Catarina M. Alves

Responsive and in situ-forming chitosan scaffolds for bone tissue engineering applications: an overview of the last decade

The use of bioabsorbable polymeric scaffolds is being investigated for use in bone tissue engineering applications, as their properties can be tailored to allow them to degrade and integrate at optimal rates as bone remodelling is completed. The main goal of this review is to highlight the “intelligent” properties exhibited by chitosan scaffolds and their use in the bone tissue engineering field. To complement the fast evolution of the bone tissue engineering field, it is important to propose the use of responsive scaffolds and take advantage of bioinspired materials and their properties as emerging technologies. There is a growing interest and need for new biomaterials, such as “smart”/responsive materials with the capability to respond to changes in the in vivo environment. This review will provide an overview of strategies that can modulate bone tissue regeneration by using in situ-forming scaffolds.

Immobilization of fibronectin in chitosan substrates improves cell adhesion and proliferation

Covalent grafting of biomolecules is a strategy to improve the biocompatibility and bioactivity of materials. However, it is critical to maintain the biological activity of the biomolecule upon its attachment to the surface. In the present study we compared the biological properties of chitosan, in which the surface was enriched with fibronectin (Fn), using two methodologies: chemical immobilization, using a water‐soluble carbodiimide; and simple adsorption. X‐ray photoelectron spectroscopy studies confirmed the successful immobilization of Fn onto modified membranes. SaOs‐2 cells were seeded onto these surfaces to assess the biological consequences of such modifications. The presence of Fn stimulated cell adhesion on chitosan. It was found that after 7 days of culture in the presence of covalently attached Fn, the cells are confluent; significantly fewer cells were detected in unmodified film and in film with adsorbed Fn. This result is consistent with the fact that considerable desorption of Fn from chitosan takes place within 24 h in culture medium. This study showed that Fn may be easily covalently attached onto chitosan substrates, improving the biological performance of the material. The technique could find applications in tissue‐engineering strategies, as the surface modification of chitosan‐based substrates could be carried out in more complex geometries, such as in scaffolds or particles. Copyright

The effect of chitosan on the in vitro biological performance of chitosan-poly(butylene succinate) blends.

Chitosan blends with synthetic biodegradable polymers have been proposed for various biomedical applications due to their versatile mechanical properties and easier processing. However, details regarding the main surface characteristics that may benefit from the blending of these two types of materials are still missing. Hence, this work aims at investigating the surface properties of chitosan-based blends, illustrating the way these properties determine the material-proteins interactions and ultimately the behavior of osteoblast-like cells. The surface characteristics of modified and nonmodified blends were assessed using complimentary techniques such as optical microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR-ATR), X-ray photoelectron spectroscopy (XPS), contact angle measurements and surface energy calculations. The adsorption of human serum albumin (HSA) and human plasma fibronectin (HFN) onto the different surfaces was quantified by association of an indirect method with a colorimetric assay. It was found that the presence of chitosan on the surface promoted the adsorption of proteins. Moreover, a preferential adsorption of albumin over fibronectin was registered. The in vitro biological performance of the studied materials was further investigated by a direct contact assay with an osteoblastic-like cell line (SaOs-2). A synergistic effect of the two components of the blend was observed. While the synthetic polyester promoted the adhesion of SaOs-2, the presence of chitosan significantly enhanced the osteoblastic activity of these cells. This work further confirmed the interest in designing polymeric blends with natural polymers as a successful strategy to enhance the biological performance of a biomaterial.

The competitive adsorption of human proteins onto natural-based biomaterials

This study aims to further the understanding of nanoscale structures relevant for cellular recognition on contact and interaction with natural-based materials. The correlation between surface characteristics and protein adsorption from unitary and complex protein systems was investigated with respect to altering the bulk chemistry of the substrate material. Polymeric blends of starch and cellulose acetate, polycaprolactone (SPCL) and ethylene vinyl alcohol (SEVA-C) were used. Different proteins, bovine serum albumin, human serum albumin (HSA) and human fibronectin (HFN), were selected for this study. The construction of adsorption isotherms is an important starting point towards characterizing the interactions between surfaces and proteins. In this study, albumin adsorption isotherms fit the Freundlich model and were correlated with the chemistry and morphology of surfaces. In addition, protein distribution, quantification and competition were measured using fluorimetry and visualized by confocal microscopy. The analysis of unitary systems demonstrated that the adsorption of HSA was generally lower than that of HFN. In the latter case, SPCL and SEVA-C blends reached adsorption values of 97 and 89 per cent, respectively. In studying the co-adsorption of proteins, an increase in both HSA and HFN on SEVA-C surfaces was observed. SPCL showed no substantial increase in the adsorption of the proteins in competitive conditions. The similarity of these materials with other polysaccharide-based materials increases the relevance of the presented results. This study provides valuable information for the development of strategies towards the control of protein orientation and functionality as the availability of cell signalling epitopes for a broader family of materials that continue to be a significant component of this field of research.

