A novel, microfluidic high-throughput single-cell encapsulation of human bone marrow mesenchymal stromal cells.

The efficacy of stem-cell therapy depends on the ability of the transplanted cells to escape early immunological reactions and to be retained at the site of transplantation. The use of tissue engineering scaffolds or injectable biomaterials as carriers has been proposed, but they still present limitations linked to a reliable manufacturing process, surgical practice and clinical outcomes. Alginate microbeads are potential candidates for the encapsulation of mesenchymal stromal cells with the aim of providing a delivery carrier suitable for minimally-invasive and scaffold-free transplantation, tissue-adhesive properties and protection from the immune response. However, the formation of stable microbeads relies on the cross-linking of alginate with divalent calcium ions at concentrations that are toxic for the cells, making control over the beads' size and a single-cell encapsulation unreliable. The present work demonstrates the efficiency of an innovative, high throughput, and reproducible microfluidic system to produce single-cell, calcium-free alginate coatings of human mesenchymal stromal cells. Among the various conditions tested, visible light and confocal microscopy following staining of the cell nuclei by DAPI showed that the microfluidic system yielded an optimal single-cell encapsulation of 2000 cells/min in 2% w/v alginate microcapsules of reproducible morphology and an average size of 28.2 ± 3.7 µm. The adhesive properties of the alginate microcapsules, the viability of the encapsulated cells and their ability to escape the alginate microcapsule were demonstrated by the relatively rapid adherence of the beads onto tissue culture plastic and the cells' ability to gradually disrupt the microcapsule shell after 24 h and proliferate. To mimic the early inflammatory response upon transplantation, the encapsulated cells were exposed to proliferating macrophages at different cell seeding densities for up to 2 days and the protection effect of the microcapsule on the cells assessed by time-lapse microscopy showing a shielding effect for up to 48 h. This work underscores the potential of microfluidic systems to precisely encapsulate cells by good manufacturing practice standards while favouring cell retention on substrates, viability and proliferation upon transplantation.

Synthetic peptides of IL-1Ra and HSP70 have anti-inflammatory activity on human primary monocytes and macrophages: Potential treatments for inflammatory diseases.

Chronic inflammatory diseases are increasing in developed societies, thus new anti-inflammatory approaches are needed in the clinic. Synthetic peptides complexes can be designed to mimic the activity of anti-inflammatory mediators, in order to alleviate inflammation. Here, we evaluated the anti-inflammatory efficacy of tethered peptides mimicking the interleukin-1 receptor antagonist (IL-1Ra) and the heat-shock protein 70 (HSP70). We tested their biocompatibility and anti-inflammatory activity in vitro in primary human monocytes and differentiated macrophages activated with two different stimuli: the TLR agonists (LPS + IFN-γ) or Pam3CSK4. Our results demonstrate that IL-1Ra and HSP70 synthetic peptides present a satisfactory biocompatible profile and significantly inhibit the secretion of several pro-inflammatory cytokines (IL-6, IL-8, IL-1ß and TNFα). We further confirmed their anti-inflammatory activity when peptides were coated on a biocompatible material commonly employed in surgical implants. Overall, our findings support the potential use of IL-1Ra and HSP70 synthetic peptides for the treatment of inflammatory conditions.

Exploiting the Features of Short Peptides to Recognize Specific Cell Surface Markers.

