Engenharia de tecidos de defeitos osteocondrais combinando estratégias de design de suportes processados por agregação de partículas e de libertação controlada de agentes bioactivos

Tese de Doutoramento em Engenharia BiomédicaOsteochondral defects are lesions of the articular cartilage where the underlying bone tissue is also damaged. Although some studies have achieved success in repairing small cartilage defects, there is no widely accepted method for complete repair of large osteochondral defects. This is due to different factors, for instances, the ones associated with mechanical instability, among others. The requirements for a successful regeneration of an osteochondral defect could potentially be met by using a tissue-engineered osteochondral hybrid construct. Therefore, the need for a simultaneous regeneration of both cartilage and subchondral bone should be considered, making osteochondral tissue engineering an interesting challenge to the present research since it requires the combination of both bone and cartilage tissue engineering principles. Some researchers suggest that osteochondral defects could be regenerated from single-layer scaffolds, engineering complex tissue grafts with gradients of molecular, structural, and functional properties. Nevertheless, it has been better accepted that a bilayered structure would be more adequate to regenerate an osteochondral defect. This structure should be able to support the growth and development of different types of cells while providing a mechanical and biochemical environment able to promote the formations of the two distinct tissues. The main goal of this thesis was to develop and assess the potential of several possible strategies for osteochondral tissue engineering. This includes: (1) the development of the cell culturing to be performed independently in the two components that are integrated before implantation; (2) the biphasic scaffold with adequate properties for both cartilage and bone parts to be used in a special bioreactor with two separate chambers where two different cell types can be seeded, and (3) the biphasic scaffold loaded with distinct differentiation agents able to provide the adequate biochemical cues to common progenitor cells. Furthermore, and as a key requisite for biomedical applications including tissue engineering, the presented work also tried to keep present the needs for (4) the in vivo biofunctionality of the new materials. These were the main challenges addressed in this thesis. The work described intends mainly to be yet another positive contribute for the design of a successful approach for osteochondral tissue engineering, having always in mind that, to date, there is no accepted method for complete repair of large osteochondral defects. In this PhD research, an innovative methodology for scaffolds production is proposed, based on the aggregation of prefabricated degradable particles. This methodology allowed to produce scaffolds with high interconnectivity and mechanical stability. The processing route was also used efficiently for the production of bilayered scaffolds by assembling both polymeric and composite particles. The obtained bilayered scaffolds present a very good integration between both components which is critical, since any discontinuity is likely to cause long-term device failure. By using the particles aggregation methodology, and as described in this work, it was possible to successfully produce polymeric, composite and bilayered scaffolds. Chitosan was selected as polymeric matrix due to its structural similarity to glycosaminoglycans (GAGs) found in extracellular matrices including native articular cartilage. On the other hand, hydroxylapatite was used as bioactive ceramic filler, since this it is the main mineral present in bone composition, approaching in this way the developed composite scaffolds to the bone composite structure. An extensive materials characterization was carried out, with a particular emphasis on the morphometric analysis and mechanical performance of the developed materials that have demonstrated to be adequate, corroborating the claimed advantages of the proposed methodology. Preliminary studies with human adipose stem cells were also carried out, clearly indicating the presence of cells with osteogenic and chondrogenic morphology in the 3D particle agglomerated scaffolds. The influence of the 3D support on the cells differentiation ability is further suggested, since no events of osteogenic or chondrogenic differentiation in the control cells without the scaffolds were detected. A specific double-chamber bioreactor was designed in order to allow, as an ultimate goal, for the simultaneous culturing of chondrocytes and osteoblasts or the same source of progenitor cells with