Biodegradable polymeric fiber structures in tissue engineering

Tissue engineering offers a promising new approach to create biological alternatives to repair or restore function of damaged or diseased tissues. To obtain three-dimensional tissue constructs, stem or progenitor cells must be combined with a highly porous three-dimensional scaffold, but many of the structures purposed for tissue engineering cannot meet all the criteria required by an adequate scaffold because of lack of mechanical strength and interconnectivity, as well as poor surface characteristics. Fiber-based structures represent a wide range of morphological and geometric possibilities that can be tailored for each specific tissue-engineering application. The present article overviews the research data on tissue-engineering therapies based on the use of biodegradable fiber architectures as a scaffold

Fiber-based structures from natural origin polymers for tissue engineering approches

Tese de doutoramento em Ciência e Tecnologia de Materiais - Área de Biomateriais.Tissue engineering is a new concept emerged as an alternative approach to tissue and organ reconstruction. It differs from organ transplantation by regenerating patient’s own tissue and organs avoiding the biocompatibility and low biofunctionality problems as well as severe immune rejection; which are the main problems of organ transplantation. Tissue engineering methods generally require the use of three main components: a porous scaffold that serves as a matrix, cells and growth factors. The architecture of the tissue engineered scaffold is an important factor to take into consideration that can modulate biological response and the clinical success of the scaffold. Fiber-based scaffolds can provide large surface area and highly interconnective porous structure for cell attachment and ingrowth as well as variety of geometric possibilities that can be regulated depending on the application. In the works presented in this thesis, we developed different fiber based structures based on two natural origin polymers, chitosan and starch, for use in tissue engineering. In Chapter III, chitosan fibers and fiber mesh scaffolds were produced by means of wet spinning technique. The tensile strength of produced fibers was around 205 MPa and Ca-P layer formation could be observed on their surfaces after 14 days of immersion in simulated body fluid (SBF). In Chapter IV, these fibers were then used in further studies for the reinforcement of the structure of a composite material which was consisting of microporous coralline origin hydroxyapatite microgranules, chitosan membranes and chitosan fibers. This composite architecture showed 88% (w/w) swelling in one hour and preserved its complex structure upon long-term incubation. Chitosan fiber meshes were obtained by moulding a predetermined amount of wet-spun fibers. After 7 days of culture, it was found that they were able to support osteoblast-like cell attachment and proliferation. A bone-like apatite layer was obtained on these scaffolds by means of using a simple biomimetic coating process. The apatite formation was determined by different techniques, including SEM, FTIR-ATR, EDS, XRD. The influence of biomimetic coating on osteoblast cell behaviour was also examined by culturing SaOs-2 cells onto scaffolds. The cell population and ALP enzyme activity were found to be higher in the biomimetic coated scaffolds than those in uncoated scaffolds. Furthermore, cell presented more spread and flat morphology when they were seeded on biomimetic coated scaffolds. Regarding starch-based fiber structures, wet spinning was used in Chapter VI as an alternative method to melt spinning for production of starch/polycaprolactone fiber mesh scaffolds. This method seemed to be a very reproducible way of obtaining the fiber mesh scaffolds, typically with 77% porosity and mean pore size 250µm. The specific surface of the scaffolds was measured around 29 mm2/mm3, which was very similar to natural bone. The surfaces of the scaffolds were then treated with plasma under Ar atmosphere. Although both treated and untreated scaffolds exhibited ability for osteoblast-like cell attachment and proliferation, DNA content and ALP enzyme activity were higher in plasma treated scaffolds. Finally, and as a new approach to mimic the natural extracellular matrix (ECM), nano- and micro-fiber combined scaffolds from starch/polycaprolactone blend were designed by means of two step methodology. Electrospinning was used to obtain nanofibers on melt-spun micro-fiber meshes. With regard to the cell culture studies with osteoblast-like