Auswahl der wissenschaftlichen Literatur zum Thema „Electroactive scaffold“

For many cells used in tissue engineering applications, the scaffolds upon which they are seeded do not entirely mimic their native environment, particularly in the case of excitable tissues. For instance, muscle cells experience contraction and relaxation driven by the electrical input of an action potential. Electroactive materials can also deform in response to electrical input; however, few such materials are currently suitable as cell scaffolds. We previously described the development of poly(ethyelene glycol) diacrylate-poly(acrylic acid) as an electroactive scaffold. Although the scaffold itself supported cell growth and attachment, the voltage (20 V) required to actuate these scaffolds was cytotoxic. Here, we describe the further development of our hydrogels into scaffolds capable of actuation at voltages (5 V) that were not cytotoxic to seeded cells. This study describes the critical next steps towards the first functional electroactive tissue engineering scaffold.

Applying electrical stimulation (ES) could affect different cellular mechanisms, thereby producing a bactericidal effect and an increase in human cell viability. Despite its relevance, this bioelectric effect has been barely reported in percolated conductive biopolymers. In this context, electroactive polycaprolactone (PCL) scaffolds with conductive Thermally Reduced Graphene Oxide (TrGO) nanoparticles were obtained by a 3D printing method. Under direct current (DC) along the percolated scaffolds, a strong antibacterial effect was observed, which completely eradicated S. aureus on the surface of scaffolds. Notably, the same ES regime also produced a four-fold increase in the viability of human mesenchymal stem cells attached to the 3D conductive PCL/TrGO scaffold compared with the pure PCL scaffold. These results have widened the design of novel electroactive composite polymers that could both eliminate the bacteria adhered to the scaffold and increase human cell viability, which have great potential in tissue engineering applications.

Traditional bone defect treatments are limited by an insufficient supply of autologous bone, the immune rejection of allogeneic bone grafts, and high medical costs. To address this medical need, bone tissue engineering has emerged as a promising option. Among the existing tissue engineering materials, the use of electroactive scaffolds has become a common strategy in bone repair. However, single-function electroactive scaffolds are not sufficient for scientific research or clinical application. On the other hand, multifunctional electroactive scaffolds are often complicated and expensive to prepare. Therefore, we propose a new tissue engineering strategy that optimizes the electrical properties and biocompatibility of carbon-based materials. Here, a hydroxyapatite/carbon nanofiber (HAp/CNF) scaffold with optimal electrical activity was prepared by electrospinning HAp nanoparticle-incorporated polyvinylidene fluoride (PVDF) and then carbonizing the fibers. Biochemical assessments of the markers of osteogenesis in human adipose-derived stem cells (h-ADSCs) cultured on HAp/CNF scaffolds demonstrate that the material promoted the osteogenic differentiation of h-ADSCs in the absence of an osteogenic factor. The results of this study show that electroactive carbon materials with a fibrous structure can promote the osteogenic differentiation of h-ADSCs, providing a new strategy for the preparation and application of carbon-based materials in bone tissue engineering.

Electroactive biomaterials are fascinating for tissue engineering applications because of their ability to deliver electrical stimulation directly to cells, tissue, and organs. One particularly attractive conductive filler for electroactive biomaterials is silver nanoparticles (AgNPs) because of their high conductivity, antibacterial activity, and ability to promote bone healing. However, production of AgNPs involves a toxic reducing agent which would inhibit biological scaffold performance. This work explores facile and green synthesis of AgNPs using extract of Cilembu sweet potato and studies the effect of baking and precursor concentrations (1, 10 and 100 mM) on AgNPs’ properties. Transmission electron microscope (TEM) results revealed that the smallest particle size of AgNPs (9.95 ± 3.69 nm) with nodular morphology was obtained by utilization of baked extract and ten mM AgNO3. Polycaprolactone (PCL)/AgNPs scaffolds exhibited several enhancements compared to PCL scaffolds. Compressive strength was six times greater (3.88 ± 0.42 MPa), more hydrophilic (contact angle of 76.8 ± 1.7°), conductive (2.3 ± 0.5 × 10−3 S/cm) and exhibited anti-bacterial properties against Staphylococcus aureus ATCC3658 (99.5% reduction of surviving bacteria). Despite the promising results, further investigation on biological assessment is required to obtain comprehensive study of this scaffold. This green synthesis approach together with the use of 3D printing opens a new route to manufacture AgNPs-based electroactive with improved anti-bacterial properties without utilization of any toxic organic solvents.

