Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

PHB-HV 3D Scaffold supports osteoblast growth

Bone tissue engineering requires a biocompatible scaffold that supports cell growth and enhances the native repair process. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-HV) is a biodegradable 3D scaffold with 88.1 â 0.3% porosity and pore size of 163.5 â 0.1 mm. Previous studies demonstrated the potential of PHB-HV as a scaffold in spinal cord repair. The aim of this study was to evaluate PHB-HV as a scaffold for bone regeneration by assessing the cytocompatability of this scaffold.

Tendon regeneration through a scaffold-free approach: development of tenogenic magnetic hASCs sheets

Tendon's regeneration is limited, demanding for cell-based strategies to fully restore their functionality upon injury. The concept of magnetic force-based TE(1), generally using magnetic nanoparticles may enable, for example, stem cell stimulation and/or remote control over TE constructs. Thus, we originally propose the development of magnetic cell sheets (magCSs) with tenogenic capability, aimed at promoting tendon's regeneration. A Tenomodulin (TNMD+) subpopulation was sorted from human adipose stem cells (hASCs), using TNMD-coated immunomagnetic beads(2) and used as cell source for the development of magCSs. Briefly, cells were labeled with iron oxide composite particles (Micromod) and cultured for 7 days in α-MEM medium with or without magnetic stimulation provided by a magnetic device (nanoTherics). CSs were retrieved from the plates using magnet attraction as contiguous sheets of cells within its own deposited ECM.

A demonstration case on the derivation of process-level logical architectures for ambient assisted living ecosystems

When representing the requirements for an intended software solution during the development process, a logical architecture is a model that provides an organized vision of how functionalities behave regardless of the technologies to be implemented. If the logical architecture represents an ambient assisted living (AAL) ecosystem, such representation is a complex task due to the existence of interrelated multidomains, which, most of the time, results in incomplete and incoherent user requirements. In this chap- ter, we present the results obtained when applying process-level modeling techniques to the derivation of the logical architecture for a real industrial AAL project. We adopt a V-Model–based approach that expresses the AAL requirements in a process-level perspec- tive, instead of the traditional product-level view. Additionally, we ensure compliance of the derived logical architecture with the National Institute of Standards and Technology (NIST) reference architecture as nonfunctional requirements to support the implementa- tion of the AAL architecture in cloud contexts.

Modelling and verifying smell-free architectures with the Archery language

Architectural (bad) smells are design decisions found in software architectures that degrade the ability of systems to evolve. This paper presents an approach to verify that a software architecture is smellfree using the Archery architectural description language. The language provides a core for modelling software architectures and an extension for specifying constraints. The approach consists in precisely specifying architectural smells as constraints, and then verifying that software architectures do not satisfy any of them. The constraint language is based on a propositional modal logic with recursion that includes: a converse operator for relations among architectural concepts, graded modalities for describing the cardinality in such relations, and nominals referencing architectural elements. Four architectural smells illustrate the approach.

Acellular Gellan-gum based bilayered structures for the regeneration of osteochondral defects: a preclinical study

 In orthopaedics, the management and treatment of osteochondral (OC) defects remains an ongoing clinical challenge. Autologous osteochondral mosaicplasty has been used as a valid option for OC treatments although donor site morbidity remains a source of concern [1]. Engineering a whole structure capable of mimicking different tissues (cartilage and subchondral bone) in an integrated manner could be a possible approach to regenerate OC defects. In our group we have been proposing the use of bilayered structures to regenerate osteochondral defects [2,3]. The present study aims to investigate the pre-clinical performance of bilayered hydrogels and spongy-like hydrogels in in vivo  models (mice and rabbit, respectively), in both subcutaneous and orthotopic models. The bilayered structures were produced from Low Acyl Gellan Gum (LAGG) from Sigma-Aldrich, USA. Cartilage-like layers were obtained from a 2wt% LAGG solution. The bone-like layers were made of 2wt% LAGG with incorporation of hydroxyapatite at 20% and 30% (w/v). Hydrogels and spongy-like were subcutaneouly implanted in mice to evaluate the inflammatory response. Then, OC defects were induced in rabbit knee to create a critical size defect (4 mm diameter and 5 mm depth), and then hydrogels and sponges implanted. Both structures followed different processing methods. The hydrogels were injected allowing in situ  crosslinking. Unlike, the spongy-like were pre-formed by freeze-drying. The studies concerning subcutaneous implantation and critical size OC defect were performed for 2 and 4 weeks time, respectively. Cellular behavior and inflammatory responses were assessed by means of histology staining and biochemical function and matrix deposition by immunohistochemistry. Additionally, both OC structures stability and new cartilage and bone formation were evaluated by using vivo- computed tomography (Scanco 80). The results showed no acute inflammatory response for both approaches. New tissue formation and integration in the adjacent tissues were also observed, which present different characteristic behaviors when comparing hydrogels and sponges response. As future insights, a novel strategy for regeneration of OC defects can be designed encompassing both, hydrogels and spongy-like structures and cellular approaches. References: 1. Espregueira-Mendes J. et al. Osteochondral transplantation using autografts from the upper tibio-fibular joint for the treatment of knee cartilage lesions. Knee Surgery, Sports Traumatology, Arthroscopy 20,1136, 2012. 2. Oliveira JM. et al, Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells. Biomaterials 27, 6123, 2006. 3. Pereira D R. et al. Gellan Gum-Based Hydrogel Bilayered Scaffolds for Osteochondral Tissue Engineering. Key Engineering Materials 587, 255, 2013.