cells and rat bone marrow stromal cells, these new architectures showed excellent cell support ability and very promising properties to make them a proper tissue engineering scaffold. In summary, results from these works showed that the designed fiberbased structures from natural origin polymers could successfully serve as a scaffold for tissue engineering.Engenharia de tecidos é um conceito novo que emergiu como uma abordagem alternativa para a reconstrução de tecidos e órgãos. Difere da transplantação de órgãos na medida em que gera os tecidos e órgãos do próprio doente melhorando desta forma a biocompatibilidade e funcionalidade, assim como reduzindo o risco de rejeição pelo sistema imunitário, que são os principais problemas associados à transplantação de órgãos. Geralmente os métodos de engenharia de tecidos requerem o uso de três componentes principais: um suporte poroso que serve de matriz, células e factores de crescimento. A arquitectura do suporte é um aspecto importante a ter em consideração que pode modular a resposta biológica e o sucesso clínico do mesmo a longo prazo. Suportes à base de fibras podem dar origem a uma grande área superficial e a uma estrutura porosa interconectada para a adesão migração celulares, assim como uma variedade de possibilidades geométricas que podem ser adaptadas dependendo da aplicação. No trabalho apresentado nesta tese foram desenvolvidas, para uso em engenharia de tecidos, diferentes estruturas à base de fibras produzidas a partir de dois polímeros de origem natural, sendo estes o quitosano e o amido. Fibras de quitosano e suportes à base de fibras foram produzidos pela técnica de “wet spinning”. A resistência à tracção das fibras produzidas foi em média 204.9 MPa e a formação da camada Ca-P foi observada nas suas superfícies após 14 dias de imersão em “simulated body fluid” (SBF). Estas fibras foram usadas em estudos posteriores para o reforço da estrutura do material compósito, que consiste em microgrânulos de hidroxiapatite de origem coralina microporosa, membranas e fibras de quitosano. Esta arquitectura compósita apresentou 88% (p/p) de inchamento ao fim de uma hora e manteve a sua estrutura complexa em incubações prolongadas. Suportes à base de fibras de quitosano foram obtidos moldando uma quantidade pré-determinada de fibras produzidas por “wetspinning”. Após 7 dias de cultura, verificou-se que eram capazes de suportar a adesão e proliferação de osteoblastos. Uma camada de apatite idêntica ao osso foi obtida nestes suportes através de um processo simples de revestimento biomimético. A formação de apatite foi determinada por diferentes técnicas, tais como: SEM, FTIR-ATR, EDS e XRD. A influência do revestimento biomimético foi também examinada na actividade celular dos osteoblastos, cultivando células SaOs-2 nos suportes. Observou-se que a população celular e a actividade da enzima ALP é maior nos suportes com revestimento biomimético do que nos suportes sem revestimento. Além disso, células semeadas nos suportes com revestimento biomimético apresentam-se mais espalhadas e com uma morfologia plana. Relativamente às estruturas de fibras à base de amido, a técnica de “wet spinning” é utilizada como uma alternativa à técnica de “melt spinning” para produção de suportes à base de fibras de amido/policaprolactona. Este método permite obter de uma forma reprodutível suportes à base de fibras com 77% de porosidade e tamanho médio de poro de 250 µm. A superfície específica dos suportes é de aproximadamente 29 mm2/mm3, que é em muito semelhante ao osso natural. As superfícies dos suportes foram então tratadas com plasma em atmosfera árgon. Embora ambos os suportes tratados e não tratados por plasma tenham exibido capacidade para adesão e proliferação dos osteoblastos, o conteúdo de DNA e a actividade da enzima ALP foram maiores em suportes tratados. Numa nova abordagem para mimetizar a matriz extracelular natural (ECM), foram concebidos, por uma metodologia em duas etapas, suportes combinados de nano- e micro-fibras a partir de uma mistura de amido/policaprolactona. A técnica de “electrospinning” é utilizada para produzir nano-fibras no topo de malhas de micro-fibras sendo estas obtidas por “melt-spun”. No que diz respeito a estudos de culturas celulares com osteoblastos e células da medula óssea de rato, estas novas arquitecturas mostraram uma excelente capacidade de suporte celular como uma estrutura de engenharia de tecidos. Em resumo, os resultados destes trabalhos demonstraram que as estruturas à base de fibras, concebidas a partir de polímeros naturais, podem servir com êxito como suporte para engenharia de tecidos

