Francesca Ravanetti

Rough surface topography enhances the activation of Wnt/β-catenin signaling in mesenchymal cells

It is known that the roughness of titanium surfaces affects cell proliferation and differentiation. However, the mechanisms mediating the cellular responses to surface topography are only partially understood. The present study investigated whether Wnt canonical signaling, an important pathway in determining cell fate, is modulated by surface roughness. This study analyzed the behavior of the murine C2C12 mesenchymal cell line on polished or acid-etched, sand-blasted (SLA) commercially pure titanium. When we transfected cells with Wnt3a or wild-type β-catenin and a reporter construct, we found that stimulation of Wnt canonical signaling was enhanced in cells on SLA surfaces. Moreover, more β-catenin translocated to the nucleus in cells on SLA surfaces after stimulation with Wnt3a as evidenced by immunofluorescence. However, when cells were transfected with constitutively active S33Y β-catenin mutant, no difference was observed between the groups. Higher levels of transcripts of Wnt target genes were detected in C2C12 cells cultured on SLA surfaces following transfection with Wnt3a, but the expression of a gene regulating β-catenin degradation, Axin 2, was reduced on SLA surfaces. Inhibition of β-catenin mediated transcription by dnTCF in murine osteoblastic MC3T3 cells, reversed the effects of topography on cell differentiation. Taken together, these results show that surface roughness modulates the responsiveness of mesenchymal cells to Wnt3a, that this requires the control of β-catenin degradation, and that the control of β-catenin signaling by surface topography is accountable for at least part of the effects of surface on cell differentiation.

In vitro cellular response and in vivo primary osteointegration of electrochemically modified titanium

Anodic spark deposition (ASD) is an attractive technique for improving the implant-bone interface that can be applied to titanium and titanium alloys. This technique produces a surface with microporous morphology and an oxide layer enriched with calcium and phosphorus. The aim of the present study was to investigate the biological response in vitro using primary human osteoblasts as a cellular model and the osteogenic primary response in vivo within a short experimental time frame (2 and 4 weeks) in an animal model (rabbit). Responses were assessed by comparing the new electrochemical biomimetic treatments to an acid-etching treatment as control. The in vitro biological response was characterized by cell morphology, adhesion, proliferation activity and cell metabolic activity. A complete assessment of osteogenic activity in vivo was achieved by estimating static and dynamic histomorphometric parameters at several time points within the considered time frame. The in vitro study showed enhanced osteoblast adhesion and higher metabolic activity for the ASD-treated surfaces during the first days after seeding compared to the control titanium. For the ASD surfaces, the histomorphometry indicated a higher mineral apposition rate within 2 weeks and a more extended bone activation within the first week after surgery, leading to more extensive bone-implant contact after 2 weeks. In conclusion, the ASD surface treatments enhanced the biological response in vitro, promoting an early osteoblast adhesion, and the osteointegrative properties in vivo, accelerating the primary osteogenic response.

Actin cytoskeleton controls activation of Wnt/β-catenin signaling in mesenchymal cells on implant surfaces with different topographies

Surface topography affects cell function and differentiation. It has been previously shown that rough surfaces can enhance the activation of canonical Wnt signaling, an important pathway for osteoblast differentiation and bone maintenance, but the underlying mechanisms are still poorly understood. The present paper investigates whether cytoskeletal organization contributes to regulating this pathway. Rho-associated protein kinase (ROCK), an important controller of actin microfilaments, was inhibited with 2mM specific antagonist Y-27632 in mesenchymal and osteoblastic cells growing on titanium discs with a polished or acid-etched, sand-blasted (SLA) surface. Y-27632 subverted the morphology of the cytoskeleton on polished and, to a lesser extent, on SLA surfaces, as evidenced by fluorescence microscopy. Although ROCK inhibition did not affect cell viability, it increased activation of Wnt signaling in uncommitted C2C12 mesenchymal cells on polished surfaces but not on SLA discs upon reporter assay. Consistently with this, real-time polymerase chain reaction analysis showed that MC3T3 cells on polished surfaces expressed higher mRNA levels for β-catenin and alkaline phosphatase, a known Wnt target gene, and for the osteoblastic differentiation marker osteocalcin after ROCK inhibition. Taken together, these data demonstrate that cytoskeletal organization mediates activation of Wnt canonical signaling in cells on titanium surfaces with different topographies.

Attachment, proliferation and osteogenic response of osteoblast-like cells cultured on titanium treated by a novel multiphase anodic spark deposition process.