Antibodies are the macromolecules of choice to ensure specific recognition of biomarkers in biological assays. However, they present a range of shortfalls including a relatively high production cost and limited tissue penetration. Peptides are relatively small molecules able to reproduce sequences of highly specific paratopes and, although they have less biospecificity than antibodies, they offer advantages like ease of synthesis, modifications of their amino acid sequences and tagging with fluorophores and other molecules required for detection. This work presents a strategy to design peptide sequences able to recognize the CD44 hyaluronic acid receptor present in the plasmalemma of a range of cells including human bone marrow stromal mesenchymal cells. The protocol of identification of the optimal amino acid sequence was based on the combination of rational design and in silico methodologies. This protocol led to the identification of two peptide sequences which were synthesized and tested on human bone marrow mesenchymal stromal cells (hBM-MSCs) for their ability to ensure specific binding to the CD44 receptor. Of the two peptides, one binds CD44 with sensitivity and selectivity, thus proving its potential to be used as a suitable alternative to this antibody in conventional immunostaining. In the context of regenerative medicine, the availability of this peptide could be harnessed to functionalize tissue engineering scaffolds to anchor stem cells as well as to be integrated into systems such as cell sorters to efficiently isolate MSCs from biological samples including various cell subpopulations. The data here reported can represent a model for developing peptide sequences able to recognize hBM-MSCs and other types of cells and for their integration in a range of biomedical applications.

Bone Regeneration Guided by a Magnetized Scaffold in an Ovine Defect Model.

The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe3O4 nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.

New Aß(1-42) ligands from anti-amyloid antibodies: Design, synthesis, and structural interaction.

Alzheimer's disease (AD), is the most common neurodegenerative disorder of the aging population resulting in progressive cognitive and functional decline. Accumulation of amyloid plaques around neuronal cells is considered a critical pathogenetic event and, in most cases, a hallmark of the pathology. In the attempt to identify anti-AD drug candidates, hundreds of molecules targeting Aß peptides have been screened. Peptide molecules have been widely explored, appreciating chemical stability, biocompatibility, and low production cost. More recently, many anti-Aß(1-42) monoclonal antibodies have been developed, given the excellent potential of immunotherapy for treating or preventing AD. Antibodies are versatile ligands that bind a large variety of molecules with high affinity and specificity; however, their extensive therapeutic application is complex and requires huge economic investments. Novel approaches to identify alternative antibody formats are considered with great interest. In this context, taking advantage of the favorable peptide properties and the availability of Aß-antibodies structural data, we followed an innovative research approach to identify short peptide sequences on the model of the binding sites of Aß(1-42)/antibodies. WAibH and SYSTPGK were designed as mimics of solanezumab and aducanumab, respectively. Circular dichroism and nuclear magnetic resonance analysis reveal that the antibody-derived peptides interact with Aß(1-42) in the soluble monomeric form. Moreover, AFM microscopy imaging shows that WAibH and SYSTPGK are capable of controlling the Aß(1-42) aggregation. The strategy to identify WAibH and SYSTPGK is innovative and can be widely applied for new anti-Aß antibody mimicking peptides.

Complexation of Injectable Biphasic Calcium Phosphate with Phosphoserine-Presenting Dendrons with Enhanced Osteoregenerative Properties.

Injectable biphasic calcium phosphates have been proposed as a solution in the treatment of a range of clinical applications including as fillers in the augmentation of osteoporotic bone. To date, various biodegradable natural or synthetic organics have been used as a polymer component of bone materials to increase their cohesiveness. Herein, a novel bone material was developed combining osteoconductive biphasic calcium phosphate (BCP) nanoparticles with phosphoserine-tethered generation 3 poly(epsilon-lysine) dendron (G3-K PS), a class of hyperbranched peptides previously shown to induce biomineralization and stem cell osteogenic differentiation. Strontium was also incorporated into the BCP nanocrystals (SrBCP) to prevent bone resorption. Within 24 h, an antiwashout behavior was observed in G3-K PS-integrated pure BCP group (BCPG3). Moreover, both in vitro tests by relevant cell phenotypes and an in vivo tissue regeneration study by an osteoporotic animal bone implantation showed that the integration of G3-K PS would downregulate Cxcl9 gene and protein expressions, thus enhancing bone regeneration measured as bone mineral density, new bone volume ratio, and trabecular microarchitectural parameters. However, no synergistic effect was found when Sr was incorporated into the BCPG3 bone pastes. Notably, results indicated a concomitant reduction of bone regeneration potential assessed as reduced Runx2 and PINP expression when bone resorptive RANKL and CTX-I levels were reduced by Sr supplementation. Altogether, the results suggest the potential of injectable BCPG3 bone materials in the treatment of osteoporotic bone defects.

