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

Unusual features of coarsening with detachment rates decreasing with cluster mass

We study conserved one-dimensional models of particle diffusion, attachment and detachment from clusters, where the detachment rates decrease with increasing cluster size as gamma(m) ~ m^{-k}, k>0. Heuristic scaling arguments based on random walk properties show that the typical cluster size scales as (t/ln(t))^z, with z=1/(k+2). The initial symmetric flux of particles between neighboring clusters is followed by an effectively assymmetric flux due to the unbalanced detachement rates, which leads to the above logarithmic correction. Small clusters have densities of order t^{-mz(1)}, with z(1) = k/(k+2). Thus, for k<1, the small clusters (mass of order unity) are statistically dominant and the average cluster size does not scale as the size of typically large clusters does. We also solve the Master equation of the model under an independent interval approximation, which yields cluster distributions and exponent relations and gives the correct dominant coarsening exponent after accounting for the effects of correlations. The coarsening of large clusters is described by the distribution P_t(m) ~ 1/t^y f(m/t^z), with y=2z. All results are confirmed by simulation, which also illustrates the unusual features of cluster size distributions, with a power law decay for small masses and a negatively skewed peak in the scaling region. The detachment rates considered here can apply in the presence of strong attractive interactions, and recent applications suggest that even more rapid rate decays are also physically realistic.Comment: 12 pages, with 9 figures include

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