Manuela E. Gomes

A new approach based on injection moulding to produce biodegradable starch-based polymeric scaffolds: morphology, mechanical and degradation behaviour.

One of the present challenges in polymer scaffold processing is the fabrication of three-dimensional (3D) architectures with an adequate mechanical performance to be used in the tissue engineering of hard tissues. This paper describes a preliminary study on the development of a new method to produce biodegradable scaffolds from a range of corn-starch-based polymers. In some cases, hydroxlapatite was also used as a reinforcement of the biodegradable polymers. The developed methodology consists of a standard conventional injection moulding process, on which a solid blowing agent based on carboxylic acids is used to generate the foaming of the bulk of the moulded part. The proposed route allows for the production of scaffolds with a compact skin and a porous core, with promising mechanical properties. By using the developed method it is possible to manufacture biodegradable polymer scaffolds in an easy (melt-based processing) and reproducible manner. The scaffolds can be moulded into complex shapes, and the blowing additives do not affect the non-cytotoxic behaviour of the starch-based materials. The materials produced using this method were evaluated with respect to the morphology of the porous structure, and the respective mechanical properties and degradation behaviour. It was demonstrated that it is possible to obtain, by a standard melt based processing route, 3D scaffolds with complex shapes that exhibit an appropriate morphology, without decreasing significantly the mechanical properties of the materials. It is believed that the optimisation of the proposed processing methodology may lead to the production of scaffolds that might be used on the regeneration of load-bearing tissues.

Alternative tissue engineering scaffolds based on starch: processing methodologies, morphology, degradation and mechanical properties

Abstract An ideal tissue engineering scaffold must be designed from a polymer with an adequate degradation rate. The processing technique must allow for the preparation of 3-D scaffolds with controlled porosity and adequate pore sizes, as well as tissue matching mechanical properties and an appropriate biological response. This communication revises recent work that has been developed in our laboratories with the aim of producing 3-D polymeric structures (from starch-based blends) with adequate properties to be used as scaffolds for bone tissue engineering applications. Several processing methodologies were originally developed and optimised. Some of these methodologies were based on conventional melt-based processing routes, such as extrusion using blowing agents (BA) and compression moulding (combined with particulate leaching). Other developed technologies included solvent casting and particle leaching and an innovative in situ polymerization method. By means of using the described methodologies, it is possible to tailor the properties of the different scaffolds, namely their degradation, morphology and mechanical properties, for several applications in tissue engineering. Furthermore, the processing methodologies (including the blowing agents used in the melt-based technologies) described above do not affect the biocompatible behaviour of starch-based polymers. Therefore, scaffolds obtained from these materials by means of using one of the described methodologies may constitute an important alternative to the materials currently used in tissue engineering.

Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells.

Novel silicate nanoplatelets that induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of any osteoinductive factor are reported. The presence of the silicate triggers a set of events that follows the temporal pattern of osteogenic differentiation. These findings underscore the potential applications of these silicate nanoplatelets in designing bioactive scaffolds for musculoskeletal tissue engineering.

Contribution of outgrowth endothelial cells from human peripheral blood on in vivo vascularization of bone tissue engineered constructs based on starch polycaprolactone scaffolds

In the present study we assessed the potential of human outgrowth endothelial cells (OEC), a subpopulation within endothelial progenitor cell cultures, to support the vascularization of a complex tissue engineered construct for bone. OEC cultured on starch polycaprolactone fiber meshes (SPCL) in monoculture retained their endothelial functionality and responded to angiogenic stimulation by VEGF (vascular endothelial growth factor) in fibrin gel-assays in vitro. Co-culture of OEC with human primary osteoblasts (pOB) on SPCL, induced an angiogenic activation of OEC towards microvessel-like structures achieved without additional supplementation with angiogenic growth factors. Effects of co-cultures with pOB on the vascularization process by OEC in vivo were tested by subcutaneous implantation of Matrigel plugs containing both, OEC and pOB, and resulted in OEC-derived blood vessels integrated into the host tissue and anastomosed to the vascular supply. In addition, morphometric analysis of the vascularization process by OEC indicated a better performance of OEC in the co-cultures with primary osteoblasts compared to monocultures of OEC. The contribution of OEC to vascular structures and the beneficial effect of the co-culture with primary human osteoblasts on the vascularization in vivo was additionally proven by subcutaneous implantation of pre-cellularized and pre-cultured SPCL constructs. OEC contributed to the vascular structures, by generating autogenic vessels or by incorporation into chimeric vessels consisting of both, human and mouse endothelial cells. The current data highlight the vasculogenic potential of OEC for bone tissue engineering applications and indicate a beneficial influence of constructs including both osteoblasts and endothelial cells for vascularization strategies.

Modified Gellan Gum hydrogels with tunable physical and mechanical properties.

Gellan Gum (GG) has been recently proposed for tissue engineering applications. GG hydrogels are produced by physical crosslinking methods induced by temperature variation or by the presence of divalent cations. However, physical crosslinking methods may yield hydrogels that become weaker in physiological conditions due to the exchange of divalent cations by monovalent ones. Hence, this work presents a new class of GG hydrogels crosslinkable by both physical and chemical mechanisms. Methacrylate groups were incorporated in the GG chain, leading to the production of a methacrylated Gellan Gum (MeGG) hydrogel with highly tunable physical and mechanical properties. The chemical modification was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR-ATR). The mechanical properties of the developed hydrogel networks, with Youngs modulus values between 0.15 and 148 kPa, showed to be tuned by the different crosslinking mechanisms used. The in vitro swelling kinetics and hydrolytic degradation rate were dependent on the crosslinking mechanisms used to form the hydrogels. Three-dimensional (3D) encapsulation of NIH-3T3 fibroblast cells in MeGG networks demonstrated in vitro biocompatibility confirmed by high cell survival. Given the highly tunable mechanical and degradation properties of MeGG, it may be applicable for a wide range of tissue engineering approaches.