A comparative in vitro study of the effect of biospecific integrin recognition processes and substrate nanostructure on stem cell 3D spheroid formation.

The in vitro study of the properties of the human mesenchymal stem cells as well as their manipulation in culture for clinical purposes depends on the elimination of artefacts caused by the lack of their natural environment. It is now widely accepted that mesenchymal stem cells should be studied when they are organised as 3D spheroids rather than fibroblast-like colonies. Although this can be achieved with the use of some extracellular matrix proteins or by non-adherent conditions these suffer of significant limitations. The recent development of synthetic substrates resembling the physicochemical and biochemical properties of the adult stem cell niche has prompted questions about the role played by nanotopography and receptor-mediated adhesion. In the present paper, the influence of two types of substrates bearing the same nanostructure, but exposing either a non-specific or an integrin-specific binding motif was studied. Carboxybetaine-tethered hyperbranched poly(ɛ-lysine) dendrons showed that the hyperbranched structure was fundamental to induce spheroid formation, but these were forming more slowly, were of reduced size and less stable than those growing on substrates based on the same hyperbranched structures that had been functionalised at their uppermost branching generation by a laminin amino acid sequence, i.e. YIGSR. The study shows that both nanostructure and biorecognition need to be combined to achieve a substrate for stem cell spheroid formation as that observed in vivo in the adult stem cell niche.

Gadolinium Tagged Osteoprotegerin-Mimicking Peptide: A Novel Magnetic Resonance Imaging Biospecific Contrast Agent for the Inhibition of Osteoclastogenesis and Osteoclast Activity.

The control of osteoblast/osteoclast cross-talk is crucial in the bone remodelling process and provides a target mechanism in the development of drugs for bone metabolic diseases. Osteoprotegerin is a key molecule in this biosignalling pathway as it inhibits osteoclastogenesis and osteoclast activation to prevent run-away bone resorption. This work reports the synthesis of a known osteoprotegerin peptide analogue, YCEIEFCYLIR (OP3-4), and its tagging with a gadolinium chelate, a standard contrast agent for magnetic resonance imaging. The resulting contrast agent allows the simultaneous imaging and treatment of metabolic bone diseases. The gadolinium-tagged peptide was successfully synthesised, showing unaltered magnetic resonance imaging contrast agent properties, a lack of cytotoxicity, and dose-dependent inhibition of osteoclastogenesis in vitro. These findings pave the way toward the development of biospecific and bioactive contrast agents for the early diagnosis, treatment, and follow up of metabolic bone diseases such as osteoporosis and osteosarcoma.

Anti-angiogenic potential of VEGF blocker dendron loaded on to gellan gum hydrogels for tissue engineering applications.

Damage of non-vascularised tissues such as cartilage and cornea can result in healing processes accompanied by a non-physiological angiogenesis. Peptidic aptamers have recently been reported to block the vascular endothelial growth factor (VEGF). However, the therapeutic applications of these aptamers are limited due to their short half-life in vivo. In this work, an enhanced stability and bioavailability of a known VEGF blocker aptamer sequence (WHLPFKC) was pursued through its tethering of molecular scaffolds based on hyperbranched peptides, the poly(ɛ-lysine) dendrons, bearing three branching generations. The proposed design allowed simultaneous and orderly-spaced exposure of 16 aptamers per dendrimer to the surrounding biological microenvironent, as well as a relatively hydrophobic core based on di-phenylalanine aiming to promote an hydrophobic interaction with the hydrophobic moieties of ionically crosslinked methacrylated gellan gum (iGG-MA) hydrogels. The VEGF blocker dendrons were entrapped in iGG-MA hydrogels, and their capacity to prevent endothelial cell sprouting was assessed qualitatively and quantitatively using 3D in vitro models and the in vivo chick chorioallantoic membrane assay. The data demonstrate that at nanoscale concentrations, the dendronised structures were able to enhance control of the biological actvity of WHLPFKC at the material/tissue interface and hence the anti-angiogenic capacity of iGG-MA hydrogels not only preventing blood vessel invasion, but also inducing their regression at the tissue/iGG-MA interface. The in ovo study confirmed that iGG-MA functionalised with the dendron VEGF blockers do inhibit angiogenesis by controlling both size and ramifications of blood vessels in the proximity of the implanted gel surface. Copyright © 2016 John Wiley & Sons, Ltd.