Nanofibrous materials generated through electrospinning have gained significant attention in tissue regeneration, particularly in the domain of bone reconstruction. There is high interest in designing a material resembling bone tissue, and many scientists are trying to create materials applicable to bone tissue engineering with piezoelectricity similar to bone. One of the prospective candidates is highly piezoelectric poly(vinylidene fluoride) (PVDF), which was used for fibrous scaffold formation by electrospinning. In this study, we focused on the effect of PVDF molecular weight (180,000 g/mol and 530,000 g/mol) and process parameters, such as the rotational speed of the collector, applied voltage, and solution flow rate on the properties of the final scaffold. Fourier Transform Infrared Spectroscopy allows for determining the effect of molecular weight and processing parameters on the content of the electroactive phases. It can be concluded that the higher molecular weight of the PVDF and higher collector rotational speed increase nanofibers’ diameter, electroactive phase content, and piezoelectric coefficient. Various electrospinning parameters showed changes in electroactive phase content with the maximum at the applied voltage of 22 kV and flow rate of 0.8 mL/h. Moreover, the cytocompatibility of the scaffolds was confirmed in the culture of human adipose-derived stromal cells with known potential for osteogenic differentiation. Based on the results obtained, it can be concluded that PVDF scaffolds may be taken into account as a tool in bone tissue engineering and are worth further investigation.

Electrostimulation and electroactive scaffolds can positively influence and guide cellular behaviour and thus has been garnering interest as a key tissue engineering strategy. The development of conducting polymers such as polyaniline enables the fabrication of conductive polymeric composite scaffolds. In this study, we report on the initial development of a polycaprolactone scaffold incorporating different weight loadings of a polyaniline microparticle filler. The scaffolds are fabricated using screw-assisted extrusion-based 3D printing and are characterised for their morphological, mechanical, conductivity, and preliminary biological properties. The conductivity of the polycaprolactone scaffolds increases with the inclusion of polyaniline. The in vitro cytocompatibility of the scaffolds was assessed using human adipose-derived stem cells to determine cell viability and proliferation up to 21 days. A cytotoxicity threshold was reached at 1% wt. polyaniline loading. Scaffolds with 0.1% wt. polyaniline showed suitable compressive strength (6.45 ± 0.16 MPa) and conductivity (2.46 ± 0.65 × 10−4 S/cm) for bone tissue engineering applications and demonstrated the highest cell viability at day 1 (88%) with cytocompatibility for up to 21 days in cell culture.

Biomimetic bioreactor systems are increasingly being developed for tissue engineering applications, due to their ability to recreate the native cell/tissue microenvironment. Regarding bone-related diseases and considering the piezoelectric nature of bone, piezoelectric scaffolds electromechanically stimulated by a bioreactor, providing the stimuli to the cells, allows a biomimetic approach and thus, mimicking the required microenvironment for effective growth and differentiation of bone cells. In this work, a bioreactor has been designed and built allowing to magnetically stimulate magnetoelectric scaffolds and therefore provide mechanical and electrical stimuli to the cells through magnetomechanical or magnetoelectrical effects, depending on the piezoelectric nature of the scaffold. While mechanical bioreactors need direct application of the stimuli on the scaffolds, the herein proposed magnetic bioreactors allow for a remote stimulation without direct contact with the material. Thus, the stimuli application (23 mT at a frequency of 0.3 Hz) to cells seeded on the magnetoelectric, leads to an increase in cell viability of almost 30% with respect to cell culture under static conditions. This could be valuable to mimic what occurs in the human body and for application in immobilized patients. Thus, special emphasis has been placed on the control, design and modeling parameters governing the bioreactor as well as its functional mechanism.

A microbial fuel cell bioanode encapsulating electroactive bacteria in core–shell fibers mixed with a conductive scaffold was electrospun. This new design opens up perspectives of storable ready-to-use anodes for portable applications.