Incorporation of a sequential BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering

The aim of this study was to develop a 3-D construct carrying an inherent sequential growth factor delivery system. Poly(lactic acid-co-glycolic acid) (PLGA) nanocapsules loaded with bone morphogenetic protein BMP-2 and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanocapsules loaded with BMP-7 made the early release of BMP-2 and longer term release of BMP-7 possible. 3-D fiber mesh scaffolds were prepared from chitosan and from chitosan–PEO by wet spinning. Chitosan of 4% concentration in 2% acetic acid (CHI4–HAc2) and chitosan (4%) and PEO (2%) in 5% acetic acid (CHI4– PEO2–HAc5) yielded scaffolds with smooth and rough fiber surfaces, respectively. These scaffolds were seeded with rat bone marrow mesenchymal stem cells (MSCs). When there were no nanoparticles the initial differentiation rate was higher on (CHI4–HAc2) scaffolds but by three weeks both the scaffolds had similar alkaline phosphatase (ALP) levels. The cell numbers were also comparable by the end of the third week. Incorporation of nanoparticles into the scaffolds was achieved by two different methods: incorporation within the scaffold fibers (NP–IN) and on the fibers (NP–ON). It was shown that incorporation on the CHI4–HAc2 fibers (NP–ON) prevented the burst release observed with the free nanoparticles, but this did not influence the total amount released in 25 days. However NP–IN for the same fibers revealed a much slower rate of release; ca. 70% released at the end of incubation period. The effect of single, simultaneous and sequential delivery of BMP-2 and BMP-7 from the CHI4–HAc2 scaffolds was studied in vitro using samples prepared with both incorporation methods. The effect of delivered agents was higher with the NP–ON samples. Delivery of BMP-2 alone suppressed cell proliferation while providing higher ALP activity compared to BMP-7. Simultaneous delivery was not particularly effective on cell numbers and ALP activity. The sequential delivery of BMP-2 and BMP-7, on the other hand, led to the highest ALP activity per cell (while suppressing proliferation) indicating the synergistic effect of using both growth factors holds promise for the production of tissue engineered bone.This project was conducted within the scope of the EU FP6 NoE Project Expertissues (NMP3-CT-2004-500283). We acknowledge the support to PY through the same project in the form of an integrated PhD grant. We also would like to acknowledge the support from Scientific and Technical Research Council of Turkey (TUBITAK) through project METUNANOBIOMAT (TBAG 105T508)

Enzymatic degradation behavior and cytocompatibility of silk fibroin–starch–chitosan conjugate membranes

The objective of this study was to investigate the influence of silk fibroin and oxidized starch conjugation on the enzymatic degradation behavior and the cytocompatability of chitosan based biomaterials. The tensile stress of conjugate membranes, which was at 50 Megapascal (MPa) for the lowest fibroin and starch composition (10 weight percent (wt.%)), was decreased significantly with the increased content of fibroin and starch. The weight loss of conjugates in α-amylase was more notable when the starch concentration was the highest at 30 wt.%. The conjugates were resistant to the degradation by protease and lysozyme except for the conjugates with the lowest starch concentration. After 10 days of cell culture, the proliferation of osteoblast-like cells (SaOS-2) was stimulated significantly by higher fibroin compositions and the DNA synthesis on the conjugate with the highest fibroin (30 wt.%) was about two times more compared to the native chitosan. The light microscopy and the image analysis results showed that the cell area and the lengths were decreased significantly with higher fibroin/chitosan ratio. The study proved that the conjugation of fibroin and starch with the chitosan based biomaterials by the use of non-toxic reductive alkylation crosslinking significantly improved the cytocompatibility and modulated the biodegradation, respectively.E.T. Baran thanks the Portuguese Foundation for Science and Technology for providing him a PostDoc scholarship (SFRH/BPD/30768/2006). This work was partially supported by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758)