Cell adhesion and proliferation on biomimetic calcium-phosphate coatings produced by a sodium silicate gel methodology

The present study describes a methodology to produce bioactive coatings on the surface of starch based biodegradable polymers or other polymeric biomaterials. As an alternative to the more typical bioactive glass percursors, a sodium silicate gel is being employed as a nucleating agent, for inducing the formation of a calcium-phosphate (Ca-P) layer. The method has the advantage of being able to coat efficiently both compact materials and porous 3D architectures aimed at being used on tissue replacement applications and as bone tissue engineering scaffolds. This treatment is also very effective in reducing the incubation periods, being possible to observe the formation of an apatite-like layer, only after 6 h of immersion in a simulated body fluid (SBF). The influence of the SBF concentration on the formation of the apatite coating was also studied. The apatite coatings formed under different conditions were analyzed and compared in terms of morphology, chemical composition and structure. After the first days of SBF immersion, the apatite-like films exhibit the typical cauliflower like morphology. With increasing immersion times, these films exhibited a partially amorphous nature and the Ca/P ratios became very closer to the value attributed to hydroxyapatite (1.67). The obtained results are very promising for pre-calcifying bone tissue engineering scaffolds. Therefore, in order to study cell behavior and response to these apatite coatings, adhesion, morphology, and proliferation of a human osteoblast cell line (SaOS-2) was also analyzed after being cultured in the coatings formed after 15 days of immersion in SBF. Results indicate a good correlation between crystallinity of the apatite like coatings formed in these conditions and respective cell spreading and morphology. In general, higher cell proliferation was observed for higher crystalline Ca-P coatings.

Surface-modified 3D starch-based scaffold for improved endothelialization for bone tissue engineering

Providing adequate vascularization is one of the main hurdles to the widespread clinical application of bone tissue engineering approaches. Due to their unique role in blood vessel formation, endothelial cells (EC) play a key role in the establishment of successful vascularization strategies. However, currently available polymeric materials do not generally support EC growth without coating with adhesive proteins. In this work we present argon plasma treatment as a suitable method to render the surface of a 3D starch-based scaffold compatible for ECs, this way obviating the need for protein pre-coating. To this end we studied the effect of plasma modification on surface properties, protein adsorption and ultimately on several aspects regarding EC behaviour. Characterization of surface properties revealed increased surface roughness and change in topography, while at the chemical level a higher oxygen content was demonstrated. The increased surface roughness of the material, together with the changed surface chemistry modulated protein adsorption as indicated by the different adsorption profile observed for vitronectin. In vitro studies showed that human umbilical vein ECs (HUVECs) seeded on plasma-modified scaffolds adhered, remained viable, proliferated, and maintained the typical cobblestone morphology, as observed for positive controls (scaffold pre-coated with adhesive proteins). Furthermore, genotypic expression of endothelial markers was maintained and neighbouring cells expressed PECAM-1 at the single-cell-level. These results indicate that Ar plasma modification is an effective methodology with potential to be incorporated in biomaterial strategies to promote the formation of vascularized engineered bone.

Preliminary study on human protein adsorption and leukocyte adhesion to starch-based biomaterials

In this study, the adsorption of human serum albumin (HSA), fibronectin (FN) and vitronectin (VN) onto the surface of novel biodegradable materials was evaluated by immunostaining. Specifically, polymeric blends of corn starch with cellulose acetate (SCA), ethylene vinyl alcohol copolymer (SEVA-C), and polycaprolactone (SPCL) were immersed in unitary and competitive systems; that is, binary and more complex protein solutions. For binary solutions, different HSA and FN protein distribution patterns were observed depending on the starch-based surface. Furthermore, the relative amount of proteins adsorbed onto starch-based surfaces was clearly affected by protein type: a preferential adsorption of VN and FN as compared to HSA was observed. On tests carried out with unitary, binary and more complex solutions, it was found that vitronectin adsorption ability was enhanced in competitive systems, which was associated with a lower amount of adsorbed albumin. In order to assess the effect of these human proteins on cell behavior, a mixed population of human lymphocytes and monocytes/macrophages was cultured over pre-coated SEVA-C surfaces. Through anti-CD3 and CD-14 monoclonal antibody labeling and cell counting, leukocyte adhesion onto pre-coated SEVA-C surfaces was analyzed. Based on the results, it was possible to detect albumin long-term effects and fibronectin short-term effects on cell adhesion proving that previously adsorbed proteins modulate leukocyte behavior.

