Ana M. Martins

Electrically Conductive Chitosan/Carbon Scaffolds for Cardiac Tissue Engineering

In this work, carbon nanofibers were used as doping material to develop a highly conductive chitosan-based composite. Scaffolds based on chitosan only and chitosan/carbon composites were prepared by precipitation. Carbon nanofibers were homogeneously dispersed throughout the chitosan matrix, and the composite scaffold was highly porous with fully interconnected pores. Chitosan/carbon scaffolds had an elastic modulus of 28.1 ± 3.3 KPa, similar to that measured for rat myocardium, and excellent electrical properties, with a conductivity of 0.25 ± 0.09 S/m. The scaffolds were seeded with neonatal rat heart cells and cultured for up to 14 days, without electrical stimulation. After 14 days of culture, the scaffold pores throughout the construct volume were filled with cells. The metabolic activity of cells in chitosan/carbon constructs was significantly higher as compared to cells in chitosan scaffolds. The incorporation of carbon nanofibers also led to increased expression of cardiac-specific genes involved in muscle contraction and electrical coupling. This study demonstrates that the incorporation of carbon nanofibers into porous chitosan scaffolds improved the properties of cardiac tissue constructs, presumably through enhanced transmission of electrical signals between the cells.

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.

Natural origin scaffolds with in situ pore forming capability for bone tissue engineering applications

This work describes the development of a biodegradable matrix, based on chitosan and starch, with the ability to form a porous structure in situ due to the attack by specific enzymes present in the human body (alpha-amylase and lysozyme). Scaffolds with three different compositions were developed: chitosan (C100) and chitosan/starch (CS80-20, CS60-40). Compressive test results showed that these materials exhibit very promising mechanical properties, namely a high modulus in both the dry and wet states. The compressive modulus in the dry state for C100 was 580+/-33MPa, CS80-20 (402+/-62MPa) and CS60-40 (337+/-78MPa). Degradation studies were performed using alpha-amylase and/or lysozyme at concentrations similar to those found in human serum, at 37 degrees C for up to 90 days. Scanning electron micrographs showed that enzymatic degradation caused a porous structure to be formed, indicating the potential of this methodology to obtain in situ forming scaffolds. In order to evaluate the biocompatibility of the scaffolds, extracts and direct contact tests were performed. Results with the MTT test showed that the extracts of the materials were clearly non-toxic to L929 fibroblast cells. Analysis of cell adhesion and morphology of seeded osteoblastic-like cells in direct contact tests showed that at day 7 the number of cells on CS80-20 and CS60-40 was noticeably higher than that on C100, which suggests that starch containing materials may promote cell adhesion and proliferation. This combination of properties seems to be a very promising approach to obtain scaffolds with gradual in vivo pore forming capability for bone tissue engineering applications.

The Role of Lipase and α-Amylase in the Degradation of Starch/Poly(ɛ-Caprolactone) Fiber Meshes and the Osteogenic Differentiation of Cultured Marrow Stromal Cells

The present work studies the influence of hydrolytic enzymes (alpha-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(epsilon-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium-expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (alpha-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with alpha-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 microm from the surface. This study demonstrates that supplementation of culture media with lipase holds great potential for the generation of bone tissue engineering constructs from MSCs seeded onto SPCL fiber meshes, because lipase enhances the osteoblastic differentiation of the seeded MSCs and promotes matrix mineralization without harming the structural integrity of the meshes over 16 days of culture.

In vitro degradation and in vivo biocompatibility of chitosan–poly(butylene succinate) fiber mesh scaffolds

In tissue engineering, the evaluation of the host response to the biomaterial implantation must be assessed to determine the extent of the inflammatory reaction. We studied the degradation of poly(butylene succinate) and chitosan in vitro using lipase and lysozyme enzymes, respectively. The subcutaneous implantation of the scaffolds was performed to assess tissue response. The type of inflammatory cells present in the surrounding tissue, as well as within the scaffold, was determined histologically and by immunohistochemistry. In the presence of lipase or lysozyme, the water uptake of the scaffolds increased. Based on the weight loss data and scanning electron microscopy analysis, the lysozyme combined with lipase had a notable effect on the in vitro degradation of the scaffolds. The in vivo implantation showed a normal inflammatory response, with presence of neutrophils, in a first stage, and macrophages, lymphocytes, and giant cells in a later stage. Vascularization in the surrounding tissue and within the implant increased with time. Moreover, the collagen deposition increased with time inside the implant. In vivo, the scaffolds maintained the structural integrity. The degradation in vitro was faster and greater compared to that observed in vivo within the same time periods.