distinct differentiation mediums. As proof of concept, dynamic bioactivity tests are described using bilayered chitosan scaffolds. Insulin-loaded chitosan particle aggregated scaffolds are proposed as a potential model system to induce chondrogenic differentiation. The in vitro release profiles and the effect of these systems on a pre-chondrogenic cell line were investigated. The most promising results were obtained for cells seeded in the higher insulin-loaded scaffolds that showed a typical chondrocytic round morphology, were positively stained for toluidine blue, presented a high GAGs production, and expressed genes encoding cartilaginous markers. This approach opens the possibility to assemble the developed systems in bilayered constructs, in order to provide the adequate biochemical cues to promote selective differentiation of cartilage and bone in osteochondral applications. The final study described in this work is focused on the assessment of the in vivo biofunctionality of polymeric chitosan scaffolds. The scaffolds in vivo performance is one obvious key requirement for tissue engineering applications. The scaffolds properties shown to be favourable to the connective tissues ingrowth into the scaffolds, demonstrating a good integration with the host tissue. Furthermore, the scaffolds were able to promote an organization of the extracellular matrix and an increasing neo-vascularization with the time of implantation, which is rather promising and not typical at all for porous biodegradable systems. As a general remark, and in the context of possible strategies for osteochondral tissue engineering, the chitosan-based scaffolds produced by particle aggregation are morphologically and mechanically competent. In addition, the developed scaffolds are able to incorporate adequate biochemical cues, and present an in vivo biocompatible behaviour, and may thereby be potential candidates for large osteochondral tissue engineering applications. Each of the individual performed work opens the possibility to better accomplish an osteochondral hybrid strategy following the different discussed options. This is the main innovation coming from this thesis.Os defeitos osteocondrais são lesões na cartilagem articular onde o tecido ósseo adjacente está também danificado. Apesar do sucesso alcançado em alguns estudos na regeneração de pequenos defeitos da cartilagem, não existe actualmente um método consensual para a reabilitação completa de grandes defeitos osteocondrais. Isto é devido a diferentes factos, entre os associados com instabilidade mecânica. Os requisitos para uma regeneração bem sucedida de um defeito osteocondral podem ser potencialmente preenchidos utilizando um suporte híbrido desenvolvido através de engenharia de tecidos. Por isso, a necessidade da regeneração simultânea da cartilagem e do osso subcondral deve ser considerada, tornando a engenharia de tecidos osteocondral num desafio interessante para a investigação actual, uma vez que requer a combinação dos princípios de engenharia de tecidos do osso e da cartilagem. Alguns investigadores sugerem que os defeitos osteocondrais podem ser regenerados a partir de suportes monofásicos, desenvolvendo implantes tecidulares complexos com gradientes de propriedades moleculares, estruturais e funcionais. No entanto, tem sido melhor aceite que uma estrutura bifásica pode ser mais adequada para regenerar um defeito osteocondral. Esta estrutura deve ser capaz de sustentar o crescimento e desenvolvimento de diferentes tipos de células, providenciando um ambiente bioquímico e mecânico capaz de promover a formação dos dois tecidos distintos. O principal objectivo desta tese foi desenvolver e avaliar o potencial de várias estratégias possíveis para engenharia de tecidos osteocondrais. Estas incluem: (i) o desenvolvimento de cultura de células para ser efectuado nos dois componentes independentemente que são integrados antes da implantação; (ii) o suporte bifásico com propriedades adequadas para a regeneração de osso e cartilagem é utilizado num bioreactor específico com duas câmaras separadas, onde os dois tipos de células diferentes podem ser cultivados; e (iii) os agentes de diferenciação são incorporados no suporte bifásico, de modo a proporcionar estímulos bioquímicos adequados às células progenitoras comuns. Para além disso, e como um précondição essencial para aplicações biomédicas incluindo engenharia de tecidos, este trabalho tentou ter sempre presente os requisitos de biofuncionalidade in vivo de novos materiais. Estes são os principais desafios propostos nesta tese. O trabalho descrito pretende principalmente ser mais um contributo positivo para o design de uma estratégia de sucesso na engenharia de tecidos osteocondrais, não esquecendo que até à data não