Microfabrication of spatially-patterned, polymer scaffolds for applications in stem cell and tissue engineering

textTissue engineering is a recently developed field that combines material science, cell biology, and engineering to create or improve functional tissues/organs. The field of tissue engineering has progressed from a fledgling science to an emerging technology, in large part due to parallel advances in the application of biomaterials and understanding stem cell behavior. Current studies have evaluated certain types of natural and synthetic biomaterials for feasibility of replicating the physio-chemical microenvironments of stem cells. Furthermore, technologies derived from micro-machining and solid free-form fabrication industries have utilized these biomaterials to create scaffolds that resemble tissue-like structures. Recent scaffold fabrication methods have attempted to overcome certain challenges in engineering tissues and organs. One of the fundamental limitations in current tissue engineering efforts has been the inability to develop multiple tissue types (i.e. bone, cartilage, muscles, ligaments) within a single scaffold structure in a predesigned manner. The differentiation of multiple cells within a three-dimensional (3D) scaffold using a single stem cell population has yet to be developed due to challenges in integrating various biochemical factors in a spatially-patterned method. This dissertation discusses scaffold micro-fabrication techniques that use layerby-layer, ultraviolet-based (UV) stereolithography systems. These approaches in microfabricating scaffolds provide an optimal, biomimetic environment for the pre-patterned differentiation of mesenchymal stem cells into skeletal-type tissues. We demonstrated both laser-based and digital micromirror device-based stereolithography systems for creating intricate scaffold architectures with multiple bio-factors encapsulated in predetermined regions. We showed that micro-stereolithography has the powerful capability of building 3D complex scaffolds with specific pore sizes and shapes in a layer-by-layer fashion using photo-crosslinkable monomers. These polymer-based scaffolds were functionalized with specific signaling proteins to create a biomimetic niche in which stem cells can respond, attach, and differentiate. The ultimate goal of this project is to integrate novel concepts of micro-manufacturing along with polymer-controlled release kinetics and stem cell biology to attain pre-designed architectures of tissue structures.Biomedical Engineerin

Innovative Biomaterials for Tissue Engineering

In the field of regenerative medicine, biomaterials play a crucial role since they may serve as a support (scaffold) to promote cell growth and differentiation in order to promote the healing of tissue lesion. The aim of this chapter will be to analyze the properties of more recent biomaterials suitable for tissue engineering strategies, to end to define better and innovative materials for scaffold production. To this purpose, we will analyze the main materials (natural and synthetic) and their characteristics, such as biocompatibility, bioactivity, and biodegradation, and it will be discussed how their chemical-physical properties (surface morphology, porosity, stiffness, and mechanical strength) could affect the interaction with cells and living system. Moreover, the chapter will be focused on methods of extraction or production of biomaterial suitable for scaffolds

骨組織再生のためのデキサメタゾンを担持した二相性リン酸カルシウムナノ粒子とコラーゲンの複合多孔質材料の作製

筑波大学 (University of Tsukuba)201

TUBULAR PERFUSION SYSTEM BIOREACTOR FOR THE DYNAMIC CULTURE OF HUMAN MESENCHYMAL STEM CELLS

In vitro culture techniques must be improved in order to increase the feasibility of cell based tissue engineering strategies. Limitations of current techniques are largely a result of the slow diffusion of molecules such as oxygen into the interior of three dimensional scaffolds in static culture. In order to enhance nutrient transport we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture human mesenchymal stem cells (hMSCs) in three dimensional scaffolds. In our design, hMSCs are cultured on scaffolds tightly packed in a tubular growth chamber. Media is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds. In the first part of the work hMSCs are encapsulated in alginate scaffolds and results demonstrate bioreactor culture enhances late osteoblastic differentiation of hMSCs. An investigation into shear stress in the system revealed that osteogenic markers increase with increasing shear stress and that the differentiation of hMSCs is dependent on cell radial position within scaffolds. In order to enhance the ability to implant these constructs in vivo, a method to create an aggregated cell containing construct in vitro in a bioreactor system was developed. In this part of the work hMSCs are cultured in individual alginate beads in the TPS bioreactor and the beads are aggregated to form one large construct. Following this the TPS bioreactor was investigated to culture synthetic poly-L-lactic acid scaffolds which were fabricated using supercritical carbon dioxide gel drying. In addition to investigating the effects of perfusion on hMSC growth in these scaffolds, the effect of microporosity was investigated. In the final part of the work, a study was completed to determine how TPS culture influenced in vivo bone regeneration. Here alginate beads as well as synthetic PLGA/PCL constructs were used as scaffolds. Results revealed the efficacy of using the tubular perfusion system for bone tissue engineering and demonstrated increased bone formation as a result of hMSC implantation in both alginate and PLGA/PCL scaffolds. These studies highlighted the need for bioreactor culture in vitro as well as scaffolds to support in vivo tissue interaction