Gradual pore formation in natural origin scaffolds throughout subcutaneous implantation

This study used a rat subcutaneous implantation model to investigate gradual in situ pore formation in a self-regulating degradable chitosan-based material, which comprises lysozyme incorporated into biomimetic calcium phosphate (CaP) coatings at the surface to control the scaffold degradation and subsequent pore formation. Specifically, the in vivo degradation of the scaffolds, the in situ pore formation, and the tissue response were investigated. Chitosan or chitosan/starch scaffolds were studied with and without a CaP coating in the presence or absence of lysozyme for a total of six experimental groups. Twenty-four scaffolds per group were implanted, and eight scaffolds were retrieved at each of three time points (3, 6, and 12 weeks). Harvested samples were analyzed for weight loss, microcomputed tomography, and histological analysis. All scaffolds showed pronounced weight loss and pore formation as a function of time. The highest weight loss was 29.8% ± 1.5%, obtained at week 12 for CaP chitosan/starch scaffolds with lysozyme incorporated. Moreover, all experimental groups showed a significant increase in porosity after 12 weeks. At all time points no adverse tissue reaction was observed, and as degradation increased, histological analysis showed cellular ingrowth throughout the implants. Using this innovative methodology, the ability to gradually generate pores in situ was clearly demonstrated in vivo.

Effecs of the Incorporation of Proteins and Active Enzymes on Biomimetic Calcium-Phosphate Coatings

In order to investigate the influence of biomolecules on the formation of a calcium phosphate (Ca-P) layer on a starch based biodegradable polymer, proteins and enzymes were incorporated on Ca-P biomimetic coatings. In this study, a biodegradable blend of corn starch with cellulose acetate (SCA) was used as substrate and, by means of dapting a biomimetic route, the formation of a bioactive layer was evaluated. Bioactive glass ( 45S5 Bioglass ) was used as nucleating agent. Two different types of conditions were studied: (i) the addition of a-amylase or bovine serum albumin (BSA) to a simulated body fluid (SBF) solution and ( ii) the addition of aamylase or BSA to the bioactive glass used to nucleate the coati ng. The obtained results indicated that both proteins and enzymes could significantly change the morphology a nd growth of biomimetic Ca-P coatings. Furthermore, this work clearly showed tha t the incorporation of active enzymes allows for controlling the degradation rate of starch-based polymeric m aterials.

Combination of enzymes and flow perfusion conditions improves osteogenic differentiation of bone marrow stromal cells cultured upon starch/poly(e-caprolactone) fiber meshes

Previous studies have shown that alpha-amylase and lipase are capable of enhancing the degradation of fiber meshes blends of starch and poly(epsilon-caprolactone) (SPCL) under dynamic conditions, and consequently to promote the proliferation and osteogenic differentiation of bone marrow stromal cells (MSCs). This study investigated the effect of flow perfusion bioreactor culture in combination with enzymes on the osteogenic differentiation of MSCs. SPCL fiber meshes were seeded with MSCs and cultured with osteogenic medium supplemented with alpha-amylase, lipase, or a combination of the two for 8 or 16 days using static or flow conditions. Lipase and its combination with alpha-amylase enhanced cell proliferation after 16 days. In addition, the flow perfusion culture enhanced the infiltration of cells and facilitated greater distribution of extracellular matrix (ECM) throughout the scaffolds in the presence/absence of enzymes. A significant amount of calcium was detected after 16 days in all groups cultured in flow conditions compared with static cultures. Nevertheless, when alpha-amylase and lipase were included in the flow perfusion cultures, the calcium content was 379 +/- 30 microg/scaffold after as few as 8 days. The highest calcium content (1271 +/- 32 microg/scaffold) was obtained for SPCL/cell constructs cultured for 16 days in the presence of lipase and flow. Furthermore, von Kossa staining and tetracycline fluorescence of histological sections demonstrated mineral deposition within the scaffolds for all groups cultured for 16 days under flow. However, all the data corroborate that lipase coupled with flow perfusion conditions improve the osteogenic differentiation of MSCs and enhance ECM mineralization.