New applications of particulate materials in tissue engineering strategies : delivery of bioactive agents and selective cell isolation

Tese de doutoramento (ramo de conhecimento em Ciências e Tecnologia de Materiais/área de especialização em Biomateriais)During the past few years, researchers working in tissue engineering (TE) have realized that drug delivery is a fundamental part of the TE strategy. Therefore, a large number of technologies have been optimized to guide the potential of drug delivery in those applications. Drug delivery systems (DDS) can be designed to provide controlled release of bioactive molecules to the site of action with minimal side-effects by reducing the exposure of the drug to other tissues. Microparticulate DDS are claimed to have several advantages, such as enhanced bioavailability, possibility for targeting delivery and minimally invasive administration, together with greater efficacy/safety. This PhD work describes attempts to develop particulate systems with potential application in TE strategies. Microparticles based on natural origin polymers (starch and chitosan and blends thereof) were used. In addition, a synthetic polymer (poly-e-caprolactone, PCL) was used alone or combined with starch. For the preparation of these particulate systems, emulsion procedures, based on evaporation of solvents or use of crosslinking agents, were optimized. The developed systems were characterized in terms of morphology, physicochemical properties, particle size and size distribution. Their potential as DDS either for antibiotics (gentamicin sulphate, GTM), steroids (Dexamethasone, DEX) or growth factors (bone morphogenetic protein 2, BMP-2) was evaluated with encapsulation efficiencies of 93% for DEX, 67% for GTM and 24% for BMP-2. The release profiles were evaluated in environments mimicking physiological conditions and were characterized by three main phases typical of drug release from biodegradable carriers: i) burst release, ii) period of minimal release, and iii) the release of the remaining active agent while the polymeric matrix is degraded. The in vitro biocompatibility of the developed system was investigated using different cell lines where aspects such as cytotoxicity, cell proliferation and morphology were evaluated. Moreover, the maintenance of the activity/function of all entrapped molecules was investigated. The antibacterial activity of released GTM was assessed by an in vitro disc diffusion susceptibility test using Gram positive bacteria (Staphylococcus aureus) whereas the bioactivity of DEX and BMP-2 was analyzed by determining their ability to induce osteogenic differentiation of precursor cells. For the tested conditions, all developed microparticles were non-cytotoxic, highly biodegradable and suitable as carriers for sustained delivery purposes. The ability to isolate living cells is an essential aspect of TE research. Magnetic cell separation methods are among the most efficient methods for cell separation. Therefore, an additional objective of this work was to develop magnetic functionalized particles for cell isolation (e.g. stem cell subpopulations). Towards this goal, surface functionalized magnetic poly-e- caprolactone microparticles (m-PCL) were fabricated. 13% of magnetite nanoparticles (core) were effectively entrapped within a poly-e-caprolactone (shell). Amino and epoxy groups were introduced on the surface of the m-PCL. The m-PCL were characterized for morphology, particle size and size distribution, physicochemical and magnetic properties. Their effectiveness for covalent coupling of protein-like molecules was evaluated by using bovine serum albumin, resulting in coupling efficiency higher than 47% for epoxy and 71% for amino functionalized m-PCL. Additionally, cell viability, proliferation and morphology upon contact with developed microparticles were evaluated. The m-PCL were shown to be non-cytotoxic and their surface functionalization did not show any detrimental influence on cell viability and proliferation. Overall, the developed microparticulate systems are versatile and very promising to be used in TE strategies.Durante os últimos anos, tem havido um crescente interesse no desenvolvimento de sistemas de libertação controlada para aplicação em estratégias de engenharia de tecidos (ET) humanos. Os sistemas de libertação controlada podem ser utilizados com a finalidade de melhorar o índice terapêutico de fármacos por alteração da sua distribuição e, consequentemente, aumentar a sua eficácia terapêutica e/ou reduzir a sua toxicidade. Diversas tecnologias têm sido desenvolvidas e optimizadas a fim de direccionar o potencial da libertação controlada em aplicações de ET. Sistemas na forma de micropartículas para libertação de agentes bioactivos apresentam diversas vantagens tais como controlo da sua biodisponibilidade, possiblidade de direccionar a terapia e administração não invasiva. O presente trabalho de doutoramento pretende avaliar e explorar a potencial de sistemas de micropartículas para libertação controlada de agentes bioactivos em estratégias de ET de osso. Para tal, foram desenvolvidas micropartículas à base de polímeros naturais, nomeadamente misturas de amido e quitosano. Foi também usado o polímero sintético policaprolactona (PCL) isolado ou combinado com amido. As partículas foram produzidas recorrendo a técnicas de emulsão utilizando diferentes agentes reticuladores ou por evaporação de solventes. As propiedades físico-químicas assim como a morfologia e o tamanho das partículas foram avaliados de forma a caracterizar os sistemas desenvolvidos. Como agentes bioactivos foram usados o antibiótico gentamicina (GTM), o corticóide dexametasona (DEX) e o factor de crescimento proteína morfogenética do osso (BMP-2), tendo estes sido incorporados nos sistemas desenvolvidos com eficiências de encapsulamento de 67%, 93% e 24%, respectivamente. Os perfis de libertação foram estudados de forma a mimetizar condições fisiológicas, tendo-se observado três fases distintas: i) uma fase inicial em que ocorre libertação súbita do agente incorporado, ii) período de libertação mínima e iii) libertação do agente activo remanescente que ocorre com a degradação da matriz polimérica. A biocompatibilidade dos sistemas desenvolvidos foi testada in vitro usando linhas celulares, sendo avaliados parâmetros como a citotoxicidade, morfologia e proliferação celular. A actividade dos agentes incorporados foi analisada por diferentes métodos. A actividade antibacteriana da gentamicina libertada foi avaliadada pelo método da difusão em agar, que se baseia na determinação dos halos de inibição do crescimento de um dado microorganismo (bactéria Gram positiva Staphylococcus aureus). A actividade osteogénica dos agentes encapsulados DEX e BMP-2 foi analisada através de estudos in vitro com células precursoras, tendo-se avaliado a sua morfologia, proliferação e viabilidade, bem como a expressão de marcadores da linhagem osteogénica (e.g. ALP, osteocalcina). Todas as partículas desenvolvidas mostraram ser biodegradáveis e não citotóxicas nas condições testadas. Além disso, apresentam uma libertação controlada do agente incorporado sem comprometer a sua acção, o que as torna adequadas para aplicações em estratégias de ET. É conhecido que algumas nanopartículas magnéticas apresentam um enorme potencial para diversas aplicações biotecnológicas, nomeadadamente no isolamento de subpopulações de células estaminais/precursoras. Estas partículas podem ser revestidas com um material biocompatível e funcionalizadas com anticorpos específicos para determinado tipo de células. De facto, este trabalho envolveu também o desenvolvimento de partículas magnéticas funcionalizadas para o isolamento de células. Nanopartículas de magnetite foram revestidas com uma solução polimérica de PCL de forma a obter partículas do tipo core-shell. As partículas foram caracterizadas em termos de morfologia, tamanho e distribuição, sendo também avaliadas as suas propriedades fisico-quimicas e magnéticas. Posteriormente foram funcionalizadas com grupos epóxi e amino para ligação de proteínas na superfície. A sua biocompatabilidade foi avaliada em estudos in vitro. As micropartículas magnéticas não apresentaram citoxicidade nem afectaram a viabilidade e proliferação celulares, podendo ser usadas para isolamento de células. Em geral, pode afirmar-se que os sistemas de micropartículas desenvolvidas neste trabalho são versáteis e apresentam grande potencial para serem aplicadas em estratégias de engenharia de tecidos

