a viable approach to improve cell response to implant surfaces.

Aims: Promoting bone formation at the tissue interface is an important step to improve implant success. This study investigated whether stimulation of Wnt signaling by GSK3b inhibitor lithium chloride (LiCl) could affect the response of mesenchymal or osteoblastic cells growing on titanium surfaces with different topography and wettability, and improve their differentiation along the osteoblastic lineage., Material and Methods: Murine mesenchymal C2C12 cells were plated on Pickled, acid-etched/sand-blasted (SLA), and hydrophilic SLA titanium disks (modSLA) and stimulated with increasing doses of LiCl. Cell viability was measured using chemiluminescence-based ATP quantitation and activation of Wnt canonical signaling was measured using a Luciferase-based reporter assay. Gene expression was measured using real time PCR in C2C12 cells, murine osteoblastic MC3T3 cells or murine primary bone marrow cells., Results: LiCl stimulated Wnt activation and expression of Wnt markers in C2C12 cells on modSLA. Addition of 1 mM LiCl increased levels for bone marker Osteocalcin in MC3T3 cells on modSLA surfaces. Similarly, LiCl potently enhanced Osteoprotegetrin levels in MC3T3 cells on modSLA. When primary bone marrow cells were stimulated with LiCl, the expression of Wnttarget genes and osteoblastic differentiation markers was increased on modSLA surfaces., Conclusions: Stimulation of the canonical Wnt pathway promoted osteoblast differentiation on hydrophilic modSLA surfaces. Taken together, these results demonstrate that Wnt activators such as LiCl should be further tested as a possible approach to improve implant osseointegration., (© 2012 John Wiley & Sons A/S.)

Endosseous implants are important tools to replace missing teeth or damaged tissue segments. Their clinical success depends on their integration in bone and, thus, on the response of bone cells to material and surface characteristics. Recent evidence has shown that surface topography and chemistry affect WNT signalling, a pivotal pathway for the commitment of mesenchymal progenitors to the osteoblast lineage and for bone homeostasis. WNT signalling comprises several cascades that, acting through different effectors, modulate several aspects of cell behaviour. It has been shown that cells growing on rough titanium surfaces display a different expression profile for WNT factors, and that surface features can alter the response of bone cells to WNT factors. Although the underlying mechanisms to this regulation are still poorly understood, the present review reports intriguing evidence that that cell cytoskeletal signalling is involved in activating WNT signalling in cells growing on rough implant surfaces.

Introduction: Mini-implants are used to improve orthodontic anchorage, but optimal composition and surface characteristics have yet to be determined. We investigated the behavior of osteoblast-like cells on grade 4 commercially pure titanium and grade 5 titanium alloy with different surface treatments for mini-implants., Methods: MC3T3 cells were plated on machined, acid-etched, or acid-etched grade 4 titanium enriched with calcium phosphate, or machined, anodized, or anodized and calcium phosphate-enriched grade 5 titanium disks. Surface and cell morphologies were assessed by scanning electron microscopy. Cell viability was measured by chemiluminescence, cytoskeletal organization was investigated by immunofluorescence, and real-time polymerase chain reaction for osteoblast-specific genes was performed to measure cell differentiation., Results: Flattened shapes and strong stress fibers were observed on the machined surfaces; cells on the rough surfaces had a spindle shape, with lower cytoskeletal polarization. Cell proliferation was highest on smooth grade 4 titanium surfaces, whereas cells quickly reached a plateau on rough grade 4 titanium; no difference was observed after 72 hours in the grade 5 titanium groups. Calcium phosphate enrichment on grade 4 titanium significantly increased the messenger RNA levels for alkaline phosphatase and osteocalcin. Osteoblastic markers were higher on the grade 5 titanium machined surfaces than on the rough surfaces, and comparable with acid-etched grade 4 titanium., Conclusions: Although the grade 4 titanium enriched with calcium phosphate had the highest level of differentiation in vitro, the grade 5 titanium machined surfaces supported cell proliferation and matrix synthesis, and induced high expression of early differentiation markers. Increased mechanical resistance of grade 5 titanium makes it a potential candidate for orthodontic mini-implants., (Copyright © 2012 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

Protein adsorption is the first and decisive step to define cell-biomaterial interaction. Guiding the adsorption of desired protein species may represent a viable approach to promote cell activities conducive to tissue regeneration. The aim of the present study was to investigate whether immobilized anti-Fibronectin aptamers could promote the attachment and growth of osteoblastic cells. Polyethyleneglycole diacrylate/thiolated Hyaluronic Acid hydrogels (PEGDA/tHA) were coated with anti-Fibronectin aptamers. Hydrogel loading and Fibronectin bonding were investigated, through spectrophotometry and Bradford assay. Subsequently, human osteoblasts (hOBs) were cultured on hydrogels for 10 days in 2D and 3D cultures. Cells were monitored through microscopy and stained for focal adhesions, microfilaments and nuclei using fluorescence microscopy. Samples were also included in paraffin and stained with Hematoxylin-Eosin. Cell number on hydrogels was quantitated over time. Cell migration into the hydrogels was also studied through Calcein AM staining. Aptamers increased the number of adherent hOBs and their cytoplasm appeared more spread and richer in adhesion complexes than on control hydrogels. Viability assays confirmed that significantly more cells were present on hydrogels in the presence of aptamers, already after 48 h of culture. When hOBs were encapsulated into hydrogels, cells were more numerous on aptamer-containing PEGDA-tHA. Cells migrated deeper in the gel in the presence of DNA aptamers, appearing on different focus planes. Our data demonstrate that anti-Fibronectin aptamers promote scaffold enrichment for this protein, thus improving cell adhesion and scaffold colonization. Statement of Significance We believe aptamer coating of biomaterials is a useful and viable approach to improve the performance of scaffold materials for both research and possibly clinical purposes, because different medical devices could be envisaged able to capture bioactive mediators from the patients’ blood and concentrate them where they are needed, on the biomaterial itself. At the same time, this technology could be used to confer 3D cell culture scaffold with the ability to store proteins, such as Fibronectin, taking it from the medium and capture what is produced by cells. This is an improvement of traditional biomaterials that can be enriched with exogenous molecules but are not able to selectively capture a desired molecule. [ABSTRACT FROM AUTHOR]