A new bioactive titanium surface treatment, labeled Ti-ASD, was developed using the electrochemical anodic spark deposition (ASD) technique and results in a thickened titanium oxide layer with higher levels of calcium and phosphorus typical of newly deposited mineral phase. This study was aimed at extending the knowledge on Ti-ASD treatment, by means of evaluation of the attachment, morphology, proliferation, metabolic activity, differentiation, and mineralization of osteoblast-like cells (SaOS-2) after growth on Ti-ASD treated titanium compared with nontreated titanium (Ti) and with chemically etched titanium (Ti-ETC). This novel type of titanium coating supported cell attachment, cell proliferation, and mineralization, revealing no cytotoxicity effects. The expression of differentiation markers on Ti-ASD treated titanium shows that genes related to the proliferation phase (Collagen type I, Coll I; Cbfa-1) were early expressed, whereas genes related to the mineralization phase (alkaline phosphatase, osteopontin, bone sialo protein) increased in a time-related way. Mineralization occurred on all analyzed surfaces, but on Ti-ASD the number of bone-like nodules and the amount of mineralized area was higher. In conclusion, Ti-ASD resulted to be a good surface for osteoblast attachment and proliferation, also promoting the maintenance of cell differentiation and matrix mineralization, a fundamental requirement for sustain the osseointegration and the clinical success of dental implants.

The Effects of Er:YAG Laser Treatment on Titanium Surface Profile and Osteoblastic Cell Activity: An In Vitro Study

BACKGROUND Laser light has been proposed as a tool to decontaminate the surface of endosseous implants. The effects of this maneuver on the interactions between cells and surface, however, are poorly known. The goal of the present study is to investigate osteoblast growth and differentiation on three commercially available surfaces untreated or after irradiation by erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser at two levels: 150 and 200 mJ/pulse at 10 Hz. METHODS Human osteoblastic Saos-2 cells were plated on machined, sandblasted and acid-etched titanium, or titanium plasma-sprayed disks. The effects of lasing were observed with a scanning electronic microscope, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Moreover, we measured the production of the osteoblast-specific protein osteocalcin and of osteoprotegerin in the supernatants by immunoenzymatic assays. RESULTS Although no visible changes were observed on machined or titanium plasma-sprayed disk samples at the tested levels, titanium peaks on sandblasted and acid-etched titanium disks appeared fused as a consequence of laser irradiation. Interestingly, cell proliferation was slower on irradiated titanium at both intensities on all the surfaces. Cell differentiation, as assessed by osteocalcin production, was generally unaffected by laser treatment, whereas the production of osteoprotegerin was decreased on all the surfaces irradiated at the intensity of 200 mJ/10Hz. CONCLUSIONS These results indicate that Er:YAG laser at energy levels used in this study can alter the surface profile of titanium implants and these changes may negatively affect the viability and the activity of osteoblastic cells. Therefore, Er:YAG lasers should be used with caution on titanium surfaces.

Inhalation of peptide-loaded nanoparticles improves heart failure

Inhalation delivers drug-loaded nanoparticles to the heart, improving cardiac function in murine and porcine models. A puff of particles for the heart Nanoparticles can be useful for imaging and drug delivery but generally require intravenous injection to reach their targets. Miragoli et al. delivered nanoparticles carrying peptides to the heart by inhalation rather than injection. The inhaled particles reached the heart faster than injected particles and were taken up by cardiomyocytes to improve cardiac function in a mouse model of diabetic cardiomyopathy. In healthy pigs, inhaled particles were also found in heart tissue, suggesting that this minimally invasive method of targeted cardiac delivery could potentially translate to humans. Peptides are highly selective and efficacious for the treatment of cardiovascular and other diseases. However, it is currently not possible to administer peptides for cardiac-targeting therapy via a noninvasive procedure, thus representing scientific and technological challenges. We demonstrate that inhalation of small (<50 nm in diameter) biocompatible and biodegradable calcium phosphate nanoparticles (CaPs) allows for rapid translocation of CaPs from the pulmonary tree to the bloodstream and to the myocardium, where their cargo is quickly released. Treatment of a rodent model of diabetic cardiomyopathy by inhalation of CaPs loaded with a therapeutic mimetic peptide that we previously demonstrated to improve myocardial contraction resulted in restoration of cardiac function. Translation to a porcine large animal model provides evidence that inhalation of a peptide-loaded CaP formulation is an effective method of targeted administration to the heart. Together, these results demonstrate that inhalation of biocompatible tailored peptide nanocarriers represents a pioneering approach for the pharmacological treatment of heart failure.