Hyperbranched poly(ϵ-lysine) substrate presenting the laminin sequence YIGSR induces the formation of spheroids in adult bone marrow stem cells.

Unlike the fibroblast-like cells formed upon monolayer culture of human mesenchymal stem cells, the natural stem cell niche of the bone marrow and other types of tissues favours the formation of 3-dimensional (3D) cell clusters. The structuring and biological activity of these clusters are regulated by the contacts established by cells with both the basement membrane and neighbour cells and results in their asymmetric division and the consequent maintenance of both a stem population and a committed progeny. The present work demonstrates the potential of a synthetic substrate to mimic the stem cell niche in vitro. The side amino groups of a linear Poly-L-lysine were modified with hyperbranched poly-(ϵ-lysine) peptides, named as dendrons, tethered with the laminin-mimicking sequence, YIGSR. These dendrons presented the YIGSR sequence at the uppermost molecular branching ensuring a controlled spacing of the bioligand. When used to coat the surface of tissue culture plates in a serum-free in vitro cell culture system, the substrate was able to mimic the most relevant features of the basement membrane of the stem cell niche, i.e. the mesh structure of Collagen Type IV and the availability of laminin bioligands relevant to integrin biorecognition. The substrate biomimetic properties were tested for their ability to support the formation of human bone marrow mesenchymal stem cells (hMSCs) 3D spheroids similar to those observed in the natural stem cell niches and their ability to maintain stem cell pluripotency markers. These features were related to the substrate-specific expression and localisation of (i) cell adhesion receptors (i.e. ß-integrin and N-cadherin), (ii) transcription factors of pluripotency markers and cytoskeleton protein and (iii) regulators of cell migration throughout cell culture passages 2 to 4. The results clearly demonstrate the formation of 3D spheroids starting from the asymmetric division of substrate-adhering spread cells, the clustering of relevant integrins and the expression of specific intracellular pathways controlling cytoskeleton formation suggesting their potential use as a substrate for the handling of stem cells prior to transplantation procedures.

Novel Superparamagnetic Microdevices Based on Magnetized PLGA/PLA Microparticles Obtained by Supercritical Fluid Emulsion and Coating by Carboxybetaine-Functionalized Chitosan Allowing the Tuneable Release of Therapeutics.

When superparamagnetic nanoparticles (MAG) are loaded within microcarriers of thermosensitive and injectable biopolymers, "smart" microdevices are obtained: they respond to an external magnetic field through the release of any co-encapsulated molecules with a remote on-off control. Creating reliable and effective fabrication technologies for the production of these smart nano/microdevices remains a challenge. In this work, supercritical emulsion extraction technology is proposed for the fabrication of microcapsules with a core of poly-lactic-co-glycolic acid (PLGA) or polylactic acid (PLA) covered by carboxybetaine-functionalized chitosan (f-chi) and loaded with MAG (mean size of 6.5 ± 3.0 nm) and water-soluble fluorescein (Fluo). Fluo is co-encapsulated as a fluorescent marker for the release study. Microcarriers showed a mean size of 800 ± 60 nm with an encapsulation efficiency of up to 90%. The inversion of surface charge, after the f-chi coating, suggested the presence of a uniform functionalized surface available for further chemical linkage. The external chitosan layer had a thickness of 200 ± 50 nm. An excellent MAG dispersion was confirmed within the biopolymer matrix that was shown to be responsive to external magnetic field; indeed, Fluo was released over 3 or 5 days from PLGA or f-chi-PLGA microdevices into phosphate-buffered saline medium at 37°C, whereas remote on-off controlled release was achieved when an alternating magnetic field was applied.

