Additive manufacturing of hydroxyapatite-chitosan-genipin composite scaffolds for bone tissue engineering applications.

Additive manufacturing holds promise for the fabrication of three-dimensional scaffolds with precise geometry, to serve as substrates for the guided regeneration of natural tissue. In this work, a bioinspired approach is adopted for the synthesis of hybrid hydroxyapatite hydrogels, which were subsequently printed to form 3D scaffolds for bone tissue engineering applications. These hydrogels consist of hydroxyapatite nanocrystals, biomimetically synthesized in the presence of both chitosan and l-arginine. To improve their mechanical properties, chemical crosslinking was performed using a natural crosslinking agent (genipin), and their rheology was modified by employing an acetic acid/gelatin solution. Regarding the 3D printing process, several parameters (flow, infill and perimeter speed) were studied in order to accurately produce scaffolds with predesigned geometry and micro-architecture, while also applying low printing temperature (15 °C). Following the printing procedure, the 3D scaffolds were freeze dried in order to remove the entrapped solvents and therefore, obtain a porous interconnected network. Evaluation of porosity was performed using micro-computed tomography and nanomechanical properties were assessed through nanoindentation. Results of both characterization techniques, showed that the scaffolds' porosity as well as their modulus values, fall within the corresponding range of the respective values of cancellous bone. The biocompatibility of the 3D printed scaffolds was assessed using MG63 human osteosarcoma cells for 7 days of culturing. Cell viability was evaluated by MTT assay as well as double staining and visualized under fluorescence microscopy, while cell morphology was analyzed through scanning electron microscopy. Biocompatibility tests, revealed that the scaffolds constitute a cell-friendly environment, allowed them to adhere on the scaffolds' surface, increase their population and maintain high levels of viability.

In vitro cytotoxicity assessment of pristine and carboxyl-functionalized MWCNTs.

The wide use of carbon nanotubes (CNTs) in consumer products, i.e. composites, coatings, food packaging, etc, raise concerns about the adverse effects that CNTs can induce in humans and environment. Yet, there is no global consensus regarding risks that CNTs may pose, while controversial evidence exists also on the toxic effects associated with chemical surface modification, a prerequisite for their incorporation in different matrices. Moreover, there is limited information available about the underlying mechanisms, especially when cells' interactions with the nanomaterial is assessed by imaging techniques. The present study aims at evaluating the in vitro cytotoxicity of pristine and oxygen functionalized multi-walled CNTs (MWCNTs) by assessing cell viability and apoptosis in combination with scanning electron microscopy (SEM) observations of stabilised cells. Direct observation of adenocarcinoma human epithelial cells (A549) was performed after incubation with 12.5, 50 and 100 µg/ml MWCNTs, for 0.5, 1 and 3 h, simulating a real exposure scenario during an accident, taking into account industrial safety issues during the production and use of the nanomaterial. Functionalized MWCNTs induced higher time- and dose-dependent toxic effects as compared to pristine. The SEM observations revealed the damaging effect on the cell membrane, offering insights about the toxic mechanism that takes place.

Increased VEGF165 expression in HCT116 colon cancer cells after transient transfection with a GFP vector encoding HIF-1 gene.

Hypoxia occurs in most solid tumors as a result of inefficient vascular development and/or abnormal vascular architecture. During hypoxia, HIF-1alpha acts as the primary transcription factor functioning to activate multiple target genes, including vascular endothelial growth factor (VEGF). Several studies have demonstrated that in tumors HIF-1alpha mediates VEGF protein expression at the transcription level. We aimed to establish whether HCT116 colon cancer cell VEGF expression is regulated by HIF-1 levels after transient transfection with a GFP vector encoding the HIF-1alpha gene. HCT116 cell VEGF expression were therefore assayed by immunohistochemistry and ELISA. After transfection with phMGFP-HIF-1alpha, VEGF immunostaining was significantly increased in transfected cells as compared with untransfected HCT116 cells (p = 0.024, Student's t test); culture media VEGF levels assayed by ELISA were also significantly increased in transfected cells (p = 0.008, Student's t-test). These data suggest that HIF-1alpha may play an important role in colon cancer angiogenesis, both as a biomarker of metastatic potential and as a novel target for gene therapy.

In vitro evaluation of bio-functional performances of Ghimas titanium implants.

Titanium is the most widely used material for dental implants. The natural formation, in presence of oxygen, of different oxide films (passivation films) is correlated to titanium implant biocompatibility, resistance to corrosion and is responsible for implant bacteriostatic action. Surface roughness is another surface property of Ti-implants that, affecting implant-to-bone contact, improves integration. In the present study data concerning composition, surface roughness and biocompatibility of Ghimas implants and mini-implants undergoing sandblasting with Calcium Magnesium Carbonate (CaMg(CO3)2) are reported. AFM, SEM/EDX, XRD analyses and morpho-functional tests (MTT and ALP) were performed. Cell actin cytoskeletal modification (fluorescence phalloidin staining) was also observed with confocal laser microscopy (CLSM). Data related to surface geometry and chemical properties, associated with evidence of high purity of all the tested materials (XRD and EDX), highlighted the elevated biocompatibility of tested implants and mini-implants. CLSM investigation confirmed osteoblast features of an active cell behavior able to fit cell to chemico-mechanical stimuli present at the bone/implant interface and suggests an effective implant/alveolar bone integration in vivo.

Cell dynamics in the correct control of bone metabolism using natural treatments.

The present study was undertaken in order to assess the efficacy of a commercial product containing calcium and silicon (Osteosil-Calcium) on cell metabolism. MG-63 osteblast-like cells were cultured in the presence of three different drug concentrations (10, 5 and 2.5 microg/mL). Either serum-free culture and standard culture with serum were investigated. Morpho-functional tests (MTT and ALP), scanning electron microscopy (SEM), microanalysis (EDAX) and time-lapse video microscopy were performed. Cell actin cytoskeletal modification with fluorescence phalloidin staining was also tested. Our data show the in vitro functional efficacy of Osteosil-Calcium on MG63 cell viability and ALP production. This study demonstrates its positive effect on the metabolism of the single cell and suggests wider uses of this drug in health protection and or in Regenerative Medicine therapies which are currently applied to the elderly.

Rapid-prototyped and salt-leached PLGA scaffolds condition cell morpho-functional behavior.

Three dimensional scaffolds microfabricated using pressure-assisted microsyringe (PAM) with controlled geometry and porous membranes obtained using salt leaching were both tested with three different cell types to identify an optimal microstructural architecture for tissue engineering. MG63 (osteoblast-like cells) were used as models of mesenchymal bone tissue and human endothelial cells and NCTC2544 (keratinocytes) represented two epithelial tissues. Both porosity and stiffness of PLGA structures were measured, and cell morphology and cytoskeletal organization analyzed using SEM and actin labeling. The results show that overall the PAM scaffolds, which have a repeated and regular microstructure, are more biocompatible than the random pore salt-leached membranes, and that surface morphology as well as substrate stiffness modulates cell behavior.