Morphology, mechanical characterization and in vivo neo-vascularization of chitosan particle aggregated scaffolds architectures

The present study intended to evaluate the performance of chitosan-based scaffolds produced by a particle aggregation method aimed to be used in tissue engineering applications addressing key issues such as morphological characteristics, mechanical performance and in vivo behaviour. It is claimed that the particle aggregation methodology may present several advantages, such as combine simultaneously a high interconnectivity with high mechanical properties that are both critical for an in vivo successful application. In order to evaluate these properties, micro-Computed Tomography (micro-CT) and Dynamical Mechanical Analysis (DMA) were applied. The herein proposed scaffolds present an interesting morphology as assessed by micro-CT that generally seems to be adequate for the proposed applications. At a mechanical level, DMA has shown that chitosan scaffolds have an elastic behaviour under dynamic compression solicitation, being simultaneously mechanically stable in the wet state and exhibiting a storage modulus of 4.21 ! 1.04 MPa at 1 Hz frequency. Furthermore, chitosan scaffolds were evaluated in vivo using a rat muscle-pockets model for different implantation periods (1, 2 and 12 weeks). The histological and immunohistochemistry results have demonstrated that chitosan scaffolds can provide the required in vivo functionality. In addition, the scaffolds interconnectivity has been shown to be favourable to the connective tissues ingrowth into the scaffolds and to promote the neo-vascularization even in early stages of implantation. It is concluded that the proposed chitosan scaffolds produced by particle aggregation method are suitable alternatives, being simultaneously mechanical stable and in vivo biofunctional that might be used in load-bearing tissue engineering applications, including bone and cartilage regeneration.The authors would like to acknowledge the Portuguese Foundation for Science and Technology for the PhD Grant to Patricia B Malafaya (SFRH/BD/11155/2002). This work was partially supported and carried out under the scope of the European STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and European NoE EXPERTISSUES (NMP3-CT-2004-500283). The authors also thank Prof. Heinz Redl for the collaboration in the in VIVO Studies, as well as Bernhard Horing for the surgical procedures both from LBI, Austria and Joao Oliveira from 3B's Research Group, Portugal for the initial assistance with the DMA equipment

Regeneration of the intervertebral disc

Degeneration of intervertebral disc (IVD) seems to be one of the main causes associated to lower back pain (LBP), one of the most common painful conditions that lead to work absenteeism, medical visits, and hospitalization in actual society [1,2]. This complex fibro-cartilaginous structure is composed by two structures, an outer multilayer fiber structure (annulus fibrosus, AF) and a gel-like inner core (nucleus pulposus, NP), which are sandwiched in part between two cartilage endplates (CEP) [1]. Existing conservative and surgical treatments for LBP are directed to pain relief and do not adequately restore disc structure and mechanical function [2]. In the last years, several studies have been focusing on the development of tissue engineering (TE) approaches aiming to substitute/regenerate the AF or NP, or both by developing an artificial disc that could be implanted in the body thus replacing the damaged disc [3]. TE strategies aiming to regenerate NP tissue often rely on the use of natural hydrogels, due to the number of advantages that these highly hydrated networks can offer. Nevertheless, several of the hydrogel systems developed still present numerous problems, such as variability of production, and inappropriate mechanical and degradation behaviour. Recently, our group has proposed the use of gellan gum (GG) and its derivatives, namely the ionic- and photo-crosslinked methacrylated gellan gum (GG-MA) hydrogels, as potential injectable scaffolds for IVD regeneration [4,5]. Work has been conducted regarding the improvement of GG mechanical properties either by chemically modifying the polymer (allowing to better control in situ gelation and hydrogel stability) [4] or by reinforcing it with biocompatible and biodegradable GG microparticles (enabling the control of degradation rate and cell distribution) [5]. Another strategy currently under investigation relies on the development of a biphasic scaffold that mimics the total disc by using a reverse engineering approach