Macroporous Scaffolds for Bone Engineering. Studies on Cell Culture and Ectopic Bone Formation

Bone engineering is a rapidly developing area of reconstructive medicine where bone inducing factors and/or cells are combined with a scaffold material to regenerate the structure and function of the original tissue. The aim of this study was to compare the suitability of different macroporous scaffold types for bone engineering applications. The two scaffold categories studied were a) the mechanically strong and stable titanium fiber meshes and b) the elastic and biodegradable porous polymers. Furthermore, bioactive modifications were applied to these basic scaffold types, and their effect on the osteogenic responses was evaluated in cell culture and ectopic bone formation studies. The osteogenic phenotype of cultured cell-scaffold constructs was heightened with a sol-gel derived titania coating, but not with a mixed titania-silica coating. The latter coating also resulted in delayed ectopic bone formation in bone marrow stromal cell seeded scaffolds. However, the better bone contact in early implantation times and more even bone tissue distribution at later times indicated enhanced osteoconductivity of both the coated scaffold types. Overall, the most promising bone engineering results were obtained with titania coated fiber meshes. Elastic and biodegradable poly(ε-caprolactone/D,L-lactide) based scaffolds were also developed in this study. The degradation rates of the scaffolds in vitro were governed by the hydrophilicity of the polymer matrix, and the porous architecture was controlled by the amount and type of porogen used. A continuous phase macroporosity was obtained using a novel CaCl2 • 6H2O porogen. Dynamic culture conditions increased cell invasion, but decreased cell numbers and osteogenicity, within the scaffolds. Osteogenic differentiation in static cultures and ectopic bone formation in cell seeded scaffolds were enhanced in composites, with 30 wt-% of bioactive glass filler.Siirretty Doriast

Combining technologies to create bioactive hybrid scaffolds for bone tissue engineering

Combining technologies to engineer scaffolds that can offer physical and chemical cues to cells is an attractive approach in tissue engineering and regenerative medicine. In this study, we have fabricated polymer-ceramic hybrid scaffolds for bone regeneration by combining rapid prototyping (RP), electrospinning (ESP) and a biomimetic coating method in order to provide mechanical support and a physico-chemical environment mimicking both the organic and inorganic phases of bone extracellular matrix (ECM). Poly(ethylene oxide terephthalate)-poly(buthylene terephthalate) (PEOT/PBT) block copolymer was used to produce three dimensional scaffolds by combining 3D fiber (3DF) deposition, and ESP, and these constructs were then coated with a Ca-P layer in a simulated physiological solution. Scaffold morphology and composition were studied using scanning electron microscopy (SEM) coupled to energy dispersive X-ray analyzer (EDX) and Fourier Tranform Infrared Spectroscopy (FTIR). Bone marrow derived human mesenchymal stromal cells (hMSCs) were cultured on coated and uncoated 3DF and 3DF + ESP scaffolds for up to 21 d in basic and mineralization medium and cell attachment, proliferation, and expression of genes related to osteogenesis were assessed. Cells attached, proliferated and secreted ECM on all the scaffolds. There were no significant differences in metabolic activity among the different groups on days 7 and 21. Coated 3DF scaffolds showed a significantly higher DNA amount in basic medium at 21 d compared with the coated 3DF + ESP scaffolds, whereas in mineralization medium, the presence of coating in 3DF+ESP scaffolds led to a significant decrease in the amount of DNA. An effect of combining different scaffolding technologies and material types on expression of a number of osteogenic markers (cbfa1, BMP-2, OP, OC and ON) was observed, suggesting the potential use of this approach in bone tissue engineerin

Thiol-ene Scaffolds as Synthetic Augments and Silicate Ceramics as Osteogenic Components for Bone Tissue Engineering Applications

