J. Miguel Oliveira

Physicochemical characterization of novel chitosan-soy protein/TEOS porous hybrids for tissue engineering applications

In this paper we report a new type of cross-linked porous structure based on a chitosansoy protein blend system developed by means of combining a sol-gel process with the freeze-drying technique. The final structure was investigated by Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR), contact angle measurements and the morphology by scanning electron microscopy (SEM). The water uptake capability and the weight loss were measured up to 14 days and their mechanical properties were assessed with compression tests. Results showed that the addition of tetraethyl orthosilicate (TEOS) to the chitosan-soy protein blend system provide specific interactions at the interface between the two polymers allowing to tailor the size and distribution as well as the degradation rate of the hybrids. Finally, TEOS incorporation induces an increase of the surface energy that influences the final physicochemical properties of the materials.

Innovative Technique for the Preparation of Porous Bilayer Hydroxyapatite/Chitosan Scaffolds for Osteochondral Applications

In this study, it is shown that it is possible to develop 3D-porous bilayer hydroxyapatite/chitosan scaffolds by means of combining a sintering and a freeze-drying technique. Scanning electron microscopy (SEM/EDS) studies revealed that the scaffolds possess a well-defined orientation and anisotropic porosity, with pore size ranging between 50-350 µm. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) patterns evidenced the formation of crystalline hydroxyapatite. Moreover, the compression tests revealed that these scaffolds have adequate mechanical properties for being used in tissue engineering of osteochondral defects.

Nanoparticles for bone tissue engineering

Tissue engineering (TE) envisions the creation of functional substitutes for damaged tissues through integrated solutions, where medical, biological, and engineering principles are combined. Bone regeneration is one of the areas in which designing a model that mimics all tissue properties is still a challenge. The hierarchical structure and high vascularization of bone hampers a TE approach, especially in large bone defects. Nanotechnology can open up a new era for TE, allowing the creation of nanostructures that are comparable in size to those appearing in natural bone. Therefore, nanoengineered systems are now able to more closely mimic the structures observed in naturally occurring systems, and it is also possible to combine several approaches ‐ such as drug delivery and cell labeling ‐ within a single system. This review aims to cover the most recent developments on the use of different nanoparticles for bone TE, with emphasis on their application for scaffolds improvement; drug and gene delivery carriers, and labeling techniques.

Structural Interpretation of the In Vitro Reactivity of SiO2-MgO-Na2O Glasses

The in vitro reactivity of different glasses, with 55mol% SiO2 and MgO/Na2O molar ratios ranging from 1/8 to 8/1, was investigated. Despite the same amount of SiO2, the glasses exposed different reactivities, from very reactive (low MgO/Na2O ratio) to inert (high MgO/Na2O ratio). These results are interpreted in terms of a cross-link disruption by Na2O. Introduction Since the pioneer studies of Prof. L.Hench on bioactive glasses, numerous compositions have been investigated, both in vitro and in vivo. However, most of studies usually rely on the results rather than the thorough interpretation. Hill has underlined this issue, when meeting difficulties in calculating the glass “network connectivity” for several bioactive glasses, due to inadequate information provided in the published reports [1]. The present work aims at providing an insight into the influence of glass structure on their in vitro reactivity, on the basis of preliminary results in the system SiO2-MgO-Na2O. Glass. Our previous investigations on SiO2-P2O5-CaO-Na2O-MgO glasses added new perception to the relationship between glass structure and properties, namely amorphous phase separation and in vitro reactivity in simulated body fluids [2-5]. Oliveira et al. have reviewed the most important studies done when MgO is incorporated in bio-glasses in ref.[5]. In general, MgO has been attributed an inhibiting effect on surface activity, although our experimental evidence supported rather a structural role, whereby MgO may actually improve mineralisation. In the present study, we have selected the ternary system 55SiO2-(45-x)MgO-xNa2O (mol%) with x=5 to 40. The SiO2 concentration chosen is that generally accepted as a threshold for bioactive behaviour in monolithic glasses [6]. Figure 1 and Table 1 present the eight compositions produced. Structural Characteristics. Structurally, silica glasses are networks of Si-centred SiO4 tetrahedra linked by oxygen corners. Tetrahedra can be either found isolated or connected with others by one, two, three or four binding oxygens, being identified, respectively, as Q, Q, Q, Q or Q structural units. Techniques, such as Raman spectroscopy or NMR [2, 3], can be employed to determine the structural configurations. A related concept is the degree of cross-linking, i.e. the average number of bridging oxygens per tetrahedron. Several indexes aim at quantifying the crosslinking. Among them, Stevel’s number Y [7], which anticipates that if Y>3 then the glass is nonbioactive. For all the investigated glasses (Table 1), Stevel’s number was calculated as Y=2.36. Materials and experimental procedure Glass blocks were prepared in 50g batches using reagent–grade MgO, Na2CO3 and SiO2 powders, homogenized in an agate mill for 30 minutes. The mixtures were then melted in Pt-crucibles in air between 1350-1550°C (depending on composition), for 3h. The melts were cast into metal moulds and annealed at ~500°C for 1h, in air. Raman spectroscopy was applied to small prismatic glass blocks (Jobin Yvon SPEX, T64000, Ar-laser source 514.5 nm). Key Engineering Materials Online: 2003-05-15 ISSN: 1662-9795, Vols. 240-242, pp 217-220 doi:10.4028/www.scientific.net/KEM.240-242.217

Basic science of osteoarthritis.