Carboxybetaine-modified succinylated chitosan-based beads encourage pancreatic ß-cells (Min-6) to form islet-like spheroids under in vitro conditions.

In vitro, pancreatic ß-cells tend to reduce their ability to aggregate into islets and lose insulin-producing ability, likely due to insufficient cell-cell and cell-matrix interactions that are essential for ß-cell retention, viability and functionality. In response to these needs, surfaces of succinylated chitosan-based beads (NSC) were modified with zwitterionic carboxy-betaine (CB) moieties, a compatible osmolyte known to regulate cellular hydration state, and used to promote the formation of ß-cell spheroids using a conventional 2D cell culture technique. The NSC were synthesised by ionic gelation and surface-functionalised with CB using carbodiimide chemistry. Scanning electron microscopy (SEM), dynamic laser scattering (DLS) and Fourier transform infrared spectroscopy (FTIR) were employed as characterisation tools to confirm the successful modification of the succinylated chitosan material into spherical beads with rough surfaces and a diameter of 0.4 µm. NSC with and without CB were re-suspended at concentrations of 0.1, 0.3 and 0.6 mg/mL in saline medium and tested in vitro with MIN6 murine pancreatic ß-cell line. Results showed that a concentration of 0.3 mg/mL, NSC-CB encouraged pancreatic MIN6 cells to proliferate and form spheroids via E-cadherin and Pdx-1 activation within 48 h in culture. These spheroids, with a size of approximately 80 µm, exhibited high cell viability and enhanced insulin protein expression and secretion when compared to cells organised by the non-modified beads.

Rapid and efficient magnetization of mesenchymal stem cells by dendrimer-functionalized magnetic nanoparticles.

AIM: Rapid and efficient magnetization of human bone marrow stromal cells (BMSC) through functionalized magnetic nanoparticles (MNP). METHODS: MNP were functionalized with poly(epsilon-lysine) dendrons exposing carboxybetaine residue (CB-MNP) to enhance binding to the cellular glycocalix. BMSC were incubated with CB-MNP or non-functionalized PAA-MNP for 5-30 min in suspension. RESULTS: CB-MNP functionalization increased the magnetization efficiency by threefold. Remarkably, 66% of cells were magnetized after only 5 min and the maximum efficiency of >80% was reached by 15 min. BMSC viability, proliferation and differentiation were not impaired: actually, adipogenic and osteogenic differentiation were even improved. CONCLUSION: Carboxybetaine-dendron functionalization ensured rapid and efficient BMSC magnetization and allowed innovative suspension labeling, with a potential for bypassing adhesion culture of progenitors for regenerative medicine.

In vivo biofunctional evaluation of hydrogels for disc regeneration.