Titanium dioxide aggregating nanoparticles induce autophagy and under-expression of microRNA 21 and 30a in A549 cell line: A comparative study with cobalt(II, III) oxide nanoparticles

The toxicity of TiO2 nanoparticles (NPs) is controversial, while it is widely accepted for Co3O4 NPs. We present a comparative study concerning the uptake of these NPs and their effect on cytoplasmic organelles and autophagy in a human lung carcinoma cell line (A549), including assays on the expression of autophagy-related microRNAs. The NP accumulation caused a fast dose- and time-dependent change of flow cytometry physical parameters particularly after TiO2 NP exposure. The intracellular levels of metals confirmed it, but the Co concentration was ten times higher than that of Ti. Both NPs caused neither necrosis nor apoptosis, but cytotoxicity was mainly evident for Co3O4 NPs in the first 72h. TiO2 NPs caused autophagy, contrarily to Co3O4 NPs. Furthermore, a significant and persistent downregulation of miRNA-21 and miRNA-30a was observed only in TiO2 NPs-treated cultures. The expression of miRNA-155 was similar for both NPs. Oxidative stress was evident only for Co3O4 NPs, while both NPs perturbed endoplasmic reticulum and p-53 expression. In conclusion, the oxidative stress caused by Co3O4 NPs can influence energy homeostasis and hamper the ability to detoxify and to repair the resulting damage, thus preventing the induction of autophagy, while TiO2 NPs elicit autophagy also under sub-toxic conditions.

Effect of Laser-Induced Dentin Modifications on Periodontal Fibroblasts and Osteoblasts: A New In Vitro Model

BACKGROUND The erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser has been shown to be a promising tool for root treatment in periodontitis, but little information is available regarding the surface characteristics after this treatment, mainly because it is difficult to obtain standardized dentin samples for in vitro studies. METHODS Commercially available standardized dentin disks were treated with an Er:YAG laser at different settings and used as a substrate for human primary osteoblastic cells (hOBs) and periodontal ligament fibroblasts (PLFs). Cell proliferation on untreated dentin was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 3, 6, 12, 24, and 48 hours of culture. The effects of the laser on dentin and cell morphology on treated and untreated samples were investigated by scanning electron microscopy after 3, 6, 24, and 48 hours of culture. RESULTS Dentin samples supported proliferation for both cell types, although growth kinetics were different. The laser dramatically affected the dentin profile, creating a rough and irregular surface. Cells grew easily on untreated dentin, but fewer cells were present on treated areas, often displaying long filopodes. hOBs showed poorer adhesion to treated dentin than PLFs. CONCLUSIONS The dentin disks provide a standardized and useful tool to study dentin surface modifications in vitro. PLFs behaved differently from hOBs on dentin, possibly because of their different affinity to this tissue and/or their differentiation state. The changes induced by the laser produced a less favorable environment for cell adhesion or growth, and treated dentin seemed to be more suitable for PLF adhesion compared to hOB adhesion.

Chitosan-based scaffold modified with D-(+) raffinose for cartilage repair: an in vivo study

BackgroundOsteochondral defects significantly affect patients’ quality of life and represent challenging tissue lesions, because of the poor regenerative capacity of cartilage. Tissue engineering has long sought to promote cartilage repair, by employing artificial scaffolds to enhance cell capacity to deposit new cartilage. An ideal biomaterial should closely mimic the natural environment of the tissue, to promote scaffold colonization, cell differentiation and the maintenance of a differentiated cellular phenotype. The present study evaluated chitosan scaffolds enriched with D-(+) raffinose in osteochondral defects in rabbits. Cartilage defects were created in distal femurs, both on the condyle and on the trochlea, and were left untreated or received a chitosan scaffold. The animals were sacrificed after 2 or 4 weeks, and samples were analysed microscopically.ResultsThe retrieved implants were surrounded by a fibrous capsule and contained a noticeable inflammatory infiltrate. No hyaline cartilage was formed in the defects. Although defect closure reached approximately 100% in the control group after 4 weeks, defects did not completely heal when filled with chitosan. In these samples, the lesion contained granulation tissue at 2 weeks, which was then replaced by fibrous connective tissue by week 4. Noteworthy, chitosan never appeared to be integrated in the surrounding cartilage.ConclusionsIn conclusion, the present study highlights the limits of D-(+) raffinose-enriched chitosan for cartilage regeneration and offers useful information for further development of this material for tissue repair.

Periostin improves cell adhesion to implantable biomaterials and osteoblastic differentiation on implant titanium surfaces in a topography‐dependent fashion

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.