3D Printing and bioprinting of osteomimetic materials for 3D in vitro models and tissue repair applications

Bone disorders have severe impact on body functions and quality life, and no satisfying therapies exist yet. The current models for bone disease study are scarcely predictive and the options existing for therapy fail for complex systems. To mimic and/or restore bone, 3D printing/bioprinting allows the creation of 3D structures with different materials compositions, properties, and designs. In this study, 3D printing/bioprinting has been explored for (i) 3D in vitro tumor models and (ii) regenerative medicine. Tumor models have been developed by investigating different bioinks (i.e., alginate, modified gelatin) enriched by hydroxyapatite nanoparticles to increase printing fidelity and increase biomimicry level, thus mimicking the organic and inorganic phase of bone. High Saos-2 cell viability was obtained, and the promotion of spheroids clusters as occurring in vivo was observed. To develop new syntethic bone grafts, two approaches have been explored. In the first, novel magnesium-phosphate scaffolds have been investigated by extrusion-based 3D printing for spinal fusion. 3D printing process and parameters have been optimized to obtain custom-shaped structures, with competent mechanical properties. The 3D printed structures have been combined to alginate porous structures created by a novel ice-templating technique, to be loaded by antibiotic drug to address infection prevention. Promising results in terms of planktonic growth inhibition was obtained. In the second strategy, marine waste precursors have been considered for the conversion in biogenic HA by using a mild-wet conversion method with different parameters. The HA/carbonate ratio conversion efficacy was analysed for each precursor (by FTIR and SEM), and the best conditions were combined to alginate to develop a composite structure. The composite paste was successfully employed in custom-modified 3D printer for the obtainment of 3D printed stable scaffolds. In conclusion, the osteomimetic materials developed in this study for bone models and synthetic grafts are promising in bone field.

Mineral Trioxide Aggregate-based Endodontic Sealer Stimulates Hydroxyapatite Nucleation in Human Osteoblast-like Cell Culture

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Study of Bone Biomineralization in 2D and 3D Cultures of a Human Osteosarcoma Cell Line As Osteoblast-like Model

This PhD project focuses on the study of the early stages of bone biomineralization in 2D and 3D cultures of osteoblast-like SaOS-2 osteosarcoma cells, exposed to an osteogenic cocktail. The efficacy of osteogenic treatment was assessed on 2D cell cultures after 7 days. A large calcium minerals production, an overexpression of osteogenic markers and of alkaline phosphatase activity occurred in treated samples. TEM microscopy and cryo-XANES micro-spectroscopy were performed for localizing and characterizing Ca-depositions. These techniques revealed a different localization and chemical composition of Ca-minerals over time and after treatment. Nevertheless, the Mito stress test showed in treated samples a significant increase in maximal respiration levels associated to an upregulation of mitochondrial biogenesis indicative of an ongoing differentiation process. The 3D cell cultures were realized using two different hydrogels: a commercial collagen type I and a mixture of agarose and lactose-modified chitosan (CTL). Both biomaterials showed good biocompatibility with SaOS-2 cells. The gene expression analysis of SaOS-2 cells on collagen scaffolds indicated an osteogenic commitment after treatment. and Alizarin red staining highlighted the presence of Ca-spots in the differentiated samples. In addition, the intracellular magnesium quantification, and the X-ray microscopy on mineral depositions, suggested the incorporation of Mg during the early stages of bone formation process., SaOS-2 cells treated with osteogenic cocktail produced Ca mineral deposits also on CTL/agarose scaffolds, as confirmed by alizarin red staining. Further studies are underway to evaluate the differentiation also at the genetic level. Thanks to the combination of conventional laboratory methods and synchrotron-based techniques, it has been demonstrated that SaOS-2 is a suitable model for the study of biomineralization in vitro. These results have contributed to a deeper knowledge of biomineralization process in osteosarcoma cells and could provide new evidences about a therapeutic strategy acting on the reversibility of tumorigenicity by osteogenic induction.

Osteoblastlike Cell Adhesion on Titanium Surfaces Modified by Plasma Nitriding

Purpose: The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Materials and Methods: Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nit riding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. Results: There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P < .05). Conclusions: Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion. INT J ORAL MAXILLOFAC IMPLANTS 2011;26:237-244

Human Alveolar Bone-Derived Cell-Culture Behaviour on Biodegradable Poly(L-lactic Acid)