PURPOSE: Regenerative strategies aim to restore the original biofunctionality of the intervertebral disc. Different biomaterials are available, which might support disc regeneration. In the present study, the prospects of success of two hydrogels functionalized with anti-angiogenic peptides and seeded with bone marrow derived mononuclear cells (BMC), respectively, were investigated in an ovine nucleotomy model. METHODS: In a one-step procedure iliac crest aspirates were harvested and, subsequently, separated BMC were seeded on hydrogels and implanted into the ovine disc. For the cell-seeded approach a hyaluronic acid-based hydrogel was used. The anti-angiogenic potential of newly developed VEGF-blockers was investigated on ionically crosslinked metacrylated gellan gum hydrogels. Untreated discs served as nucleotomy controls. 24 adult merino sheep were used. After 6 weeks histological, after 12 weeks histological and biomechanical analyses were conducted. RESULTS: Biomechanical tests revealed no differences between any of the implanted and nucleotomized discs. All implanted discs significantly degenerated compared to intact discs. In contrast, there was no marked difference between implanted and nucleotomized discs. In tendency, albeit not significant, degeneration score and disc height index deteriorated for all but not for the cell-seeded hydrogels from 6 to 12 weeks. Cell-seeded hydrogels slightly decelerated degeneration. CONCLUSIONS: None of the hydrogel configurations was able to regenerate biofunctionality of the intervertebral disc. This might presumably be caused by hydrogel extrusion. Great importance should be given to the development of annulus sealants, which effectively exploit the potential of (cell-seeded) hydrogels for biological disc regeneration and restoration of intervertebral disc functioning.

Poly(Epsilon-lysine) dendrons tethered with phosphoserine increase mesenchymal stem cell differentiation potential of calcium phosphate gels.

Calcium phosphates (CaP) are considered as biomaterials of choice for the treatment of critical-sized bone defects. Novel injectable CaP materials integrating poly(epsilon-lysine) generation 3 dendrons tethered with phosphoserine were obtained by sol-gel synthesis. This type of dendron was integrated to mimic the biochemical structure of noncollagenous proteins present in the forming osteoids during bone repair. Sol-gel synthesis was coupled with a dialysis process able to equilibrate the materials at a physiological pH value. Fourier transform infrared spectroscopy (FTIR) showed the successful retention of the dendrons after gel dialysis, whereas X-ray diffraction analysis demonstrated both the pH-tuned formation of a hydroxyapatite crystalline phase within the gel and the complete removal of ammonium nitrate deriving from the sol-gel reaction solvent. Scanning electron microscopy images confirmed the presence of crystalline domains in gels synthesized at pH 9.0. Injectability tests showed that the optimized formulations fulfilled the rheological properties required to minimally invasive surgical procedures. Cytotoxicity tests on osteoblast-like MG-63 cells as well as morphology and viability studies showed that the dendrons induced a significantly higher level of cell proliferation at early incubation time. Differentiation of the cell was also clearly enhanced at longer incubation time as demonstrated by both alkaline phosphatase activity and expression of typical markers. Altogether, the data from this work indicate the clinical potential of the osteoid-mimicking CaP cements in minimally invasive bone surgery.

Pre-clinical evaluation of soybean-based wound dressings and dermal substitute formulations in pig healing and non-healing in vivo models.

In the last decade, a new class of natural biomaterials derived from de-fatted soybean flour processed by either thermoset or extraction procedures has been developed. These biomaterials uniquely combine adaptability to various clinical applications to proven tissue regeneration properties. In the present work, the biomaterials were formulated either as hydrogel or as paste formulation and their potential as wound dressing material or as dermal substitute was assessed by two in vivo models in pig skin: The healing full-thickness punch biopsy model and the non-healing full-thickness polytetrafluoroethylene (PTFE) chamber model. The results clearly show that collagen deposition is induced by the presence of these biomaterials. A unique pattern of early inflammatory response, eliciting neutrophils and controlling macrophage infiltration, is followed by tissue cell colonization of the wound bed with a significant deposition of collagen fibers. The study also highlighted the importance in the use of optimal formulations and appropriate handling upon implantation. In large size, non-healing wounds, wound dermis was best obtained with the paste formulation as hydrogels appeared to be too loose to ensure lasting scaffolding properties. On the contrary, packing of the granules during the application of paste reduced biomaterial degradation rate and prevent the penetration of newly vascularized tissue, thus impeding grafting of split-thickness autologous skin grafts on the dermal substitute base.

A novel antibacterial modification treatment of titanium capable to improve osseointegration.