Bone defect repair remains a critical challenge in current orthopedic clinical practice, as the available therapeutic strategies only offer suboptimal outcomes. Therefore, bone tissue engineering (BTE) approaches, involving the development of biomimetic implantable scaffolds combined with osteoprogenitor cells and native-like physical stimuli, are gaining widespread interest. Electrical stimulation (ES)-based therapies have been found to actively promote bone growth and osteogenesis in both in vivo and in vitro settings. Thus, the combination of electroactive scaffolds comprising conductive biomaterials and ES holds significant promise in improving the effectiveness of BTE for clinical applications. The aim of this study was to develop electroconductive polyacrylonitrile/poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PAN/PEDOT:PSS) nanofibers via electrospinning, which are capable of emulating the native tissue’s fibrous extracellular matrix (ECM) and providing a platform for the delivery of exogenous ES. The resulting nanofibers were successfully functionalized with apatite-like structures to mimic the inorganic phase of the bone ECM. The conductive electrospun scaffolds presented nanoscale fiber diameters akin to those of collagen fibrils and displayed bone-like conductivity. PEDOT:PSS incorporation was shown to significantly promote scaffold mineralization in vitro. The mineralized electroconductive nanofibers demonstrated improved biological performance as observed by the significantly enhanced proliferation of both human osteoblast-like MG-63 cells and human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs). Moreover, mineralized PAN/PEDOT:PSS nanofibers up-regulated bone marker genes expression levels of hBM-MSCs undergoing osteogenic differentiation, highlighting their potential as electroactive biomimetic BTE scaffolds for innovative bone defect repair strategies.

This two-part project undertaken at QUT and the University of Wurzburg focused on a 3D printing technology known as Melt-electrospinning. In the first part, a melt electrospinner device was designed and assembled to fabricate tissue engineering scaffolds. An air ioniser was then then used to investigate the effects of reduced surface charge on the scaffolds such as to improve the output and height of the printed structure. The goal of the second project was to fabricate scaffolds using an electro-active polymer capable of altering its shape when placed within an electric field.

In vivo, les cellules sont situées dans un microenvironnement poreux et dynamique en 3D qui fournit des signaux biochimiques et biophysiques ainsi que des signaux dynamiques influençant le comportement des cellules dans des contextes physiologiques et pathologiques. Afin de mieux reproduire ces conditions in vitro pour des applications en biologie cellulaire fondamentale, en ingénierie tissulaire et en dépistage de médicaments, cette thèse présente le développement d'échafaudages 4D électro-actifs, combinant une architecture 3D passive et microporeuse nommée polyHIPE et un polymère électroactif, PEDOT. Ces échafaudages servent de plateforme de culture cellulaire dynamique capable de délivrer une stimulation électromécanique. L'étude s'est d'abord concentrée sur la synthèse et la caractérisation des échafaudages polyHIPE-PEDOT électroactifs, qui ont présenté une structure hautement poreuse (10 à 100 µm) et interconnectée, bénéfique pour une colonisation cellulaire rapide. Notamment, ces échafaudages peuvent subir des changements volumétriques en réponse à une stimulation électrique. La deuxième partie de ce travail a porté sur l’évaluation de la compatibilité des échafaudages polyHIPE-PEDOT avec les exigences de la culture cellulaire. Les échafaudages se sont révélés cytocompatibles, favorisant l'adhésion, la migration et la prolifération des cellules. Les cellules à l'intérieur de l'échafaudage ont adopté une morphologie cellulaire fusiforme typique des micro-environnements cellulaires 3D et ont synthétisé de la fibronectine, une protéine de la matrice extracellulaire essentielle pour les interactions cellule-matrice. Dans la troisième partie de cette thèse, un dispositif de stimulation électromécanique adapté aux études de culture cellulaire in vitro (plaque à 6 puits) et à l'imagerie des cellules vivantes (boîte de Petri à fond de verre) a été développé. Un protocole de stimulation a été déterminé et n'a pas induit d'effets cytotoxiques aigus. Après stimulation, les cellules présentent une morphologie hétérogène, cependant, elles sont restées attacher dans la structure poreuse de l'échafaudage. Différentes sondes employées pour marquer des cellules vivantes ont permis de suivre en temps réel la dynamique cellulaire pendant la stimulation électromécanique. En outre, les cellules stimulées présentaient un profil de cytokines différent de celui des cellules non stimulées. Ainsi, cette thèse a démontré la preuve de concept de l'échafaudage polyHIPE-PEDOT électroactif en tant qu'outil pour la culture cellulaire 4D et pour de futures études de mécanobiologie
In vivo, cells are situated within a 3D porous and dynamic microenvironment that provides biochemical and biophysical cues as well as dynamic signals influencing cell behavior across physiological and pathological contexts. To better replicate these conditions in vitro for applications in fundamental cell biology, tissue engineering, and drug screening this thesis presents the development of 4D electroactive scaffolds, combining a 3D passive microporous polyHIPE architecture and an electroactive polymer, PEDOT. These scaffolds serve as a dynamic cell culture platform capable to deliver electromechanical stimulation. The study first focused on the synthesis and characterization of electroactive polyHIPE-PEDOT scaffolds, which demonstrated a highly porous (10 to 100 µm) and interconnective structure beneficial for rapid cell colonization. Notably, these scaffolds could undergo volumetric changes in response to electrical stimulation. The second part of this work focused the polyHIPE-PEDOT scaffolds were found to be suitable for cell culture applications. The scaffolds were found to be cytocompatible, supporting cell adhesion, migration and proliferation. Cells within the scaffold adopted a spindle-like cell morphology typical of 3D cell microenvironments and synthesized fibronectin, an extracellular matrix protein essential for cell-matrix interactions. In the third part of this thesis, an electromechanical stimulation device suitable for in vitro cell culture studies (6-well cell culture plate) and live cell imaging (glass bottomed petri dish) was developed. A stimulation protocol was established and did not induce acute cytotoxic effects. After stimulation, cells exhibited heterogenic cell morphology, however, remained spread within the porous structure of the scaffold. Different live cell probes allowed the real-time monitoring of the cell dynamics during electromechanical stimulation. Furthermore, the stimulated cells exhibited different cytokine profile compared to non-stimulated cells. Thus, this thesis demonstrated the proof of concept of the electroactive polyHIPE-PEDOT scaffold as a tool for 4D cell culture and for future mechanobiological studies