Osteoarthritis (OA) is a prevalent, disabling disorder of the joints that affects a large population worldwide and for which there is no definitive cure. This review provides critical insights into the basic knowledge on OA that may lead to innovative end efficient new therapeutic regimens. While degradation of the articular cartilage is the hallmark of OA, with altered interactions between chondrocytes and compounds of the extracellular matrix, the subchondral bone has been also described as a key component of the disease, involving specific pathomechanisms controlling its initiation and progression. The identification of such events (and thus of possible targets for therapy) has been made possible by the availability of a number of animal models that aim at reproducing the human pathology, in particular large models of high tibial osteotomy (HTO). From a therapeutic point of view, mesenchymal stem cells (MSCs) represent a promising option for the treatment of OA and may be used concomitantly with functional substitutes integrating scaffolds and drugs/growth factors in tissue engineering setups. Altogether, these advances in the fundamental and experimental knowledge on OA may allow for the generation of improved, adapted therapeutic regimens to treat human OA.

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 were 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 by two fold the mineralization capacity, 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.

Preparation of Bioactive Coatings on the Surface of Bioinert Polymers through an Innovative Auto-Catalytic Electroless Route

The aim of this research was to develop a new methodology to obtain bioactive coatings on bioinert and biodegradable polymers that are not intrinsically bioactive. In this study, three types of materials were used as substrates: (i) high molecular weight polyethylene (HMWPE) and two different types of starch based blends (ii) starch/ethylene vinyl alcohol blends, SEVA-C, and (iii)starch/cellulose acetate blends, SCA. These materials were obtained by injection moulding and by extrusion with blowing agents in order to obtain compact/porous 3D architectures. Three types of baths were developed in order to produce the newly proposed auto-catalytic Ca-P coatings: (i)alkaline, (ii) acid, and (iii) oxidant bath. The obtained results indicated that it was possible to coat the materials surfaces with calcium phosphate (Ca-P) layer with only 60 min of immersion in the different types of auto-catalytic solutions. These innovative auto-catalytic electroless route allows for the production of an adherent bioactive film on the polymeric surfaces. Furthermore, it was possible observe by SEM/EDS the clear bioactive nature of the Ca-P coatings after different immersion periods, in a simulated body fluid (SBF).

Carboxymethylchitosan/Calcium Phosphate Hybrid Materials Prepared by an Innovative Auto-Catalytic Co-Precipitation Method

In this study, it is shown that it is possible to prepare carboxymethyl-chitosan/Ca-P hybrids using an innovative “auto-catalytic” co-precipitation method, namely by using an acid and an oxidant bath. The X-ray diffraction (XRD) patterns evidenced the formation of crystalline calcium-phosphate precipitates when using an acid bath, while amorphous ones were obtained for those produced in the oxidant bath. The Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscopy (SEM/EDS) studies revealed that the extent of the polymer precipitation and formation of calcium-phosphates is directly dependent on the pH and composition of the baths. Furthermore, by conducting bioactivity tests in a simulated body fluid (SBF) followed by the SEM/EDS analysis it was possible to detect the formation of an apatite layer with a cauliflower-like morphology on the surface of hybrids prepared by the acid bath, after 7 days of immersion. These results are quite promising because they can allow for the production of bioactive and biodegradable 3D porous scaffolds to be used in bone tissue engineering applications.

Hydrogel-based scaffolds to support intrathecal stem cell transplantation as a gateway to the spinal cord: clinical needs, biomaterials, and imaging technologies

The prospects for cell replacement in spinal cord diseases are impeded by inefficient stem cell delivery. The deep location of the spinal cord and complex surgical access, as well as densely packed vital structures, question the feasibility of the widespread use of multiple spinal cord punctures to inject stem cells. Disorders characterized by disseminated pathology are particularly appealing for the distribution of cells globally throughout the spinal cord in a minimally invasive fashion. The intrathecal space, with access to a relatively large surface area along the spinal cord, is an attractive route for global stem cell delivery, and, indeed, is highly promising, but the success of this approach relies on the ability of cells (1) to survive in the cerebrospinal fluid (CSF), (2) to adhere to the spinal cord surface, and (3) to migrate, ultimately, into the parenchyma. Intrathecal infusion of cell suspension, however, has been insufficient and we postulate that embedding transplanted cells within hydrogel scaffolds will facilitate reaching these goals. In this review, we focus on practical considerations that render the intrathecal approach clinically viable, and then discuss the characteristics of various biomaterials that are suitable to serve as scaffolds. We also propose strategies to modulate the local microenvironment with nanoparticle carriers to improve the functionality of cellular grafts. Finally, we provide an overview of imaging modalities for in vivo monitoring and characterization of biomaterials and stem cells. This comprehensive review should serve as a guide for those planning preclinical and clinical studies on intrathecal stem cell transplantation.

Silk Fibroin-Based Hydrogels and Scaffolds for Osteochondral Repair and Regeneration

Osteochondral lesions treatment and regeneration demands biomimetic strategies aiming physicochemical and biological properties of both bone and cartilage tissues, with long-term clinical outcomes. Hydrogels and scaffolds appeared as assertive approaches to guide the development and structure of the new osteochondral engineered tissue. Moreover, these structures alone or in combination with cells and bioactive molecules bring the mechanical support after in vitro and in vivo implantation. Moreover, multilayered structures designed with continuous interfaces furnish appropriate features of the cartilage and subchondral regions, namely microstructure, composition, and mechanical properties. Owing the potential as scaffolding materials, natural and synthetic polymers, bioceramics, and composites have been employed. Particularly, significance is attributed to the natural-based biopolymer silk fibroin from the Bombyx mori silkworm, considering its unique mechanical and biological properties. The significant studies on silk fibroin-based structures, namely hydrogels and scaffolds, towards bone, cartilage, and osteochondral tissue repair and regeneration are overviewed herein. The developed biomimetic strategies, processing methodologies, and final properties of the structures are summarized and discussed in depth.