Conjugation of fibroin and starch to chitosan for increasing cell proliferation capacity

[Excerpt] In this study, chitosan conjugates with starch and fibroin were produced for increasing degradability in the presence of physiological enzymes and cell proliferation capacities of biomaterials. The degradation profile was monitored over prolonged time periods and characterization of chemical changes during degradation periods were investigated by spectroscopic methods. Various ratios of starch, fibroin and chitosan (%, (weight/weight)) were prepared. The in vitro cell culture studies were conducted to evaluate biocompatibility and proliferation capacities of conjugate materials. [...]info:eu-repo/semantics/publishedVersio

Production and characterization of chitosan fibers and 3-D fiber mesh scaffolds for tissue engineering applicattions

This study reports on the production of chitosan fibers and 3-D fiber meshes for the use as tissue engineering scaffolds. Both structures were produced by means of a wet spinning technique. Maximum strain at break and tensile strength of the developed fibers were found to be 8.5% and 204.9 MPa, respectively. After 14 d of immersion in simulated body fluid (SBF), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and inductively coupled plasma emission (ICP) spectroscopy analyses showed that a bioactive Ca-P layer was formed on the surface of the fibers, meaning that they exhibit a bioactive behavior. The samples showed around 120% max. swelling in physiological conditions. The pore sizes of 3-D chitosan fiber mesh scaffolds were observed to be in the range of 100-500 m by SEM. The equilibrium-swelling ratio of the developed scaffolds was found to be around 170% (w/w) in NaCl solution at 37 °C. Besides that, the limit swelling strain was less than 30%, as obtained by mechanical spectroscopy measurements in the same conditions. The viscoelastic properties of the scaffolds were also evaluated by both creep and dynamic mechanical tests. By means of using short-term MEM extraction test, both types of structures (fibers and scaffolds) were found to be non-cytotoxic to fibroblasts. Furthermore, osteoblasts directly cultured over chitosan fiber mesh scaffolds presented good morphology and no inhibition of cell proliferation could be observed.FCT Foundation for Science and Technology, through funds from the POCTI and/or FEDER programmes

Investigation of osteoblast response to biodegradable bacterial cellulose scaffolds

[Excerpt] Aim of this project is to investigate the ability of bacterial cellulose (BC) and oxidized Bacterial cellulose (OBC) use as a scaffold in tissue engineering. Bacterial cellulose was produced with being provided optimum conditions from Acetobacter xylinus. BC was transformed dialdehyde cellulose (DAC) as biodegradable form by treating with periodate. Oxidation was carried out in aqueous solution at 508C in the dark for 24 hours. The mole-to-mole ratio of sodium metaperiodate to anhydroglucose repeat unit (AGU) of cellulose was 0.5, 1.0 and 1.5. [...]info:eu-repo/semantics/publishedVersio

Design of nano- and microfiber combined scaffolds by electrospinning of collagen onto starch-based fiber meshes : a man-made equivalent of natural extracellular matrix