Stimulatory effects of inorganic ions on osteogenesis in vitro

Introduction: Several studies demonstrated the effect of silicate ions (Si) on differentiation of bone precursor cells1,2, although its exact role in processes related to bone formation and remodeling is still incompletely understood. The focus of this work is to explore the effect of calcium and silicate ions on growth and osteogenic differentiation of human mesenchymal stem cells (hMSCs). This strategy may reduce the need for growth factors required to stimulate bone formation in regenerative approaches, decreasing the associated costs and overcoming stability issues. Materials and Methods In order to define the range of Si concentrations that are not toxic to cells, we performed a preliminary study varying Si concentrations from 0.00357mM to 4mM. The concentration of the Ca ions was selected based on the earlier study by Barradas et. al3. Cell culture media were supplemented by using sodium silicate (Na2SiO3) and/or calcium chloride dehydrate (CaCl2*2H2O) as Si and Ca precursors, respectively. hMSCs derived from bone marrow were seeded at a seeding density of 2.000 cells/cm2 and allowed to adhere overnight. Then, the medium was replaced by the appropriate supplemented medium and cells were cultured for 3, 7, 14 and 18 days. Basic and osteogenic media were used as negative and positive controls. Cell proliferation was evaluated by DNA quantification. hMSCs osteogenic gene expression was evaluated by Q-PCR. Results DNA quantification indicated an increase in cell number during the culture time for all the conditions. Results obtained by Q-PCR revealed a significantly higher expression of osteocalcin (OC) and bone morphogenetic protein-2 (BMP2) in cells cultured in media supplemented by both ions, as compared to media containing either Ca or Si alone. Discussion and Conclusions DNA quantification studies indicated that none of the selected concentrations had a negative influence on cell proliferation. The increase in osteogenic gene expression for cells cultured with both Ca and Si suggested a synergistic effect of the two ions on osteogenic differentiation of hMSCs. We further showed that cells cultured in the medium with the highest concentration of Ca (7.8mM) revealed a higher expression of the selected genes, which is in accordance with the earlier results by Barradas et al3. The obtained results suggest the importance of combining both ions, Ca and Si, for promoting the osteogenic differentiation of hMSCs. References 1. Hoppe A, Biomaterials 32: 2757-2774, 2011. 2. Beck Jr GR, Nanomedicine: Nanotechnology, Biology, and Medicine,1-11, 2011 3. Barradas AMC et al., Biomaterials 3205-3215, 2012. Acknowledgments The author thanks the Portuguese Foundation for Science and Technology (FCT) for the grant (SFRH/BD/69962/2010). Disclosures The authors have nothing to disclose

Soy matrix drug delivery systems obteined by melt-processing techniques

The aim of this study was to develop new soy protein drug delivery matrix systems by melt-processing techniques, namely, extrusion and injection moulding. The soy matrix systems with an encapsulated drug (theophylline, TH) were previously compounded by extrusion performed at two different pH values, (i) pH 4 (SIpDtp) and (ii) pH 7 (SIDtp), and further injection-moulded into a desired shape. During the extrusion process the matrixes SIDtp were also cross-linked with glyoxal (0.6X-SIDtp) and reinforced with a bioactive filler, hydroxylapatite (SI-HADtp). The obtained mouldings were used to study the drug-release mechanisms from the plastic soy-TH matrixes. In an isotonic saline solution (ISS) buffered at pH 5.0 (200 mM acetate buffer), the resulting release kinetics could be described using the Fick’s second law of diffusion. Because the diffusion coefficients were found to be constant and the boundary conditions to be stationary, these systems are drug-diffusion controlled. Conversely, the dominant phenomena in an isotonic saline solution buffered at pH 7.4 (200 mM Tris/HCl buffer) are more complex. In fact, because of the higher polymer solubility, the resulting matrix is time-variant. So, the drug release is affected by swelling, drug diffusion, and polymer dissolution, being faster when compared to ISS-200 mM acetate buffer, pH 5.0. The changes in the formulation composition affecting the correspondent release rates were also investigated. At pH 7.4, increasing the cross-linking degree of the polymer matrix (via reaction with glyoxal or heat treatment) or decreasing the net charge (extruding at pH near its isoelectric point) led to lower release rates. The incorporation of ceramic filler caused the opposite effect. Because of the low solubility of the matrix at pH 5.0, no significant variations were detected with variations in the selected formulations. These systems, based on a nonstandard protein-based material, seem to be very promising to be used as carriers for drug delivery.Portuguese Foundation for Science and Technology (FCT), Ministry of Science and Technology, Portugal