Stem cell based therapy has the potential to treat several severe diseases; Parkinson’s disease is one well- known example. Transplantation of stem cell derived cells into animal models is unfortunately often associated with tumour formation or- uncontrolled growth of the transplanted cells. One strategy to suppress this tumour formation might be to induce differentiation of these cells, which in turn would prevent them from dividing.   Neuroblastoma tumors are known to demonstrate the complete transition from an undifferentiated state to a completely harmful, differentiated appearance and derived cells can be used as a model for cell differentiation and tumor suppression.   In this Master Thesis’s the conducting polymers PEDOT and PPy, that upon formation can be doped with biologically active compounds which in- turn can be released in a controlled manner through electrical stimulation, were formed together with various drugs (e.g. Methotrexate and Mycophenolic Acid), here shown to have effect on Neuroblastoma cells. Neuroblastoma- derived cell line SH- SY5Y was used as a model system for neuronal differentiation and tumour inhibition. Release profiles of neuroblastoma active drugs following electrical stimulation were evaluated and the effects from electrochemical processes on simultaneously growing SH- SY5Y cells were investigated.   The methods to deposit and release the drugs were based on electropolymerization and electrochemically controlled release, respectively. Controlled release of various drugs and compounds was monitored using Vis- and UV- spectroscopy and on some occasions using HPLC.   The electrochemically controlled release of a biologically inactive compound that can be used as a negative control for electrochemical release in future experiments was shown and that resulting electrochemical processes have negative effects on neuroblastoma cell growth.

Bone is a highly hierarchical tissue which is able to heal and remodel in case of small defects and damage. For critical-size defects, the most commonly used approach requires the use of synthetic grafts. These grafts, also known as scaffolds, are physical substrates designed for cell attachment, proliferation and differentiation. Scaffolds for bone applications must be biocompatible, biodegradable, and highly porous, presenting mechanical properties similar to bone and surface characteristics that promote cell-scaffold interactions. The final properties of a scaffold strongly depend on both material compositions and process conditions. This research project investigates different aspects related to the design fabrication and characterization of bioactive electro-active scaffolds. Scaffolds were produced using an extrusion-based additive manufacturing system and different material compositions based on Poly (ε-caprolactone) (PCL) mixed with hydroxyapatite (HA), β-tri-calcium phosphate (TCP) and multi-wall carbon nanotubes (MWCNTs) were investigated. HA and TCP are biocompatible and degradable ceramics related to improve the bioactivity of the scaffolds and MWCNTs were selected to improve mechanical properties and due to their excellent electrical conductivity characteristics, to promote both cell-cell and cell-substrate communication. Experimental work was conducted to characterize both pre-processed materials and produced scaffolds evaluating the rheological, mechanical, thermal, chemical and biological properties. Rheological tests show that printability strongly depends on the concentration of the inorganic fillers (MWCNTs, HA and TCP) and processing parameters such as temperature, screw rotational velocity and deposition velocity. The addition of MWCNTs, HA and TCP can enhance the compressive modulus of PCL scaffolds from 48 MPa to 75 MPa in the case of PCL/HA, or 88 MPa in the case of PCL/TCP and PCL/MWCNTs. Biological results show that all scaffolds containing MWCNTs, HA and TCP are biocompatible (more than 80% cell viability), bioactive (40% increase for TCP, 60% increase for HA and 86% increase for MWCNTs) and osteoconductive (significant increase of ALP activity). Results also show that the addition of MWCNTs improves the osteoinductive properties and the presence of nano-sized HA improves the mineralization process. This research shows that PCL/HA/MWCNTs can be viable scaffolds for bone tissue engineering, providing a promising way for bone tissue regeneration.

