Electrospinning is a process by which micro and nanofibrous scaffolds can be easily fabricated to mimic structures such as the extracellular matrix of bone. A number of materials have been used to fabricate such scaffolds making the process an extremely versatile tool in the field of bone tissue engineering. Many scaffolds however are hydrophobic, leading to poor cellular attachment and proliferation, whilst the actual process of electrospinning is highly variable, producing irregular scaffolds that can ultimately influence cell invasion and differentiation. The focus of this thesis was to address the issues of poor biocompatibility and irregular scaffold production in three commonly used polymers each with different mechanical properties and degradation profiles. Poly (ε-caprolactone) (PCL), polyethylene terephthalate (PET) and poly lactic-co-glycolic acid (PLGA) were functionalised with surfactants in order to improve the biocompatibility and osteoinductive properties of electrospun scaffolds, whilst electrospinning equipment was modified to improve uniformity of scaffold production. Reducing variables known to affect scaffold formation such as temperature and humidity through the use of an environmental stability cabinet improved the reproducibility of scaffolds. The introduction of a Faraday cage, a larger electrode and a negative mandrel potential also improved the quality and quantity of electrospun fibres collected. Lecithin was selected as an appropriate additive for both improving biocompatibility and uniformity of electrospun fibres as it is naturally occurring and induced dose dependent reductions in water contact angle, allowing tailored hydrophobicity. Through gravimetric determination of pore sizes coupled with mathematical modelling, the addition of lecithin was found to reduce both mean fibre diameter and pore size in all scaffolds, improving scaffold homogeneity. At low concentrations (i.e. 2 %) lecithin generally did not affect the mechanical properties of scaffolds, however significant improvements in tensile strength for PCL and nanoindentation for PET were evident, indicating these scaffolds remained suitably strong for bone regeneration purposes. Reduced hydrophobicity acted to improve cellular attachment of Saos-2 osteoblasts to polymers, whilst proliferation on all scaffolds was similar to TCP controls. Furthermore, lecithin incorporation induced osteoinduction, as bone marrow mesenchymal stem cells seeded on these hybrid scaffolds expressed upregulated gene expression for alkaline phosphatase, collagen 1, osteocalcin and osteopontin. In conclusion, these scaffolds, functionalised with lecithin, improve the homogeneity of fibrous mats allowing increased reproducibility and efficiency of the electrospinning process. Furthermore, the improved biocompatibility and osteoinductivity that lecithin presents, allows for the production of more suitable electrospun scaffolds in the field of bone tissue engineering.

<p>The time when it will be possible to grow complex organs in a lab environment comes closerdue to the rapid progress taking place in the area of biotechnology and tissue engineering.Various tissue engineering methods of creating artificial scaffolds has evolved, one of thosebeing electrospinning. Electrospun scaffolds are beneficial in tissue engineering applicationsforemost in regard to their body-mimicking structure. Small pore sizes and low porosities mayhowever limit cell infiltration and thereby creation of 3D functional tissues. The issue of cellinfiltration in electrospun constructs such as nonwoven polymer scaffolds for use in tissueengineering may be solved by a method of simultaneous integration i.e. integrating particlesduring the phase of production in the electrospinning process. In this thesis investigation of aproof-of-concept to the idea of in the future distributing living cells within the threedimensionalstructure during the process of electrospinning of a polymeric biomaterial weremade. To be able to conduct simple experiments glass particles with proper sizes are used tosubstitute living cells. During this thesis a novel method called spray electrospinning tookshape enabling a fine distribution of particles in an electrospun material.The work in this thesis shows that there are methods to simultaneously integrate particles inproduction of scaffold materials, one of these composed of spraying particles whileelectrospinning on a rotating collector. The experiments were done in order to compare thedifferent methods; Double, Coaxial and Spray electrospinning pointing out similarities anddifferences between the three. The methods used to characterize the materials include scalemeasurements and SEM image analysis to determine morphology, fibre diameter, layerthickness and distance between particles. Glass particles were used as substitutes for livingcells for the sake of proof of concept which showed that these can successfully be integratedsimultaneously in an electrospun material. However porosity and the number of particles haveto be further optimized for the material to be ready for use in tissue engineering.The time when it will be possible to grow complex organs in a lab environment comes closerdue to the rapid progress taking place in the area of biotechnology and tissue engineering.Various tissue engineering methods of creating artificial scaffolds has evolved, one of thosebeing electrospinning. Electrospun scaffolds are beneficial in tissue engineering applicationsforemost in regard to their body-mimicking structure. Small pore sizes and low porosities mayhowever limit cell infiltration and thereby creation of 3D functional tissues. The issue of cellinfiltration in electrospun constructs such as nonwoven polymer scaffolds for use in tissueengineering may be solved by a method of simultaneous integration i.e. integrating particlesduring the phase of production in the electrospinning process. In this thesis investigation of aproof-of-concept to the idea of in the future distributing living cells within the threedimensionalstructure during the process of electrospinning of a polymeric biomaterial weremade. To be able to conduct simple experiments glass particles with proper sizes are used tosubstitute living cells. During this thesis a novel method called spray electrospinning tookshape enabling a fine distribution of particles in an electrospun material.The work in this thesis shows that there are methods to simultaneously integrate particles inproduction of scaffold materials, one of these composed of spraying particles whileelectrospinning on a rotating collector. The experiments were done in order to compare thedifferent methods; Double, Coaxial and Spray electrospinning pointing out similarities anddifferences between the three. The methods used to characterize the materials include scalemeasurements and SEM image analysis to determine morphology, fibre diameter, layerthickness and distance between particles. Glass particles were used as substitutes for livingcells for the sake of proof of concept which showed that these can successfully be integratedsimultaneously in an electrospun material. However porosity and the number of particles haveto be further optimized for the material to be ready for use in tissue engineering.</p>