Silk fibroin microparticles as carriers for delivery of human recombinant bone morphogenetic protein-2 : in vitro and in vivo bioactivity

The in vitro and in vivo efficiency of fibroin microparticles as a delivery carrier for bone morphogenetic protein-2 (BMP-2) was evaluated. BMP-2 was encapsulated in silk fibroin particles that were produced by a simple and very mild processing method. The dose-response of BMP-2-loaded fibroin particles was examined in C2C12 cells, after 5 days of culture. The BMP-2 retained most of its activity as observed by the increase in alkaline phosphatase activity, which was much higher when BMP-2 was encapsulated into the particles rather than just surface-adsorbed. After 2 weeks of culture, increased mineralization was observed with BMP-2-loaded particles in comparison to soluble added growth factor. No significant cytotoxicity was detected. When implanted in a rat ectopic model, bone formation was observed by in vivo micro-computed tomography after 2 and 4 weeks postimplantation, with particles loaded with 5 or 12.5 microg BMP-2. An increase in bone density was observed over time. Histology revealed further evidence of ectopic bone formation, observed by strong alizarin red staining and osteocalcin immunostaining. Our findings show that fibroin microparticles may present an interesting option for future clinical applications in the bone tissue engineering field, and therefore, further studies have been planned.The authors acknowledge Anna Hofmann and Anna Khadem for additional help with some experiments, and Karin Brenner for animal maintenance. This work was supported by Fundacao para a Ciencia e Tecnologia (Ph.D. grant SFRH/BD/17049/2004), project ElastM (POCI/CTM/57177/2004 funded by FEDER and the Fundacao para a Ciencia e Tecnologia), Marie Curie Alea Jacta EST short-term grant (MEST-CT-2004-8104), and European STREP Project HIP-POCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)