existe um método consensual para a reabilitação completa de grandes defeitos osteocondrais. Nesta investigação de doutoramento, é proposta uma metodologia inovadora para a produção de suportes para engenharia de tecidos com base na agregação de partículas biodegradáveis pré-processadas. Esta metodologia permitiu a produção de suportes para engenharia de tecidos com elevada interconectividade e estabilidade mecânica. Mais ainda, foi aplicada eficazmente na produção de suportes bifásicos combinando partículas poliméricas e compósitas. Os suportes bifásicos obtidos apresentam uma boa integração entre os dois componentes. Este facto é crítico, dado que é provável que qualquer descontinuidade provoque a falha do dispositivo a longo prazo. Utilizando a metodologia de agregação de partículas, e tal como é descrito neste trabalho, foi possível produzir com sucesso estruturas poliméricas, compósitas e bifásicas. O quitosano foi seleccionado como matriz polimérica devido à sua semelhança estrutural com os glicosaminoglicanos (GAGs) presentes nas matrizes extra-celulares, incluindo na cartilagem articular. Por outro lado, a hidroxiapatite foi utilizada como reforço cerâmico bioactivo, dado que é o principal mineral presente na composição do osso, aproximando deste modo os materiais compósitos desenvolvidos à estrutura compósita do osso. Uma extensa caracterização dos materiais foi realizada, com particular ênfase na análise morfométrica e desempenho mecânico dos materiais desenvolvidos que demonstraram ser adequados, comprovando assim as vantagens reivindicadas da metodologia proposta. Estudos preliminares com células estaminais do tecido adiposo foram efectuados, indicando claramente a presença de células com morfologia osteogénica e condrogénica nos suportes desenvolvidos. A influência da estrutura tridimensional na capacidade de diferenciação das células é também sugerida, uma vez que não foram detectadas evidências de diferenciação osteogénica e condrogénica nas células sem os suportes utilizadas como controle. Um bioreactor específico de câmara dupla foi também desenhado de modo a permitir, como objectivo final, a cultura simultânea de condrócitos e osteoblastos ou o mesmo tipo de células progenitoras com diferentes meios de diferenciação. Como prova de conceito foram efectuados estudos dinâmicos de bioactividade utilizando suporte bifásicos de quitosano. Suportes produzidos por agregação de partículas carregados com insulina são propostos como um potencial modelo para induzir a diferenciação condrogénica. Os perfis de libertação in vitro e o efeito destes sistemas numa linha celular pré-condrogénica foram investigados. Os resultados mais promissores foram obtidos com células cultivadas nos suportes com maior concentração de insulina tendo as células demonstrado uma morfologia típica de condrócitos e marcado positivamente para o azul de toluidina. Verificou-se também um aumento na produção de glicosaminoglicanos e na expressão de genes associados a marcadores típicos de cartilagem. Esta aproximação abre a possibilidade de associar os sistemas desenvolvidos em suportes bifásicos, fornecendo sinais bioquímicos adequados à promoção selectiva da diferenciação em cartilagem e osso, tendo em vista aplicações osteocondrais. O estudo final descrito neste trabalho foca-se na avaliação da biofuncionalidade in vivo dos suportes poliméricos de quitosano. O comportamento destes materiais in vivo é um pré-requisito óbvio para aplicações no campo de engenharia de tecidos. As propriedades dos suportes mostraram ser favoráveis ao crescimento dos tecidos conectivos para o interior dos mesmos, apresentando uma boa integração com o tecido hospedeiro. Os materiais foram ainda capazes de promover a organização da matriz extra-celular e um aumento da neo-vascularização durante a implantação, o que é bastante promissor e de todo invulgar para sistemas porosos biodegradáveis. Como comentário final, e no contexto de possíveis estratégias para engenharia de tecidos osteocondrais, os suportes à base de quitosano processados por agregação de partículas são morfológica e mecanicamente competentes. Para além disso, os materiais desenvolvidos são capazes de incorporar sinais bioquímicos adequados apresentando um comportamento biocompatível in vivo, podendo deste modo ser considerados potenciais candidatos para aplicações de engenharia de tecidos de grandes defeitos osteocondrais. Cada um dos trabalhos realizados abre uma possibilidade de alcançar uma estratégia híbrida osteocondral tendo em conta as opções discutidas. Esta é a principal inovação resultante desta tese.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/11155/2002.INTERREG IIIA project PROTEUS.União Europeia (UE) - NMP3-CT-2003-505758.European NoE EXPERTISSUES - NMP3-CT-2004-500283