In scaffold-based tissue engineering, control of scaffold degradation turns out to be a critical issue for reliable clinical applications. Degradation in this thesis refers to mass loss. Most of the present control methods take the approach of scaffold material modification and/or scaffold work environment adjustment to address this issue. The latter can easily get to its limit, and the former is not promising in the in-vivo implementation. This thesis proposed a new approach to control of scaffold degradation, that is, closed-loop and real-time control. To realize this approach, this thesis has tackled three important problems, namely (1) effects on degradation, (2) modeling of degradation, and (3) real-time measurement of degradation. This thesis is grounded to a biomaterial called chitosan, as it is widely used for building scaffolds. For the first problem, a statistical experiment was designed and a factorial analysis was conducted. For the second problem, a combined empirical-based and probabilistic-based approach was taken. For the third problem, a prototype of a sensor, which is based on the concept of carbon nanotube (CNT) conductive polymer, was built and tested. This thesis concludes (1) a joint thermal and enzymatic effect is significant on chitosan degradation, (2) the model for chitosan degradation is accurate, and (3) real-time measurement of mass loss of scaffold by means of carbon nanotube film is feasible. The major contributions of this thesis are (i) the proposal of the concept of the closed-loop control of degradation, (ii) a finding that there is a significant conjoint thermal and enzymatic effect on chitosan degradation in terms of mass loss, and (iii) a prototype of the novel CNT (carbon nanotube) chitosan film sensor for real-time measurement of mass loss of the scaffold. The significance of these contributions is that they give us confidence to a full development of the closed-loop and real-time degradation control approach. This approach appears promising to bring forth a transformative impact to clinic applications of scaffold-based tissue regeneration.

Elastin is an essential extracellular protein that is a key component of elastic fibres, providing elasticity to cardiac, dermal, and arterial tissues. During the development of the human cardiovascular system, elastin self-assembles before being integrated into fibres, undergoing no significant turnover during the human lifetime. Abnormalities in elastin can adversely affect its self-assembly, and may lead to malformed elastic fibres. Due to the longevity required of these fibres, even minor abnormalities may have a large cumulative effect over the course of a lifetime, leading to late-onset vascular diseases. This thesis project has identified important, over-represented repetitive elements in elastin which are believed to be important for the self-assembly and elastomeric properties of elastin. Initial studies of single nucleotide polymorphisms (SNPs) from the HapMap project and dbSNP resulted in a set of genetic variation sites in the elastin gene. Based on these studies, glycine to serine and lysine to arginine substitutions were introduced in elastin-like polypeptides. The self-assembly properties of the resulting elastin-like polypeptides were observed under microscope and measured using absorbance at 440nm. Assembled polypeptides were also cross-linked to form thin membranes whose mechanical and physical properties were measured and compared. These mutations resulted in markedly different behavior than wild-type elastin-like proteins, suggesting that mutations in the repetitive elements of the elastin sequence can lead to adverse changes in the physical and functional properties of the resulting protein. Using next-generation sequencing, patients with thoracic aortic aneurysms are being genotyped to discover polymorphisms which may adversely affect the self-assembly properties of elastin, providing a link between genetic variation in elastin and cardiovascular disease.