Bone tissue engineering approaches using polymer/ceramic composites show promise as effective biocompatible, absorbable, and osteoinductive materials. A novel class of in situ polymerizing thiol-acrylate based copolymers synthesized via an amine-catalyzed Michael addition was studied for its potential to be used in bone defect repair. Both pentaerythritol triacrylate-co-trimethylolpropane tris(3-mercaptopropionate) (PETA-co-TMPTMP) and PETA-co-TMPTMP with hydroxyapatite composites were fabricated in solid cast and foamed forms. These materials were characterized chemically and mechanically followed by an in vitro evaluation of the biocompatibility and chemical stability in conjunction with human adipose-derived mesenchymal pluripotent stem cells (hASC). The solid PETA-co-TMPTMP with and without hydroxyapatite (HA) exhibited compressive strength in the range of 7-20 MPa, while the cytotoxicity and biocompatibility results demonstrate higher metabolic activity of hASC on PETA-co-TMPTMP than on a polycaprolactone control. SEM imaging of hASC show expected spindle shaped morphology when adhered to copolymer. Micro-CT analysis indicates open cell interconnected pores. Foamed PETA-co-TMPTMP HA composite shows promise as an alternative to FDA-approved biopolymers for bone tissue engineering applications. The results of the six week in vivo biocompatibility study using a posterior lumbar spinal fusion model demonstrate that PETA:HA can be foamed in vivo without serious adverse effects at the surgical site. Additionally, it was demonstrated that cells migrate into the interconnected pore volume are found within centers of ossification. Because the natural mechanical strength of materials is highly dependent on the crystal structure, four different silicate-derived ceramics—diopside, akermanite, monticellite, and merwinite have been synthesized and evaluated for their potential as bone augments and grafts. This sparks our interest in the fabrication of polycaprolactone (PCL)/ceramic composites for potential use as scaffolds. Akermanite and monticellite exhibit better osteogenic properties than diopside and merwinite, suggesting that they might be the optimal material for fabricating bone scaffolds

Bioreator de perfusão para a análise high-throughput de combinações de biomateriais/células estaminais

Mestrado em Biotecnologia - Biotecnologia Industrial e AmbientalA engenharia de tecidos combina células humanos, materiais e engenharia de modo a induzir respostas biológicas com o objetivo de proporcionar uma regeneração rápida e correta do tecido danificado. O uso de matrizes tridimensionais (3D) para suportar o crescimento celular, ao contrário dos convencionais materiais 2D, é de grande importância para a simulação da organização estrutural de tecidos biológicos. Outros aspetos da matriz extracelular (ECM), para além da sua arquitetura são conhecidos por afetar a resposta celular. Fatores biomecânicos apresentados às células através das proteínas da ECM influenciam a adesão celular e fenómenos tais como manutenção do fenótipo, diferenciação celular e proliferação. Estudos in vitro muitas vezes falham na apresentação de fatores fisiológicos que incluem dinâmica de fluidos, o qual pode levar a uma correta oxigenação do biomaterial com células incorporadas, bem como a fenómenos de mecanotransdução. Neste trabalho, propomos um sistema que revela o efeito de 32 combinações de proteínas da ECM na adesão e expressão da alcalina fosfatasse (ALP) em células estaminais derivadas da coluna óssea (MSCs), tanto em ambiente estático como dinâmico. Um bioreator foi desenhado de modo a permitir um estudo high-throughput, para que fossem analisadas 32 combinações biomaterial-célula simultaneamente. Este bioreactor foi construído a partir de material de laboratório comum e de baixo custo (incluindo tubos e seringas descartáveis). As MSCs foram semeadas em scaffolds de quitosano poroso, modificado covalentemente com proteínas da ECM do osso, assim como proteínas responsáveis por contacto célula-célula e componentes do esmalte. Uma análise fatorial permitiu correlacionar a presença das várias combinações proteicas com melhor adesão celular ao biomaterial, assim como uma expressão de ALP após 24 horas e 5 dias de cultura. Os dados foram analisados tanto para ambiente estático, como dinâmico na presença de um pequeno fluxo, previamente comprovado como potenciador da diferenciação osteogénica de MSCs. O sistema desenvolvido foi útil na interpretação da grande complexidade das interações célula-ECM, e poderá ter possível aplicação no desenvolvimento de biomateriais para regeneração óssea, bem como em futuras aplicações como modelos de doença.Tissue engineering combines human cells, materials and engineering to induce biological responses seeking the rapid and accurate healing of damaged tissues. The use of three-dimensional (3D) matrices to support cellular growth, in opposition to traditionally used two dimensional (2D) materials, are of utmost importance to emulate the structural organization of biological tissues. Other aspects of the extracellular matrix (ECM) beyond its architecture are known to affect cell response. The biochemical cues presented to cells by ECM proteins influence cell adhesion and phenomena as cell phenotype maintenance, cell differentiation and proliferation. In vitro studies often lack physiological-like cues that include slow fluid dynamics, which may impair the correct oxygenation of the biomaterial-cells construct. Here, we engineered a system to disclose the effect of 32 different ECM protein combinations on the adhesion and alkaline phosphatase (ALP) expression of bone marrow-derived mesenchymal stem cells (MSCs), both under static and flow perfusion conditions. A novel bioreactor was designed to enable a high-throughput study, that allowed to withdraw data from 32 biomaterial-cell combinations in one single test. The bioreactor was assembled from widely available affordable labware (including plastic tubes and disposable syringes). MSCs were seeded on chitosan porous scaffolds covalently modified with bone ECM proteins, as well as cell-cell contact proteins and enamel components. A factorial analysis study allowed correlating the presence of single and combinations of proteins with improved cell adhesion to biomaterials, as well as improved ALP quantification after 24 hours and 5 days of culture. The data was analyzed both for static culture conditions, as well as in the presence of a slow perfusion rate, previously shown to potentiate MSCs osteogenic differentiation. The developed system has proven to be useful in the interpretation of the wide complexity of cells-ECM interactions, and may find application in the development of biomaterials for tissue regeneration or as disease model platforms