Porous bioactive composites from marine origin based in chitosan and hydroxylapatite particles

An optimal carrier for bone tissue engineering should be both a controlled release system and a scaffold. In the former role, the carrier must prevent rapid factor clearance and ideally meter out the growth factor in a predictable manner, allowing therapeutic doses to stimulate target cells for the appropriate duration. In the latter role, the material should act as a permissive environment into which bone cells would be attracted to migrate and begin the process of depositing bone matrix. Therefore the direct incorporation of growth factor in porous scaffolds should be a desirable goal. The inclusion of a bioactive ceramic on the scaffold design will confer to the systems a bone bonding behaviour that will guide bone formation. This work reports the development of composite chitosan/HA (from algal origin) porous structures produced by means of freeze-drying processing routes that can be further loaded with a biologically active agent. The developed bioactive 3D structures (completely from marine origin) have potential application as tissue engineering scaffolds and drug delivery systems due to their morphological and bioactive properties.(undefined

Bilayered chitosan-based scaffolds for osteochondral tissue engineering : influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor

Osteochondral tissue engineering presents a current research challenge due to the necessity of combining both bone and cartilage tissue engineering principles. In the present study, bilayered chitosan-based scaffolds are developed based on the optimization of both polymeric and composite scaffolds. A particle aggregation methodology is proposed in order to achieve an improved integrative bone–cartilage interface needed for this application, since any discontinuity is likely to cause long-term device failure. Cytotoxicity was evaluated by the MTS assay with the L929 fibroblast cell line for different conditions. Surprisingly, in composite scaffolds using unsintered hydroxyapatite, cytotoxicity was observed in vitro. This work reports the investigation that was conducted to overcome and explain this behaviour. It is suggest that the uptake of divalent cations may induce the cytotoxic behaviour. Sintered hydroxyapatite was consequently used and showed no cytotoxicity when compared to the controls. Microcomputed tomography (micro-CT) was carried out to accurately quantify porosity, interconnectivity, ceramic content, particle and pore sizes. The results showed that the developed scaffolds are highly interconnected and present the ideal pore size range to be morphometrically suitable for the proposed applications. Dynamical mechanical analysis (DMA) demonstrated that the scaffolds are mechanically stable in the wet state even under dynamic compression. The obtained elastic modulus was, respectively, 4.21 ± 1.04, 7.98 ± 1.77 and 6.26 ± 1.04 MPa at 1 Hz frequency for polymeric, composite and bilayered scaffolds. Bioactivity studies using both a simulated body fluid (SBF) and a simulated synovial fluid (SSF) were conducted in order to assure that the polymeric component for chondrogenic part would not mineralize, as confirmed by scanning electron microscopy (SEM), inductively coupled plasma-optical emission spectroscopy (ICP) and energy-dispersive spectroscopy (EDS) for different immersion periods. The assays were carried out also under dynamic conditions using, for this purpose, a specifically designed double-chamber bioreactor, aiming at a future osteochondral application. It was concluded that chitosan-based bilayered scaffolds produced by particle aggregation overcome any risk of delamination of both polymeric and composite parts designed, respectively, for chondrogenic and osteogenic components that are mechanically stable. Moreover, the proposed bilayered scaffolds could serve as alternative, biocompatible and safe biodegradable scaffolds for osteochondral tissue engineering applications

Optimization of chitosan-based composite and bi-layered scaffolds produced by particles aggregation for osteochondral tissue engineering: Influence of hydroxylapatite

[Excerpt] Osteochondral tissue engineering presents a challenge to the present research due to requirements’ combination of both bone and cartilage tissue engineering. In the present study, bilayered chitosan scaffolds are proposed based in the optimization of polymeric and composite scaffolds. µ-CT was carried out for accurate morphometric characterization quantifying porosity, interconnectivity, ceramic content, particles and pores size. The results showed that the developed scaffolds are highly interconnected and present ideal pore size range, being morphometrically adequate for the proposed applications. [...]info:eu-repo/semantics/publishedVersio

Starch-based microspheres produced by emulsion crosslinking with a potential media dependent responsive behavior to be used as drug delivery carriers

This paper describes the development and characterization of starch microspheres for being used as drug delivery carriers in tissue engineering applications. The developed starch microspheres can be further loaded with specific growth factors and immobilized in scaffolds, or administrated separately with scaffolds. Furthermore and due to the processing conditions used, it is expected that these microspheres can be also used to encapsulate living cells. The aim of this study was to evaluate the efficacy of this methodology for further studies with biologically active agents or living cells. The starch microspheres were prepared using an emulsion crosslinking technique at room temperature to allow for the loading of biologically active agents. A preliminary study was performed to evaluate the incorporation of a model drug (nonsteroidal anti-inflammatory drug-NSAID) and investigate its release profile as function of changes in the medium parameters, such as ionic strength and pH. The developed starch-based drug delivery system has shown to be dependent on the ionic strength of the release medium. From preliminary results, the release seems to be pH-dependent due to the drug solubility. It was found that the developed microspheres and the respective processing route are appropriate for further studies. In fact, and based in the processing conditions and characterization, the developed system present a potential for the loading of different growth factors or even living cells on future studies with these systems for improving bone regeneration in tissue engineering, especially because the crosslinking reaction of the microspheres takes place at room temperature

Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications

The present paper intends to overview a wide range of natural–origin polymers with special focus on proteins and polysaccharides (the systems more inspired on the extracellular matrix) that are being used in research, or might be potentially useful as carriers systems for active biomolecules or as cell carriers with application in the tissue engineering field targeting several biological tissues. The combination of both applications into a single material has proven to be very challenging though. The paper presents also some examples of commercially available natural–origin polymers with applications in research or in clinical use in several applications. As it is recognized, this class of polymers is being widely used due to their similarities with the extracellular matrix, high chemical versatility, typically good biological performance and inherent cellular interaction and, also very significant, the cell or enzyme-controlled degradability. These biocharacteristics classify the natural–origin polymers as one of the most attractive options to be used in the tissue engineering field and drug delivery applications