Poly(L-lactic acid) (PLA) is a polymer of great technological interest, whose excellent mechanical properties, thermal plasticity and bioresorbability render it potentially useful for environmental applications, as a biodegradable plastic and as a biocompatible material in biomedicine. The interactions between an implant material surface and host cells play central roles in the integration, biological performance and clinical success of implanted biomedical devices. Osteoblasts from human alveolar bone were chosen to investigate the cell behaviour when in contact with PLA discs. Cell morphology and adhesion through osteopontin (OPN) and fibronectin (FN) expression were evaluated in the initial osteogenesis, as well as cell proliferation, alkaline phosphatase activity and bone nodule formation. It was shown that the polymer favoured cell attachment. Cell proliferation increased until 21 days but in a smaller rate when compared to the control group. On the other hand, ALP activity and bone mineralization were not enhanced by the polymer. It is suggested that this polymer favours cell adhesion in the early osteogenesis in vitro, but it does not enhance differentiation and mineralization. (C) Koninklijke Brill NV, Leiden, 2009

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells

Objective: The aim of this study was to investigate the effects of PRP on SAOS-2 cells in terms of cytokine expression, cell activity and oxidative stress. Design: Cell line SAOS-2 (1 x 10(5) cells/mL) were grown in culture medium alpha-MEM with 10% FBS for 24 h and stimulated (or not) with PRP at concentrations of 3, 10 and 20%, LPS (E. coli, 10 g/mL) and IL-1 beta (1 mg/mL) for 24 h. The supernatant was collected and analyzed for the expression of cytokines in a panel array, ALP using a commercial kit and NO2- with Griess reaction method. Also, the cells were analyzed using Western blot for RANKL and slot blotting for nitrotyrosine expression. Result: There were no significant differences amongst the groups in terms of NO2-, protein nitrotyrosine content and RANKL expression. However, all stimuli increased ALP activity and in case of PRP, it was in a dose-dependent manner (p < 0.001). Also, all stimuli induced an increase in cytokines and chemokines expression, but only PRP promoted an increase of component C5, sICAM-1 and RANTES expression. Whilst IL-1 receptor antagonist (IL-1ra) expression was down-regulated by PRP, both LPS and IL-1 beta caused up-regulation of this cytokine. Conclusions: PRP can stimulate osteoblast activity and cytokine/chemokine release, as well as indicate some of the mediators that can (and cannot) be involved in this activation. (C) 2012 Elsevier Ltd. All rights reserved.

Osteoblast responses to different oxide coatings produced by the sol-gel process on titanium substrates

In order to improve the osseointegration of endosseous implants made from titanium, the structure and composition of the surface were modified. Mirror-polished commercially pure (cp) titanium substrates were coated by the sol-gel process with different oxides: TiO(2), SiO(2), Nb(2)O(5) and SiO(2)-TiO(2). The coatings were physically and biologically characterized. Infrared spectroscopy confirmed the absence of organic residues. Ellipsometry determined the thickness of layers to be approximately 100nm. High resolution scanning electron microscopy (SEM) and atomice force microscopy revealed a nanoporous structure in the TiO(2) and Nb(2)O(5) layers, whereas the SiO(2) and SiO(2)-TiO(2) layers appeared almost smooth. The R(a) values, as determined by white-light interferometry, ranged from 20 to 50nm. The surface energy determined by the sessile-drop contact angle method revealed the highest polar component for SiO(2) (30.7mJm(-2)) and the lowest for cp-Ti and 316L stainless steel (6.7mJm(-2)). Cytocompatibility of the oxide layers was investigated with MC3T3-E1 osteoblasts in vitro (proliferation, vitality, morphology and cytochemical/immunolabelling of actin and vinculin). Higher cell proliferation rates were found in SiO(2)-TiO(2) and TiO(2), and lower in Nb(2)O(5) and SiO(2); whereas the vitality rates increased for cp-Ti and Nb(2)O(5). Cytochemical assays showed that all substrates induced a normal cytoskeleton and well-developed focal adhesion contacts. SEM revealed good cell attachment for all coating layers. In conclusion, the sol-gel-derived oxide layers were thin, pure and nanostructured; consequent different osteoblast responses to those coatings are explained by the mutual action and coadjustment of different interrelated surface parameters.

Development of hydroxyapatite-based nanomaterials to enhance biological response of osteoblast cells for clinical application

Purpose: To develop a novel chitosan/gelatin-hydroxyapatite (CGHaP) microspheres for evaluating the biological response of pre-osteoblast cells. Methods: The microsphere was prepared by water-in-oil emulsion method. Cell proliferation was studied using AlamarBlue colorimetric assay and DAPI staining while alkaline phosphatase assay was carried out by colorimetric assay method. Chitosan microspheres as well as chitosan-hydroxyapatite microspheres was prepared and tested for biological response from MC3T3-E1 cell line. Results: The results showed that CGHaP promotes MC3T3-E1 cell proliferation and spread on the surface of microspheres. The cells were clustered with more actin filaments and well-linked with neighbouring cells or adjacent cells when cultured in CGHaP microspheres whereas fewer cells were spread on chitosan (CH) microspheres. CGHaP microspheres significantly (p < 0.05) promoted cell attachment, proliferation and extracellular matrix mineralization. CGHaP microspheres presented significantly (p < 0.02) higher calcium deposition (0.5 ng) than CH microspheres (0.28 ng). Specifically, CGHaP microspheres exhibited high ALP activity (8 units; 2-fold) compared to CH with 3 units, after 7 days of incubation. The results suggest that CGHaP possesses a great ability to facilitate bone ingrowth formation and possibility of good osteointegration in vivo. Conclusion: The nanomaterial enhances the proliferation of pre-osteoblast cells in tissue engineering microspheres. The outcome of this study may have a major impact on the development of novel nanomaterials for bone tissue engineering.