BACKGROUND: Among the different causes of orthopedic and dental implant failure, infection remains the most serious and devastating complication associated with biomaterial devices. PURPOSE: The aim of this study was to develop an innovative osteointegrative and antibacterial biomimetic coating on titanium and to perform a chemical-physical and in vitro biological characterization of the coating using the SAOS-2 cell line. We also studied the antibacterial properties of the coating against both Gram-positive and Gram-negative bacteria strains. METHODS: An electrochemical solution containing silicon, calcium, phosphorous, sodium, and silver nanoparticles was used to obtain the antibacterial by Anodic Spark Deposition (ASD) treatment. Surface morphology was characterized using SEM and laser profilometry. A qualitative analysis of the chemical composition of the coating was assessed by EDS. The adhesion properties of the coating to the titanium bulk were performed with a 3-point bending test. SAOS-2 osteoblastic cell line spreading and morphology and viability were investigated. The bacterial adhesion and the antibacterial properties were investigated after 3 h and 24 h of incubation with Streptococcus mutans, Streptococcus epidermidis, and Escherichia coli bacterial strains. RESULTS: The proposed anodization treatment created a chemically and morphologically modified, adherent titanium oxide layer, characterized by a microporous morphology enriched by calcium, silicon, phosphorous, and silver. The preliminary biological characterization showed optimal SAOS-2 cell adhesion and proliferation as well as a strong antibacterial effect. CONCLUSIONS: Based on the results of this study, we believe that this novel biomimetic and antibacterial treatment hold promise for enhancing osteointegration while conferring strong antibacterial properties to titanium.

Synthesis, characterisation and in vitro anti-angiogenic potential of dendron VEGF blockers.

To overcome the lack of in vivo stability of certain peptides used in cancer treatment and to increase their retention time in the extracellular matrix of the target tissue, the anti-angiogenic WHLPFKC sequence is synthesised at the uppermost branching generation of a poly(ε-lysine) dendron. The root of these dendrons is designed to interact preferentially with macromolecules of the extracellular matrix, whilst the uppermost branching generation of the dendron increased the exposed density of the bioactive peptide. Bioactivity testing of the blockers is performed on HUVECs. The results show that the dendron tethered with VEGF blockers was still able to inhibit proliferation and angiogenesis. Their relatively larger structure did not prevent the interaction with VEGF.

Substrate-induced phenotypic switches of human smooth muscle cells: an in vitro study of in-stent restenosis activation pathways.

In-stent restenosis is a clinical complication following coronary angioplasty caused by the implantation of the metal device in the atherosclerotic vessel. Histological examination has shown a clear contribution of both inflammatory and smooth muscle cells (SMCs) to the deposition of an excess of neointimal tissue. However, the sequence of events leading to clinically relevant restenosis is unknown. This paper aims to study the phenotype of SMCs when adhering on substrates and exposed to biochemical stimuli typical of the early phases of stent implantation. In particular, human SMC phenotype was studied when adhering on extracellular matrix-like material (collagen-rich gel), thrombus-like material (fibrin gel) and stent material (stainless steel) in the presence or absence of a platelet-derived growth factor (PDGF) stimulus. Cells on the collagen and fibrin-rich substrates maintained their contractile phenotype. By contrast, cells on stainless steel acquired a secretory phenotype with a proliferation rate 50 per cent higher than cells on the natural substrates. Cells on stainless steel also showed an increase in PDGF-BB receptor expression, thus explaining the increase in proliferation observed when cells were subject to PDGF-BB stimuli. The stainless steel substrate also promoted a different pattern of ß1-integrin localization and an altered expression of hyaluronan (HA) synthase isoforms where the synthesis of high-molecular-weight HA seemed to be favoured. These findings highlighted the induction of a phenotypic pattern in SMC by the stainless steel substrate whereby the formation of a HA-rich neointimal tissue is enhanced.