Sodium silicate gel as a precursor for the in vitro nucleation and grow of a bone-like apatite coating in compact and porous polymeric structures

In the present work, a new methodology to produce bioactive coatings on the surface of starch-based biodegradable polymers or other polymeric biomaterials is proposed. A sodium silicate gel is employed as an alternative nucleating agent to the more typical bioactive glasses 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 tissue engineering scaffolds. By means of this treatment, it is possible to observe the formation of an apatite-like layer, only after 6 hours of simulated body fluid immersion. For the porous materials, this layer could also be observed inside the pores, clearly covering the cell walls. Furthermore, an increase of the surface hydrophilicity (higher amount of polar groups in the surface) might contribute to the formation of silanol groups that also act as apatite inductors. After 30 days of SBF immersion, the apatite-like films exhibit a partially amorphous nature and the Ca/P ratios became much closer to the value attributed to hydroxyapatite (1.67). The obtained results are very promising for the development of cancellous bone replacement materials and for pre-calcifying bone tissue engineering scaffolds

Morphology, mechanical characterization and in vivo neo-vascularization of chitosan particle aggregated scaffolds architectures

The present study intended to evaluate the performance of chitosan-based scaffolds produced by a particle aggregation method aimed to be used in tissue engineering applications addressing key issues such as morphological characteristics, mechanical performance and in vivo behaviour. It is claimed that the particle aggregation methodology may present several advantages, such as combine simultaneously a high interconnectivity with high mechanical properties that are both critical for an in vivo successful application. In order to evaluate these properties, micro-Computed Tomography (micro-CT) and Dynamical Mechanical Analysis (DMA) were applied. The herein proposed scaffolds present an interesting morphology as assessed by micro-CT that generally seems to be adequate for the proposed applications. At a mechanical level, DMA has shown that chitosan scaffolds have an elastic behaviour under dynamic compression solicitation, being simultaneously mechanically stable in the wet state and exhibiting a storage modulus of 4.21 ! 1.04 MPa at 1 Hz frequency. Furthermore, chitosan scaffolds were evaluated in vivo using a rat muscle-pockets model for different implantation periods (1, 2 and 12 weeks). The histological and immunohistochemistry results have demonstrated that chitosan scaffolds can provide the required in vivo functionality. In addition, the scaffolds interconnectivity has been shown to be favourable to the connective tissues ingrowth into the scaffolds and to promote the neo-vascularization even in early stages of implantation. It is concluded that the proposed chitosan scaffolds produced by particle aggregation method are suitable alternatives, being simultaneously mechanical stable and in vivo biofunctional that might be used in load-bearing tissue engineering applications, including bone and cartilage regeneration.The authors would like to acknowledge the Portuguese Foundation for Science and Technology for the PhD Grant to Patricia B Malafaya (SFRH/BD/11155/2002). This work was partially supported and carried out under the scope of the European STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and European NoE EXPERTISSUES (NMP3-CT-2004-500283). The authors also thank Prof. Heinz Redl for the collaboration in the in VIVO Studies, as well as Bernhard Horing for the surgical procedures both from LBI, Austria and Joao Oliveira from 3B's Research Group, Portugal for the initial assistance with the DMA equipment

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 (a-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 ± 33 MPa, CS80-20 (402 ± 62 MPa) and CS60-40 (337 ± 78 MPa). Degradation studies were performed using a-amylase and/or lysozyme at concentrations similar to those found in human serum, at 37 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.This work was supported by the European NoE EXPERTISSUES (NMP3-CT-2004-500283), the European STREP HIPPOCRATES (NMP3-CT-2003-505758) and the Portuguese Foundation for Science and Technology (FCT) through POCTI and/or FEDER programmes

Bone, cartilage and osteochondral tissue engineering strategies using natural origin polymers and ceramics, growth factors and progenitor cells

[Excerpt] Tissue engineering (TE) has emerged in the last decade of the 20th century as an alternative approach to circumvent the existent limitations in the current therapies for organ failure or replacement. The EU HIPPOCRATES project joined academic and industrial partners to develop novel products and concepts that can be used for bone, cartilage or osteochondral TE strategies. Several issues were addressed including i) the choice and study of adequate human cell cultures, (ii) the development of culture technology with which human tissues can be grown ex vivo in three dimensional biodegradable matrices, (iii) the development of a material technology with which polymeric matrices can be produced, being suitable for cell culture (proliferation, differentiation), (iv) to assess the in-vivo functionality and clinical relevance of the tissue engineering strategies. [...]info:eu-repo/semantics/publishedVersio