Human osteoblastic cell response to a Ca- and P-enriched titanium surface obtained by anodization

The aim of the present study was to evaluate the in vitro osteogenic potential of subcultured human osteoblastic cells derived from alveolar bone on a titanium (Ti) surface produced by an anodized alkali treatment (BSP-AK). Primary osteoblastic cells were subcultured on BSP-AK and machined Ti discs (control) and grown for periods of up to 21 days under osteogenic conditions. Morphologic and biochemical methods were used to assess important parameters of in vitro bone-like tissue formation. Although no major differences were observed between the BSP-AK and the control Ti surface in terms of cell attachment and mineralized matrix formation, a significant increase in cell population, ALP activity, and collagen content was detected in cultures on BSP-AK surface. Our results demonstrate that human osteoblastic cells are sensitive to the BSP-AK-modified Ti surface during the transitional stage between the end of the proliferative phase and the onset of the differentiation /matrix maturation ones. Together with the good mechanical properties exhibited by the Ca- and P- coating, our findings suggest that BSP-AK treatment could be useful for the development of a new surface for dental and orthopedic implants. (c) 2008 Wiley Periodicals, Inc.J Biomed Mater Res 88A: 841-848, 2009

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains

Monocyte macrophages (M phi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to M phi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca2+/Mg2+ enhanced DV binding. This argues against involvement of beta (2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains. (C) 2001 Elsevier Science B.V. All rights reserved.

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. Contractile state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha -SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta -NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha -actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In synthetic state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta -non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic chan-es in their distribution. The distinct compartmentalisation of structural proteins observed in contractile state SMC was no longer obvious, with proteins more evenly distributed throughout die cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. (C) 2001 Wiley-Liss, Inc.

Gellan gum-coated gold nanorods: an intracellular nanosystem for bone tissue engineering

Gold nanorods (AuNRs) have emerged as an exceptional nanotool for a myriad of applications ranging from cancer therapy to tissue engineering. However, their surface modification with biocompatible and stabilizing biomaterials is crucial to allow their use in a biological environment. Herein, low-acyl gellan gum (GG) was used to coat AuNRs surface, taking advantage of its stabilizing, biocompatible and gelling features. The layer-by-layer based strategy implied the successive deposition of poly(acrylic acid), poly(allylamine hydrochloride) and GG, which allowed the formation of a GG hydrogel-like shell with 7 nm thickness around individual AuNRs. Stability studies in a wide range of pH and salt concentrations showed that the polysaccharide coating can prevent AuNRs aggregation. Moreover, a reversible pH-responsive feature of the nanoparticles was observed. Cytocompatibility and osteogenic ability of GG-coated AuNRs was also addressed. After 14 days of culturing within SaOS-2, an osteoblast-like cell line, in vitro studies revealed that AuNRs-GG exhibit no cytotoxicity, were internalized by the cells and localized inside lysosomes. AuNRs-GG combined with osteogenic media enhanced the mineralization capacity two-fold, as compared to cells exposed to osteogenic media alone. The proposed system has shown interesting features for osteogenesis, and further insights might be relevant for drug delivery, tissue engineering and regenerative medicine.

A fucan from the brown seaweed Spatoglossum schröederi inhibits Chinese hamster ovary cell adhesion to several extracellular matrix proteins

Fucans, a family of sulfated polysaccharides present in brown seaweed, have several biological activities. Their use as drugs would offer the advantage of no potential risk of contamination with viruses or particles such as prions. A fucan prepared from Spatoglossum schröederi was tested as a possible inhibitor of cell-matrix interactions using wild-type Chinese hamster ovary cells (CHO-K1) and the mutant type deficient in xylosyltransferase (CHO-745). The effect of this polymer on adhesion properties with specific extracellular matrix components was studied using several matrix proteins as substrates for cell attachment. Treatment with the polymer inhibited the adhesion of fibronectin to both CHO-K1 (2 x 10(5))()and CHO-745 (2 x 10(5) and 5 x 10(5)) cells. No effect was detected with laminin, using the two cell types. On the other hand, adhesion to vitronectin was inhibited in CHO-K1 cells and adhesion to type I collagen was inhibited in CHO-745 cells. In spite of this inhibition, the fucan did not affect either cell proliferation or cell cycle. These results demonstrate that this polymer is a new anti-adhesive compound with potential pharmacological applications.