Regenerative medicine holds the promise of providing relief for people suffering from diseases where treatment has been unattainable. The research is advancing rapidly; however, there are still many hurdles to overcome before the therapeutic potential of regenerative medicine and cell therapy can be realized. Low in frequency in all tissues, stem cell number is often a limiting factor. Approaches that can control the proliferation and direct the differentiation of stem cells would significantly impact the field. Developing an adequate environment that mimics in vivo conditions is an intensively studied topic for this purpose. Collaboratively, researchers have come close to incorporating nearly all biological cues representative of the human body. Arguably the most overlooked aspect is the influence of electrical stimulation. In this dissertation, we examined polyvinylidene fluoride (PVDF) as a new biomaterial and developed a 3D scaffold capable of providing mechanical and electrical stimuli to cells in vitro.

The fabrication of a 3D scaffold was performed using electrospinning. To obtain highly aligned fibers and scaffolds with controlled porosity, the set-up was modified by incorporating an auxiliary electrode to focus the electric field. Highly aligned fibers with diameters ranging from 500 nm to 15 µm were fabricated from colorless polyimide (CP2) and polyglycolic acid (PGA) and used to construct multilayer scaffolds. This experimental set-up was used to electrospin α-phase PVDF into the polar β-phase. We demonstrated the transition to the β-phase by examining the crystalline structure using x-ray diffraction (XRD), differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR) and polarized light optical microscopy (PLOM). We confirmed these results by observing a polarization peak at 80°C using the thermally stimulated current (TSC) method. Our results proved the electrospinning process used in our investigation poled the PVDF polymer in situ.

TThe influence of architecture and topographical cues was examined on 3D scaffolds and films of CP2 polyimide and PVDF. Culture of human mesenchymal stem cells (hMSCs) for 7 and 14 days demonstrated a significant difference in gene expression. The fibers upregulated the neuronal marker microtubule associated protein (MAP2), while downregulation of this protein was observed on films. Gap junction formation was observed by the expression of connexin-43 after 7 days on PVDF films attributed to its inherent pyroelectric properties. Connexin-43 expression on fibers showed cell-cell contact across the fibers indicating good communication in our 3D scaffold.

A scaffold platform was designed using PVDF fibers that allowed us to apply electrical stimulation to the cells through the fibers. The electrically stimulated PVDF fibers resulted in enhanced proliferation compared to TCPS as evidenced by a 10% increase in the uptake of EdU. Protein expression revealed upregulation of neuronal marker MAP2. Our findings indicate this new platform capable of delivering mechanical, electrical, topographical and biochemical stimuli during in vitro culture holds promise for the advancement of stem cell differentiation and tissue engineering.