Mimicking the structural organization and biologic function of natural extracellular matrix has been one of the main goals of tissue engineering. Nevertheless, the majority of scaffolding materials for bone regeneration highlights biochemical functionality in detriment of mechanical properties. In this work we present a rather innovative construct that combines in the same structure electrospun type I collagen nanofibers with starchbased microfibers. These combined structures were obtained by a two-step methodology and structurally consist in a type I collagen nano-network incorporated on a macro starch-based support. The morphology of the developed structures was assessed by several microscopy techniques and the collagenous nature of the nanonetwork was confirmed by immunohistochemistry. In addition, and especially regarding the requirements of large bone defects, we also successfully introduced the concept of layer by layer, as a way to produce thicker structures. In an attempt to recreate bone microenvironment, the design and biochemical composition of the combined structures also envisioned bone-forming cells and endothelial cells (ECs). The inclusion of a type I collagen nano-network induced a stretched morphology and improved the metabolic activity of osteoblasts. Regarding ECs, the presence of type I collagen on the combined structures provided adhesive support and obviated the need of precoating with fibronectin. It was also importantly observed that ECs on the nano-network organized into circular structures, a three-dimensional arrangement distinct from that observed for osteoblasts and resembling the microcappillary-like organizations formed during angiogenesis. By providing simultaneously physical and chemical cues for cells, the herein-proposed combined structures hold a great potential in bone regeneration as a man-made equivalent of extracellular matrixK. Tuzlakoglu and M. I. Santos thank the Portuguese Foundation for Science and Technology for their Ph.D. scholarship (SFRH/BD/8502/2002 and SFRH/BD/13428/2003). This work was partially supported by FCT Foundation for Science and Technology, through funds from the POCTI and/or FEDER programs and by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283). Work developed under the cooperation agreement between UM-3B's research group and the Hospital de S. Marcos, Braga. The authors thank to L. Goreti Pinto for her help on confocal microscopy studies

Nano- and micro-fiber combined scaffolds : a new architecture for bone tissue engineering

One possible interesting way of designing a scaffold for bone tissue engineering is to base it on trying to mimic the biophysical structure of natural extracellular matrix (ECM). This work was developed in order to produce scaffolds for supporting bone cells. Nano and micro fiber combined scaffolds were originally produced from starch based biomaterials by means of a fiber bonding and a electrospinning, two step methodology. The cell culture studies with SaOs-2 human osteoblast-like cell line and rat bone marrow stromal cells demonstrated that presence of nanofibers influenced cell shape and cytoskeletal organization of the cells on the nano/micro combined scaffolds. Moreover, cell viability and Alkaline Phosphatase (ALP) activity for both cell types was found to be higher in nano/micro combined scaffolds than in control scaffolds based on fiber meshes without nanofibers. Consequently, the developed structures are believed have a great potential on the 3D organization and guidance of cells that is provided for engineering of 3-dimensional bone tissues

Endothelial cell colonization and angiogenic potential of combined nano- and micro-fibrous scaffolds for bone tissue engineering

Presently the majority of tissue engineering approaches aimed at regenerating bone relies only on postimplantation vascularization. Strategies that include seeding endothelial cells (ECs) on biomaterials and promoting their adhesion, migration and functionality might be a solution for the formation of vascularized bone. Nano/micro-fiber-combined scaffolds have an innovative structure, inspired by extracellular matrix (ECM) that combines a nano-network, aimed to promote cell adhesion, with a micro-fiber mesh that provides the mechanical support. In this work we addressed the influence of this nano-network on growth pattern, morphology, inflammatory expression profile, expression of structural proteins, homotypic interactions and angiogenic potential of human EC cultured on a scaffold made of a blend of starch and poly(caprolactone). The nano-network allowed cells to span between individual micro-fibers and influenced cell morphology. Furthermore, on nano-fibers as well as on micro-fibers ECs maintained the physiological expression pattern of the structural protein vimentin and PECAM-1 between adjacent cells. In addition, ECs growing on the nano/micro-fiber-combined scaffold were sensitive to pro-inflammatory stimulus. Under pro-angiogenic conditions in vitro, the ECM-like nano-network provided the structural and organizational stability for ECs’ migration and organization into capillary-like structures. The architecture of nano/micro-fiber-combined scaffolds elicited and guided the 3D distribution of ECs without compromising the structural requirements for bone regeneration.M.I. Santos would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her PhD scholarship (SFRH/BD/13428/2003). This work was partially supported by FCT through funds from POCTI and/or FEDER programs and by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283)