Chitosan scaffolds incorporating lysozyme into CaP coatings produced by a biomimetic route: A novel concept for tissue engineering combining a self-regulated degradation system with in situ pore formation

This study describes an innovative self-regulated degrading material with gradual in situ pore formation ability for bone tissue engineering applications. This approach is based on the incorporation of the lysozyme enzyme into calcium phosphate (CaP) coatings, prepared on the surface of chitosan scaffolds by means of a biomimetic coating technique with the aim of controlling their degradation rate and subsequent formation of pores. However, because lysozyme has antibacterial properties, these coatings may act as a carrier for its sustained release, preventing infection upon implantation. In order to prove the concept of in situ pore formation, the coated scaffolds (with and without lysozyme) were incubated in two different solutions at different pH to simulate normal physiological conditions (pH 7.4) and inflammatory response (pH 5). The weight loss and morphology of the scaffolds was monitored over time. At pH 7.4, the scaffolds remained more stable than at pH 5. The scaffolds incubated at pH 5 showed a rapid decrease in their initial weight, and scanning electron microscopy imaging revealed the formation of a highly porous structure. Furthermore, evaluation of the activity of the incorporated lysozyme revealed that the enzyme was able to hydrolyse the peptidoglycan of the bacteria cell walls (as detected by the decrease in optical density with time), indicating that the enzyme remained active after being incorporated into the CaP coating.

The Current Status of iPS Cells in Cardiac Research and Their Potential for Tissue Engineering and Regenerative Medicine

The recent availability of human cardiomyocytes derived from induced pluripotent stem (iPS) cells opens new opportunities to build in vitro models of cardiac disease, screening for new drugs, and patient-specific cardiac therapy. Notably, the use of iPS cells enables studies in the wide pool of genotypes and phenotypes. We describe progress in reprogramming of induced pluripotent stem (iPS) cells towards the cardiac lineage/differentiation. The focus is on challenges of cardiac disease modeling using iPS cells and their potential to produce safe, effective and affordable therapies/applications with the emphasis of cardiac tissue engineering. We also discuss implications of human iPS cells to biological research and some of the future needs.

Natural stimulus responsive scaffolds/cells for bone tissue engineering: influence of lysozyme upon scaffold degradation and osteogenic differentiation of cultured marrow stromal cells induced by CaP coatings.

This work proposes the use of nonporous, smart, and stimulus responsive chitosan-based scaffolds for bone tissue engineering applications. The overall vision is to use biodegradable scaffolds based on chitosan and starch that present properties that will be regulated by bone regeneration, with the capability of gradual in situ pore formation. Biomimetic calcium phosphate (CaP) coatings were used as a strategy to incorporate lysozyme at the surface of chitosan-based materials with the main objective of controlling and tailoring their degradation profile as a function of immersion time. To confirm the concept, degradation tests with a lysozyme concentration similar to that incorporated into CaP chitosan-based scaffolds were used to study the degradation of the scaffolds and the formation of pores as a function of immersion time. Degradation studies with lysozyme (1.5 g/L) showed the formation of pores, indicating an increase of porosity ( approximately 5-55% up to 21 days) resulting in porous three-dimensional structures with interconnected pores. Additional studies investigated the influence of a CaP biomimetic coating on osteogenic differentiation of rat marrow stromal cells (MSCs) and showed enhanced differentiation of rat MSCs seeded on the CaP-coated chitosan-based scaffolds with lysozyme incorporated. At all culture times, CaP-coated chitosan-based scaffolds with incorporated lysozyme demonstrated greater osteogenic differentiation of MSCs, bone matrix production, and mineralization as demonstrated by calcium deposition measurements, compared with controls (uncoated scaffolds). The ability of these CaP-coated chitosan-based scaffolds with incorporated lysozyme to create an interconnected pore network in situ coupled with the demonstrated positive effect of these scaffolds upon osteogenic differentiation of MSCs and mineralized matrix production illustrates the strong potential of these scaffolds for application in bone tissue engineering strategies.

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.

DAYLIGHT IN A CISTERCIAN HERITAGE CHURCH IN LISBON, FROM RURAL TO URBAN CONTEXT

ABSTRACT Light levels in the churches of the Cistercian Order are mostly related to the fulfilment of liturgical needs. The compound building of Bernardas’ Convent includes a church at the utmost southern corner that dates back to the 17th century. It only has one facade facing due southeast. This paper analyses the relationship between daylight conditions within the building before and after the urbanisation of the surrounding area; taking into account the relationship between the church, its main activities, and solar trajectory. A comparative analysis of the relationship between the actual surrounding context and the initial period after it was built (open field) is given. The highest reflectance of the street canyon has augmented the levels of available daylight.