Phosphorous pentoxide-free bioactive glass exhibits dose-dependent angiogenic and osteogenic capacities which are retained in glass polymeric composite scaffolds

Bioactive glasses (BGs) are attractive materials for bone tissue engineering because of their bioactivity andosteoinductivity. In this study, we report the synthesis of a novel phosphorous pentoxide-free, silicatebasedbioactive glass (52S-BG) composed of 52.1% SiO2, 23.2% Na2O and 22.6% CaO (wt%). The glasswas thoroughly characterized. The biocompatibility and osteogenic properties of 52S-BG particles wereanalyzed in vitro with human adipose-derived mesenchymal stem cells (AdMSCs) and human osteoblasts.52S-BG particles were biocompatible and induced mineralized matrix deposition and the expression ofosteogenic markers (RunX2, alkaline phosphatase, osteocalcin, osteopontin, collagen I) and the angiogenicmarker vascular endothelial growth factor (VEGF). Angiogenic properties were additionallyconfirmed in a zebrafish embryo model. 52S-BG was added to poly-ε-caprolactone (PCL) to obtain acomposite with 10 wt% glass content. Composite PCL/52S-BG scaffolds were fabricated by additive manufacturingand displayed high porosity (76%) and pore interconnectivity. The incorporation of 52S-BG particlesincreased the Young’s modulus of PCL scaffolds from 180 to 230 MPa. AdMSC seeding efficiencyand proliferation were higher in PCL/52S-BG compared to PCL scaffolds, indicating improved biocompatibility.Finally, 52S-BG incorporation improved the scaffolds’ osteogenic and angiogenic properties byincreasing mineral deposition and inducing relevant gene expression and VEGF protein secretion. Overall,52S-BG particles and PCL/52S-BG composites may be attractive for diverse bone engineering applicationsrequiring concomitant angiogenic properties

A novel enzymatically-mediated drug delivery carrier for bone tissue engineering applications: combining biodegradable starch-based microparticles and differentiation agents

In many biomedical applications, the performance of biomaterials depends largely on their degradation behavior. For instance, in drug delivery applications, the polymeric carrier should degrade under physiological conditions slowly releasing the encapsulated drug. The aim of this work was, therefore, to develop an enzymaticmediated degradation carrier system for the delivery of differentiation agents to be used in bone tissue engineering applications. For that, a polymeric blend of starch with polycaprolactone (SPCL) was used to produce a microparticle carrier for the controlled release of dexamethasone (DEX). In order to investigate the effect of enzymes on the degradation behavior of the developed system and release profile of the encapsulated osteogenic agent (DEX), the microparticles were incubated in phosphate buffer solution in the presence of a-amylase and/or lipase enzymes (at physiological concentrations), at 37 C for different periods of time. The degradation was followed by gravimetric measurements, scanning electron microscopy (SEM) and Fourier transformed infrared (FTIR) spectroscopy and the release of DEX was monitored by high performance liquid chromatography (HPLC). The developed microparticles were shown to be susceptible to enzymatic degradation, as observed by an increase in weight loss and porosity with degradation time when compared with control samples (incubation in buffer only). For longer degradation times, the diameter of the microparticles decreased significantly and a highly porous matrix was obtained. The in vitro release studies showed a sustained release pattern with 48% of the encapsulated drug being released for a period of 30 days. As the degradation proceeds, it is expected that the remaining encapsulated drug will be completely released as a consequence of an increasingly permeable matrix and faster diffusion of the drug. Cytocompatibility results indicated the possibility of the developed microparticles to be used as biomaterial due to their reduced cytotoxic effects

Synthetic mRNA - emerging new class of drug for tissue regeneration

mRNA has the potential to be the next generation drug for tissue restoration in regenerative medicine. The variety of mRNAs that could be synthesized with the aim of increasing the expression of any required protein offers new opportunities. However, the intrinsic immunogenicity and lack of stability of mRNA has long restricted the potential of mRNA therapeutics. Fortunately, considerable progress has been made on synthetic mRNA modifications and relevant purification steps that have overcome these limitations. However, there remains a lack of efficient mRNA delivery strategies. Additionally, mRNA may need to be administered in situ via three-dimensional biomaterials. These materials, also known as transcript-activated matrices, require further consideration in terms of mRNA loading and release, immunogenicity, and other features. In this article, various limiting factors in mRNA synthesis, vector formulation, and local delivery to tissues are highlighted together with current developments and the future outlook for mRNA therapeutics in tissue regeneration

Gene therapy for bone engineering

Bone has an intrinsic healing capacity that may be exceeded when the fracture gap is too big or unstable. In that moment, osteogenic measures need to be taken by physicians. It is important to combine cells, scaffolds and growth factors, and the correct mechanical conditions. Growth factors are clinically administered as recombinant proteins. They are, however, expensive and needed in high supraphysiological doses. Moreover, their half-life is short when administered to the fracture. Therefore, gene therapy may be an alternative. Cells can constantly produce the protein of interest in the correct folding, with the physiological glycosylation and in the needed amounts. Genes can be delivered in vivo or ex vivo by viral or non-viral methods. Adenovirus is mostly used. For the non-viral methods, hydrogels and recently sonoporation seem to be promising means. This review will give an overview of recent advancements in gene therapy approaches for bone regeneration strategies

Can we accelerate the fracture healing response?