Controlled delivery achieved with bi-layer matrix devices produced by co-injection moulding

The aim of this study was to design new soy protein-based bi-layered co-injection moulded matrix systems aimed to achieve controlled drug delivery. The devices consisted of a drug-free outer layer (skin) and a drug-containing core. The systems overcame the inherent disadvantage of non-linear release associated with diffusion-controlled single-layer matrix devices by providing additional releasing area with time to compensate for the decreasing release rate. As expected, the bi-layer devices presented a significant decrease in drug release rate when compared with a correspondent single layer matrix system. The skin thickness and the degree of crosslinking of the core appeared to be very important tools to tailor the release patterns. Furthermore, due to the amphoteric nature of the soy protein, the developed devices evidenced a pH-dependent behaviour. The mechanisms of drug release were also elucidated at two different pH values: i) pH 5.0, near the isoelectric point of soy (low matrix solubility); and ii) pH 7.4, physiological pH (high matrix solubility). Consequently, changing the release medium from pH 5.0 to pH 7.4 after two hours, led to an abrupt increase in drug release and the devices presented a typical controlled drug delivery profile: slow release/fast release. These evidences may provide for the development of individual systems with different release onsets that in combination may exhibit drug releases at predetermined times in a pre-programmed way. Another possibility is the production of three-layer devices presenting bimodal release profiles (fast release/slow release/fast release) by similar technologies.Portuguese Foundation for Science and Technology (FCT), Ministry of Science and Technology, Portugal

Cytocompatibility and response of osteoblastic-like cells to starch-based polymers : effect of several additives and processing conditions

This work reports on the biocompatibility evaluation of new biodegradable starch-based polymers that are under consideration for use in orthopaedic temporary applications and as tissue engineering scaffolds. It has been shown in previous works that by using these polymers it is both possible to produce polymer/hydroxyapatite (HA) composites (with or without the use of coupling agents) with mechanical properties matching those of the human bone, and to obtain 3D structures generated by solid blowing agents, that are suitable for tissue engineering applications. This study was focused on establishing the influence of several additives (ceramic fillers, blowing agents and coupling agents) and processing methods/conditions on the biocompatibility of the materials described above. The cytotoxicity of the materials was evaluated using cell culture methods, according to ISO/EN 109935 guidelines. A cell suspension of human osteosarcoma cells (HOS) was also seeded on a blend of corn starch with ethylene vinyl alcohol (SEVA-C) and on SEVA-C/HA composites, in order to have a preliminary indication on cell adhesion and proliferation on the materials surface. In general, the obtained results show that all the different materials based on SEVA-C, (which are being investigated for use in several biomedical applications), as well as all the additives (including the novel coupling agents) and different processing methods required to obtain the different properties/products, can be used without inducing a cytotoxic behaviour to the developed biomaterial

Development and design of double-layer co-injection moulded soy protein based drug delivery devices

Novel double-layer delivery devices based on soy protein derived materials were designed and produced using an innovative two material co-injection moulding technique. It was demonstrated that the viscosity ratio between core and skin layer materials played an important role in the formation of the interfacial shape, namely the skin thickness and uniformity of the bi-materials. The adequate selection of the materials used and the optimisation of the respective processing conditions enabled an accurate control of the relative thickness of the layers of the device. The preliminary results confirmed the potential of these systems to achieve a controlled drug delivery

The sovereign-bank diabolic loop and ESBies

We propose a simple model of the sovereign-bank diabolic loop, and establish four results. First, the diabolic loop can be avoided by restricting banks’ domestic sovereign exposures relative to their equity. Second, equity requirements can be lowered if banks only hold senior domestic sovereign debt. Third, such requirements shrink even further if banks only hold the senior tranche of an internationally diversified sovereign portfolio – known as ESBies in the euro-area context. Finally, ESBies generate more safe assets than domestic debt tranching alone; and, insofar as the diabolic loop is defused, the junior tranche generated by the securitization is itself risk-free