Artificial thymus: a tissue engineering strategy

The thymus is the central organ responsible for the generation of T lymphocytes (1). Various diseases cause the thymus to produce in- sufficient T cells, which can lead to immune-suppression (2). Since T cells are essential for the protection against pathogens, it is crucial to promote de novo differentiation of T cells on diseased individuals. The available clinical solutions are: 1) one protocol involving the transplant of thymic stroma from unrelated children only applicable for athymic children (3); 2) for patients with severe peripheral T cell depletion and reduced thymic activity, the administration of stimu- lating molecules stimulating the activity of the endogenous thymus (4). A scaffold (CellFoam) was suggested to support thymus regen- eration in vivo (5), although this research was discontinued. Herein, we propose an innovative strategy to generate a bioartificial thymus. We use a polycaprolactone nanofiber mesh (PCL-NFM) seeded and cultured with human thymic epithelial cells (hTECs). The cells were obtained from infant thymus collected during pediatric cardio-tho- racic surgeries. We report new data on the isolation and characterization of those cells and their interaction with PCL-NFM, by expanding hTECs into relevant numbers and by optimizing cell seeding methods.

Step towards of a homemade business intelligence solution: A case study in textile industry

Nowadays, organizations are increasingly looking to invest in business intelligence solutions, mainly private companies in order to get advantage over its competitors, however they do not know what is necessary. Business intelligence allows an analysis of consolidated information in order to obtain more specific outlets and certain indications in order to support the decision making process. You can take the right decision based on the data collected from different information systems present in the organization and outside of them. The textile sector is a sector where concept of Business Intelligence it is not many explored yet. Actually there are few textile companies that have a BI platform. Thus, the article objective is present an architecture and show all the steps by which companies need to spend to implement a successful free homemade Business Intelligence system. As result the proposed approach it was validated using real data aiming assess the steps defined.

Refinement and variability techniques in model transformation of software requirements

Tese de Doutoramento em Tecnologias e Sistemas de Informação

Generating timed trajectories foran autonomous robot

Tese de Doutoramento Programa Doutoral em Engenharia Electrónica e Computadores

Piezoelectric polymers as biomaterials for tissue engineering applications

Tissue engineering often rely on scaffolds for supporting cell differentiation and growth. Novel paradigms for tissue engineering include the need of active or smart scaffolds in order to properly regenerate specific tissues. In particular, as electrical and electromechanical clues are among the most relevant ones in determining tissue functionality in tissues such as muscle and bone, among others, electroactive materials and, in particular, piezoelectric ones, show strong potential for novel tissue engineering strategies, in particular taking also into account the existence of these phenomena within some specific tissues, indicating their requirement also during tissue regeneration. This referee reports on piezoelectric materials used for tissue engineering applications. The most used materials for tissue engineering strategies are reported together with the main achievements, challenges and future needs for research and actual therapies. This review provides thus a compilation of the most relevant results and strategies and a start point for novel research pathways in the most relevant and challenging open questions.

Design and validation of a biomechanical bioreactor for cartilage tissue culture

Specific tissues, such as cartilage undergo mechanical solicitation under their normal performance in human body. In this sense, it seems necessary that proper tissue engineering strategies of these tissues should incorporate mechanical solicitations during cell culture, in order to properly evaluate the influence of the mechanical stimulus. This work reports on a user-friendly bioreactor suitable for applying controlled mechanical stimulation - amplitude and frequency - to three dimensional scaffolds. Its design and main components are described, as well as its operation characteristics. The modular design allows easy cleaning and operating under laminar hood. Different protocols for the sterilization of the hermetic enclosure are tested and ensure lack of observable contaminations, complying with the requirements to be used for cell culture. The cell viability study was performed with KUM5 cells.

Piezoelectric poly(vinylidene fluoride) microstructure and poling state in active tissue engineering

Tissue engineering often rely on scaffolds for supporting cell differentiation and growth. Novel paradigms for tissue engineering include the need of active or smart scaffolds in order to properly regenerate specific tissues. In particular, as electrical and electromechanical clues are among the most relevant ones in determining tissue functionality in tissues such as muscle and bone, among others, electroactive materials and, in particular, piezoelectric ones, show strong potential for novel tissue engineering strategies, in particular taking also into account the existence of these phenomena within some specific tissues, indicating their requirement also during tissue regeneration. This referee reports on piezoelectric materials used for tissue engineering applications. The most used materials for tissue engineering strategies are reported together with the main achievements, challenges and future needs for research and actual therapies. This review provides thus a compilation of the most relevant results and strategies and a start point for novel research pathways in the most relevant and challenging open questions.

Mechanical fatigue performance of PCL-chondroprogenitor constructs after cell culture under bioreactor mechanical stimulus

In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in Poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements.

In vitro mechanical fatigue behavior of poly-ε-caprolactone macroporous scaffolds for cartilage tissue engineering: Influence of pore filling by a poly(vinyl alcohol) gel

Polymeric scaffolds used in regenerative therapies are implanted in the damaged tissue and subjected to repeated loading cycles. In the case of articular cartilage engineering, an implanted scaffold is typically subjected to long term dynamic compression. The evolution of the mechanical properties of the scaffold during bioresorption has been deeply studied in the past, but the possibility of failure due to mechanical fatigue has not been properly addressed. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. In this work fatigue studies of polycaprolactone scaffolds were carried by subjecting the scaffold to repeated compression cycles in conditions simulating the scaffold implanted in the articular cartilage. The behaviour of the polycaprolactone sponge with the pores filled with a poly(vinyl alcohol) gel simulating the new formed tissue within the pores was compared with that of the material immersed in water. Results were analyzed with Morrow’s criteria for failure and accurate fittings are obtained just up to 200 loading cycles. It is also shown that the presence of poly(vinyl alcohol) increases the elastic modulus of the scaffolds, the effect being more pronounced with increasing the number of freeze/thawing cycles.