The application of agent-based modeling and fuzzy-logic controllers for the study of magnesium biomaterials

Agent-based modeling (ABM) is a powerful approach for studying complex systems and their underlying properties by explicitly modeling the actions and interactions of individual agents. Over the past decade, numerous software programs have been developed to address the needs of the ABM community. However, these solutions often suffer from limitations in design, a lack of comprehensive documentation, or poor performance. As the first objective of this thesis, we introduce CppyABM-a general-purpose software for ABM that provides simulation tools in both Python and C++. CppyABM also enables ABM development using a combination of C++ and Python, taking advantage of the computational performance of C++ and the data analysis and visualization tools of Python. We demonstrate the capabilities of CppyABM through its application to various problems in ecology, virology, and computational biology. As the second objective of this thesis, we use ABM and fuzzy logic controllers (FLCs) to numerically study the effects of magnesium (Mg2+) ions on osteogenesis. Mg-based materials have emerged as the next generation of biomaterials that degrade in the body after implantation and eliminate the need for secondary surgery. We develop two computer models using ABM and FLC and calibrate them based on cell culture experiments. The models were able to capture the regulatory effects of Mg2+ ions and other important factors such as inflammatory cytokines on mesenchymal stem cells (MSC) activities. The models were also able to shed light on the fundamental differences in the cells cultured in different experiments such as proliferation capacity and sensitivity to environmental factors

Strontium Functionalization of Biomaterials for Bone Tissue Engineering Purposes: A Biological Point of View

Strontium (Sr) is a trace element taken with nutrition and found in bone in close connection to native hydroxyapatite. Sr is involved in a dual mechanism of coupling the stimulation of bone formation with the inhibition of bone resorption, as reported in the literature. Interest in studying Sr has increased in the last decades due to the development of strontium ranelate (SrRan), an orally active agent acting as an anti-osteoporosis drug. However, the use of SrRan was subjected to some limitations starting from 2014 due to its negative side effects on the cardiac safety of patients. In this scenario, an interesting perspective for the administration of Sr is the introduction of Sr ions in biomaterials for bone tissue engineering (BTE) applications. This strategy has attracted attention thanks to its positive effects on bone formation, alongside the reduction of osteoclast activity, proven by in vitro and in vivo studies. The purpose of this review is to go through the classes of biomaterials most commonly used in BTE and functionalized with Sr, i.e., calcium phosphate ceramics, bioactive glasses, metal-based materials, and polymers. The works discussed in this review were selected as representative for each type of the above-mentioned categories, and the biological evaluation in vitro and/or in vivo was the main criterion for selection. The encouraging results collected from the in vitro and in vivo biological evaluations are outlined to highlight the potential applications of materials’ functionalization with Sr as an osteopromoting dopant in BTE