Dissertation

Tese de Doutoramento em Engenharia de Materiais
Biomaterials play an increasingly prominent important role in the development and success of tissue engineering, particularly in the regeneration or reestablishment of tissue functions and organs. The improvement in the understanding of the role of biomaterials in the formation and regeneration of new tissue has promoted faster and more effective developments in this area. Biomaterials based on electroactive polymers have gained special interest in the scientific community for applications in tissue engineering, in particular for mechanosensitive tissues (bone, ligaments/tendons) and electroative tissues (brain cells, heart and muscles). Among them, piezoelectric materials show a strong application potential due to their ability to mimic specific biological environments through electrical stimulation. The main objective of this study was to produce scaffolds with different morphologies (fibers, particles and three-dimensional scaffolds) based on piezoelectric polymers, poly(vinylidene fluoride) (PVDF), poly(hydroxybutyrate) (PHB) and poly(L- lactic acid) (PLLA) for tissue engineering applications. Plasma treatments were also used to modify the wettability of the materials. Thus, PVDF samples were processed by electrospinning technique and plasma treatments were performed under oxygen atmosphere for different times and applied power, in order to modify the wettability of the hydrophobic fiber surface. It was observed that plasma treatments didn´t significantly change the average fiber diameter (~ 400 ± 200 nm) or the physicochemical properties of the membranes, in particularly the β-phase content (~ 80-85 %) and the crystallinity degree (42 ± 2 %), showing that this is a suitable method to obtain superhydrophilic membranes. PVDF microspheres were processed by electrospray technique. Among the different processing parameters, polymer concentration was the one that most influenced the microspheres formation. Microspheres with average diameter ranging between 0.81±0.34 μm and 5.55±2.34 μm with a β-phase content between 63-74 % and a degree of crystallinity between 45 and 55% were obtained from dilute or semi-dilute solutions. Cell viability assays demonstrated the potential of the PVDF microspheres for tissue engineering applications. Three dimensional scaffolds based on PVDF with different porosities were produced using three different methods: solvent casting with sodium chloride (NaCl), solvent casting and freeze extraction using nylon and poly (vinyl alcohol) (PVA) templates. Regardless of the processing method, the scaffolds showed polymer crystallization in the β-phase and a degree of crystallinity of ~ 45%. Mechanical tests demonstrated the suitability of the materials for tissue engineering applications. PHB membranes were processed by electrospinning and the influence of processing parameters on the size and distribution of fibers was studied. It was found that the average fiber diameter of the PHB membranes decreased with decreasing internal diameter of the needle and increased with increasing applied electric field and flow rate up to ~ 2.0 µm. The processing parameters didn´t affect the crystalline phase of the PHB membranes yielding a degree of crystallinity of 53%. Further, cell viability studies proved the suitability of the material for tissue engineering applications. Plasma treatments under argon and oxygen atmospheres were performed with thin films and PLLA membranes obtained by solvent casting and electrospinning, respectively. The average diameter of the fibers didn´t change significantly for argon (866 ± 361 nm) or oxygen (1179 ± 397 nm) treatments. However, it was found an increase of the roughness of the films. Surface wettability studies proved that plasma treatments allowed to obtain superhydrophilic or low contact angles on membranes and films, with no influence on cell viability. PLLA microspheres with sizes between 0.16 and 3.9 μm and a degree of crystallinity of 40% and composite PLLA microspheres with cobalt ferrite nanoparticles (CoFe2O4) in the range of 0.8 to 2.2 μm were produced by emulsifying an oil (PVA solution) in water. PLLA spheres proved to be more stable in alkaline environments compared to magnetic composite PLLA microspheres. Moreover, it was found that the introduction of nanoparticles promoted the amorphous state in PLLA. It was shown that PLLA microspheres with and without CoFe2O4 particles didn´t inhibit cellular viability. In conclusion, it was demonstrated the possibility of processing different electroactive polymers in the form of microspheres, fibers, membranes and three-dimensional scaffolds, as well as evaluated the possibility to modify their wettability. This work represents thus a relevant contribution for increasing the use of these materials in innovative strategies for tissue engineering.
Os biomateriais desempenham um papel cada vez mais proeminente no desenvolvimento e sucesso da engenharia de tecidos, nomeadamente na regeneração ou no restabelecimento da função de tecidos/órgãos do corpo humano. Os avanços registados relativamente à compreensão do papel dos biomateriais na formação de novos tecidos e na sua regeneração têm promovido uma maior rapidez e eficácia nos estudos desenvolvidos nesta área. Biomateriais à base de polímeros eletroativos têm despertado especial interesse na comunidade científica, para aplicações em engenharia de tecidos, nomeadamente para tecidos mecano-sensitivos (osso, ligamentos/tendões) e tecidos eletroativos (neurónios, coração e músculos). Em particular, materiais eletroativos à base de polímeros piezoelétricos apresentam uma forte potencialidade por serem capazes de mimetizar o ambiente biológico do tecido através de estímulos eletromecânicos. O principal objetivo do presente trabalho consistiu na produção de scaffolds com diferentes morfologias (fibras, partículas e scaffolds tridimensionais) baseados em polímeros piezoelétricos, o poli(fluoreto de vinilideno) (PVDF), poli(hidroxibutirato) (PHB) e o poli(L-ácido láctico) (PLLA) para aplicações de engenharia de tecidos. Igualmente, foram utilizados tratamentos de plasma para modificar a hidrofobicidade dos materiais. Deste modo, foram processadas membranas de PVDF pela técnica de electrospinning e realizados tratamentos de plasma sobre atmosfera de oxigénio para diferentes tempos de tratamento e potência aplicada de modo a modificar a molhabilidade da superfície hidrofóbica das fibras. Foi observado que o plasma não altera significativamente o diâmetro médio das fibras (~400±200 nm) nem as suas propriedades físico-químicas nomeadamente o conteúdo de fase β (~80-85%) e o seu grau de cristalinidade (42±2 %) demonstrando ser um método eficaz na obtenção de membranas superhidrofílicas. Microesferas de PVDF foram processadas pela técnica de electrospray. De todos os parâmetros estudados (concentração de polímero e parâmetros de processamento) verificou-se que a concentração de polímero é aquela que mais influência a formação de microesferas. Microesferas com diâmetros médios variando entre os 0,81±0,34 μm e 5,55±2,34 μm com um conteúdo de fase β entre os 63-74% e um grau de cristalinidade entre 45 e 55% foram obtidas através de soluções diluídas ou semi-diluídas. Ensaios de viabilidade celular demonstraram a potencialidade destas microesferas para aplicações m engenharia de tecidos. Scaffolds tridimensionais à base de PVDF com diferentes porosidades foram produzidos recorrendo a três métodos distintos: solvent casting – com cloreto de sódio (NaCl), solvent casting e extração a frio utilizando telas de nylon e poli(vinil álcool) (PVA). Independentemente do método de processamento utilizado, os scaffolds apresentam a fase β e um grau de cristalinidade de ~ 45 %. Ensaios mecânicos demostraram a viabilidade dos materiais para a aplicação em causa. Membranas de PHB foram produzidas por electrospinning, realizando-se igualmente um estudo da influência dos parâmetros de processamento no diâmetro e distribuição de fibras. Assim, verificou-se que o diâmetro médio das fibras de PHB diminui com o do diâmetro interno da agulha e aumenta com o aumento do campo elétrico aplicado e taxa de fluxo até ~2,0 μm. Os parâmetros de processamento não influenciaram a fase cristalina das membranas de PHB tendo sido obtido um grau de cristalinidade de 53%. Estudos de viabilidade celular comprovaram a sua potencialidade para aplicações na área de engenharia de tecidos. Tratamentos de plasma sobre atmosferas de árgon e oxigénio foram efetuados em filmes e membranas de PLLA obtidas por solvent casting e por electrospinning, respetivamente. O diâmetro médio das fibras não sofreu uma alteração significativa para o árgon (866±361 nm) nem para o oxigénio (1179±397 nm) tendo-se, no entanto, verificado um aumento da rugosidade dos filmes. Estudos de molhabilidade de superfície demonstraram ser possível obter membranas superhidrofílicas e filmes com um menor valor de ângulo de contacto, não influenciando a viabilidade celular. Microesferas de PLLA com tamanhos compreendidos entre os 0,16 -3,9 μm e um grau de cristalinidade de 40% e microesferas compósitas de PLLA com nanopartículas de ferrita de cobalto (CoFe2O4) na ordem dos 0,8-2,2 μm foram produzidas pelo método de emulsão de um óleo (solução de PVA) em água. Esferas de PLLA demonstraram ser mais estáveis em ambientes alcalinos comparativamente às esferas de PLLA magnéticas. Verificou-se que a introdução de nanopartículas promove o estado amorfo no PLLA. Foi demonstrado que as microesferas de PLLA com e sem partículas de CoFe2O4 não inibem a viabilidade celular. Em conclusão, testou-se a possibilidade de processar diferentes polímeros eletroativos nas formas de microesferas, fibras, membranas e scaffolds tridimensionais, sendo igualmente provada a possibilidade de modificar a sua molhabilidade. Este trabalho representa um contributo relevante para a crescente utilização destes materiais em estratégias inovadoras de engenharia de tecidos.