Osteoporosis is a systemic skeletal disease. It is characterized by a disbalance of bone formation and bone resorption. It is treated by escalating steps of drug therapy. This is influencing bone density. However, therapies for fractures in osteoporotic patients are typically surgical without really treating the osteoporotic phenotype locally. Several possibilities exist. Some drugs used for systemic therapy can be administered locally to the defect space using drug delivery systems. Other local therapies have been applied to fracture and nonunion treatments. An important asset are stem cells that can be obtained from the bone marrow or adipose tissue. A more complete form including natural extracellular matrix and supporting cells is constituted by Reamer Irrigator Aspirator (RIA). Finally, an innovative possibility is taking molecular biology techniques into consideration. Osteoporotic fractures have a specific microRNA signature. These upregulated microRNAs can be counteracted by antagomirs. Using them, a broad range of effector messenger RNAs and thus proteins can be modulated. In summary, treatment of osteoporotic fractures should take more the osteoporotic pathogenetic pathways into consideration for locally treating and accelerating fracture healing

Controlled delivery systems : from pharmaceuticals to cells and genes

During the last few decades, a fair amount of scientific investigation has focused on developing novel and efficient drug delivery systems. According to different clinical needs, specific biopharmaceutical carriers have been proposed. Micro-and nanoparticulated systems, membranes and films, gels and even microelectronic chips have been successfully applied in order to deliver biopharmaceuticals via different anatomical routes. The ultimate goal is to deliver the potential drugs to target tissues, where regeneration or therapies (chemotherapy, antibiotics, and analgesics) are needed. Thereby, the bioactive molecule should be protected against environmental degradation. Delivery should be achieved in a dose-and time-correct manner. Drug delivery systems (DDS) have been conceived to provide improvements in drug administration such as ability to enhance the stability, absorption and therapeutic concentration of the molecules in combination with a long-term and controlled release of the drug. Moreover, the adverse effects related with some drugs can be reduced, and patient compliance could be improved. Recent advances in biotechnology, pharmaceutical sciences, molecular biology, polymer chemistry and nanotechnology are now opening up exciting possibilities in the field of DDS. However, it is also recognized that there are several key obstacles to overcome in bringing such approaches into routine clinical use. This review describes the present state-of-the-art DDS, with examples of current clinical applications, and the promises and challenges for the future in this innovative field.This work was supported through the European Union funded projects Marie Curie Host Fellowships for Early Stage Research Training (EST) "Alca Jacta EST" (MEST-CT-2004-008104), which provided E. R. Balmayor with a PhD fellowship, and the European Network of Excellence EXPERTISSUES (NMP3-CT-2004-500283)

The addition of zinc ions to polymer-ceramic composites accelerated osteogenic differentiation of human mesenchymal stromal cells

Critical-sized bone defects, caused by congenital disorders or trauma, are defects that will not heal spontaneously and require surgical intervention. Recent advances in biomaterial design for the treatment of such defects focus on improving their osteoinductive properties. Here, we propose a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50 % beta-tricalcium phosphate (ß-TCP) with the addition of zinc in a form of a coating on the TCP particles. Due to its essential role in bone homeostasis, we hypothesised that the addition of zinc to the polymer-ceramic composite will further enhance its osteogenic properties. ß-TCP particles were immersed in a zinc solution with a concentration of 15 or 45 mM. The addition of zinc did not alter the ß-TCP composition or the release of calcium or phosphate ions. 3D porous 1000PEOT70PBT30 - ß-TCP scaffolds were additively manufactured by “3D fibre deposition” and their ability to support the osteogenic differentiation was assessed by culturing clinically relevant human mesenchymal stromal cells (hMSCs) on the scaffolds for 3, 7, 14 and 28 days. The expression of osteogenic gene markers was increased in the presence of both zinc concentrations. Remarkably, upregulation of osteocalcin (OCN), a late osteogenic marker, was observed after three days of culture. Furthermore, enhanced extracellular matrix (ECM) production and mineralization was observed. These findings support the existing evidence on the osteogenic properties of zinc and further demonstrate that the incorporation of zinc into a polymer-ceramic composite could be a promising strategy in the field of regeneration of critical-sized bone defects