The Potential of Cellulose Nanocrystals in Tissue Engineering Strategies

Cellulose nanocrystals (CNCs) are a renewable nanosized raw material that is drawing a tremendous level of attention from the materials community. These rod-shaped nanocrystals that can be produced from a variety of highly available and renewable cellulose-rich sources are endowed with exceptional physicochemical properties which have promoted their intensive exploration as building blocks for the design of a broad range of new materials in the past few decades. However, only recently have these nanosized substrates been considered for bioapplications following the knowledge on their low toxicity and ecotoxicological risk. This Review provides an overview on the recent developments on CNC-based functional biomaterials with potential for tissue engineering (TE) applications, focusing on nanocomposites obtained through different processing technologies usually employed in the fabrication of TE scaffolds into various formats, namely, dense films and membranes, hierarchical three-dimensional (3D) porous constructs (micro/nanofibers mats, foams and sponges), and hydrogels. Finally, while highlighting the major achievements and potential of the reviewed work on cellulose nanocrystals, alternative applications for some of the developed materials are provided, and topics for future research to extend the use of CNCs-based materials in the scope of the TE field are identified.

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 biomaterials.

Distinct Stem Cells Subpopulations Isolated from Human Adipose Tissue Exhibit Different Chondrogenic and Osteogenic Differentiation Potential

Recently adipose tissue has become a research topic also for the searching for an alternative stem cells source to use in cell based therapies such as tissue engineer. In fact Adipose Stem Cells (ASCs) exhibit an important differentiation potential for several cell lineages such as chondrogenic, osteogenic, myogenic, adipogenic and endothelial cells. ASCs populations isolated using standard methodologies (i.e., based on their adherence ability) are very heterogeneous but very few studies have analysed this aspect. Consequently, several questions are still pending, as for example, on what regard the existence/ or not of distinct ASCs subpopulations. The present study is originally aimed at isolating selected ASCs subpopulations, and to analyse their behaviour towards the heterogeneous population regarding the expression of stem cell markers and also regarding their osteogenic and chondrogenic differentiation potential. Human Adipose derived Stem Cells (hASCs) subpopulations were isolated using immunomagnetic beads coated with several different antibodies (CD29, CD44, CD49d, CD73, CD90, CD 105, Stro-1 and p75) and were characterized by Real Time RT-PCR in order to assess the expression of mesenchymal stem cells markers (CD44, CD73, Stro-1, CD105 and CD90) as well as known markers of the chondrogenic (Sox 9, Collagen II) and osteogenic lineage (Osteopontin, Osteocalcin). The obtained results underline the complexity of the ASCs population demonstrating that it is composed of several subpopulations, which express different levels of ASCs markers and exhibit distinctive differentiation potentials. Furthermore, the results obtained clearly evidence of the advantages of using selected populations in cell-based therapies, such as bone and cartilage regenerative medicine approaches.

Cartilage Tissue Engineering Using Electrospun PCL Nanofiber Meshes and MSCs

Mesenchymal stem cells (MSCs) have been recognized for their ability to differentiate into cells of different tissues such as bone, cartilage, or adipose tissue, and therefore are of great interest for potential therapeutic strategies. Adherent, colony-forming, fibroblastic cells were isolated from human bone marrow aspirates, from patients undergoing knee arthroplasties, and the MSCs phenotype characterized by flow cytometry. Afterward, cells were seeded onto electrospun polycaprolactone nanofiber meshes and cultured in a multichamber flow perfusion bioreactor to determine their ability to produce cartilagineous extracellular matrix. Results indicate that the flow perfusion bioreactor increased the chondrogenic differentiation of hBM-MSCs, as confirmed either by morphological and RT-PCR analysis. Cartilage-related genes such as aggrecan, collagen type II, and Sox9 were expressed. ECM deposition was also detected by histological procedures. Collagen type II was present in the samples, as well as collagen type I. Despite no statistically significant values being obtained for gene expression, the other results support the choice of the bioreactor for this type of culture.

Preliminary study on the adhesion and proliferation of human osteoblasts on starch-based scaffolds

Up to today, several techniques have been used to produce biodegradable porous scaffolds for tissue engineering. In this work, a new technique based on extrusion by using blowing agents in combination with a 50:50 (wt.%) blend of starch/cellulose acetate (SCA) was studied. The results show that by using this technique it was possible to obtain scaffolds with 70% of porosity and a fully interconnected network of pores, with sizes ranging from 200 to 500 Am. After their production, the mechanical properties of these scaffolds were tested, presenting a compressive modulus of 124.6F27.2 MPa and a compressive strength of 8.0F0.9 MPa. These values are within the best found in the literature and show that by using this technique, it is possible to produce scaffolds that, from a mechanical standpoint, may be suitable for bone tissue engineering. Cell culturing experiments showed that cells were viable and that there were no signs of cellular death after 3 weeks of culture. Finally, biochemical assays demonstrate that cells maintained the osteogenic phenotype throughout the experiment and deposition of mineralized extracellular matrix could be detected. D 2002 Elsevier Science B.V. All rights reserved.