博士
中原大學
化學研究所
98
This dissertation provides a study of related phenomena (electroactivity and conductivity) which is induced by micro- and nano-structuring polyaniline (PANI) architecture. In the construction of classification approaches, including sacrificial core template for the hollow spheres and electrospinning technique for non-woven nanofibers-mat are presented. N-[3-(trimethoxysilyl)-propyl]aniline simply functioned as a coupling agent was successfully the polyaniline-coated SiO2 core-shell (SiO2@PANI) hybrid micro-capsules and prepared hollow PANI spheres after HF etching. The raspberry-like hollow PANI spheres with wall thickness of 60 and 120 nm can be observed by SEM image. The electrical properties of hollow spheres incorporated PANI thickness was also examined and compared to corresponding core-shell micro-particles. The neat electroactive free-standing nonwoven mat was first prepared through without blending with or grafting onto poly(o-methoxyaniline) (POMA) using an electrospinning technique. The studies showed that continuous fiber structure was obtained due to the higher molecular wight of POMA is synthesized in the CaCl2 presence and an alkoxyl ring-substituted structure on POMA forced to be more soluble. Comparing with governing parameters, uniform POMA fibers produced from 5 wt % POMA solution at 20 kV, feeding rate of 0.02 ml•min-1, and 12 cm of nozzle-to-collector distance. The electrospinning parameters decided the morphological changes through SEM. In addition, electroactivity and mechanical strength of neat electractive electrospun nonwoven mat were also studied by electrochemical CV and DMA. Furthermore, the electrospun POMA fibers will be as a bio-scaffold and study the influence of fiber structure about cortical neural stem cells (NSCs) of proliferation and differentiation. The electrospun POMA scaffold is less harmful and no adverse effects in the long-term proliferation of NSCs, which retained the ability to proliferate, form neurospheres, self-renew, and exhibit multipotentiality. The study of interaction between cells and scaffold were carried out culturing NSCs on electrospun POMA scaffold and assessing their growth, cell viability and differentiation. The results of trypan blue staining cell viability assay, immunofluorescence staining, and SEM images studies confirmed, not only did POMA spun scaffolds showed better NSCs attachment but also enhanced and accelerated differentiation, proving that electrospinning technique produced superior and more suitable biocompatible nsnofibrous scaffolds for NSCs tissue engineering.

The porosity of fibrillar scaffolds plays a pivotal role in the regeneration of living tissue, significantly influencing cell spreading, proliferation, and differentiation, thereby impacting the overall efficiency of regenerative processes. A genuine fibrillar scaffold comprises fibers and pores of diverse sizes and scales. The interconnectivity of the fiber network is intrinsically linked to its mechanical properties. When the scaffold material exhibits piezoelectric characteristics, the application of an electric field to enhance regeneration induces microelectromechanical movement among the fibers, both in relation to one another and in alignment with the applied field. This results in alterations to the connectivity parameters of the network, with notable fluctuations in the distances between electroactive fibers and pore sizes. Consequently, the pathways for signal transduction within the piezoelectric scaffold adapt according to changes in the equivalent circuits of the network. Therefore, a straightforward mathematical approach (time-resolved) is essential for assessing field-induced variations in interfilament distances, network connectivity parameters, and the orientation of fiber ensembles composed of various filaments. This extends to the formation of multiscale networks and tree-like structures with lateral branches for signal routing, which is critical for the engineering and optimization of electroactive scaffolds. We propose employing multifractal analysis for this purpose. We recommend utilizing the multifractal spectra parameters D(q) and f(α), while advising against direct analysis of the scaling behavior for the dynamics of fiber structures for scaffold engineering applications.

After loss of skeletal muscle function due to traumatic injuries, muscle healing may result in scar tissue formation and reduced function. A restoration method is needed to create a bioartificial muscle that supports cell growth. An electroactive, coaxial electrospun scaffold was created using PCL, MWCNT, and a PAA/PVA hydrogel. This scaffold was conductive and displayed an actuation response when electrically stimulated. Rat primary skeletal muscle cells were biocompatible with the scaffold and displayed multi-nucleated constructs with actin interaction. MWCNT toxicity was tested using a single exposure method on rat primary skeletal muscle cells. A decrease in cellular activity was found on day 14, but a recovering trend was observed on days 21 and 28. Scaffolds were implanted in the quadriceps muscle of rats for in vivo biocompatibility investigation. Muscle cells were found to have attached and infiltrated the PCL-MWCNT-PAA/PVA scaffolds over the 28 day period. Further development of this scaffold would lead to a viable option for a bioartificial muscle as it is biocompatible and may provide some functional movement to the patient.