Elastin is an essential extracellular protein that is a key component of elastic fibres, providing elasticity to cardiac, dermal, and arterial tissues. During the development of the human cardiovascular system, elastin self-assembles before being integrated into fibres, undergoing no significant turnover during the human lifetime. Abnormalities in elastin can adversely affect its self-assembly, and may lead to malformed elastic fibres. Due to the longevity required of these fibres, even minor abnormalities may have a large cumulative effect over the course of a lifetime, leading to late-onset vascular diseases. This thesis project has identified important, over-represented repetitive elements in elastin which are believed to be important for the self-assembly and elastomeric properties of elastin. Initial studies of single nucleotide polymorphisms (SNPs) from the HapMap project and dbSNP resulted in a set of genetic variation sites in the elastin gene. Based on these studies, glycine to serine and lysine to arginine substitutions were introduced in elastin-like polypeptides. The self-assembly properties of the resulting elastin-like polypeptides were observed under microscope and measured using absorbance at 440nm. Assembled polypeptides were also cross-linked to form thin membranes whose mechanical and physical properties were measured and compared. These mutations resulted in markedly different behavior than wild-type elastin-like proteins, suggesting that mutations in the repetitive elements of the elastin sequence can lead to adverse changes in the physical and functional properties of the resulting protein. Using next-generation sequencing, patients with thoracic aortic aneurysms are being genotyped to discover polymorphisms which may adversely affect the self-assembly properties of elastin, providing a link between genetic variation in elastin and cardiovascular disease.

The time when it will be possible to grow complex organs in a lab environment comes closerdue to the rapid progress taking place in the area of biotechnology and tissue engineering.Various tissue engineering methods of creating artificial scaffolds has evolved, one of thosebeing electrospinning. Electrospun scaffolds are beneficial in tissue engineering applicationsforemost in regard to their body-mimicking structure. Small pore sizes and low porosities mayhowever limit cell infiltration and thereby creation of 3D functional tissues. The issue of cellinfiltration in electrospun constructs such as nonwoven polymer scaffolds for use in tissueengineering may be solved by a method of simultaneous integration i.e. integrating particlesduring the phase of production in the electrospinning process. In this thesis investigation of aproof-of-concept to the idea of in the future distributing living cells within the threedimensionalstructure during the process of electrospinning of a polymeric biomaterial weremade. To be able to conduct simple experiments glass particles with proper sizes are used tosubstitute living cells. During this thesis a novel method called spray electrospinning tookshape enabling a fine distribution of particles in an electrospun material.The work in this thesis shows that there are methods to simultaneously integrate particles inproduction of scaffold materials, one of these composed of spraying particles whileelectrospinning on a rotating collector. The experiments were done in order to compare thedifferent methods; Double, Coaxial and Spray electrospinning pointing out similarities anddifferences between the three. The methods used to characterize the materials include scalemeasurements and SEM image analysis to determine morphology, fibre diameter, layerthickness and distance between particles. Glass particles were used as substitutes for livingcells for the sake of proof of concept which showed that these can successfully be integratedsimultaneously in an electrospun material. However porosity and the number of particles haveto be further optimized for the material to be ready for use in tissue engineering.The time when it will be possible to grow complex organs in a lab environment comes closerdue to the rapid progress taking place in the area of biotechnology and tissue engineering.Various tissue engineering methods of creating artificial scaffolds has evolved, one of thosebeing electrospinning. Electrospun scaffolds are beneficial in tissue engineering applicationsforemost in regard to their body-mimicking structure. Small pore sizes and low porosities mayhowever limit cell infiltration and thereby creation of 3D functional tissues. The issue of cellinfiltration in electrospun constructs such as nonwoven polymer scaffolds for use in tissueengineering may be solved by a method of simultaneous integration i.e. integrating particlesduring the phase of production in the electrospinning process. In this thesis investigation of aproof-of-concept to the idea of in the future distributing living cells within the threedimensionalstructure during the process of electrospinning of a polymeric biomaterial weremade. To be able to conduct simple experiments glass particles with proper sizes are used tosubstitute living cells. During this thesis a novel method called spray electrospinning tookshape enabling a fine distribution of particles in an electrospun material.The work in this thesis shows that there are methods to simultaneously integrate particles inproduction of scaffold materials, one of these composed of spraying particles whileelectrospinning on a rotating collector. The experiments were done in order to compare thedifferent methods; Double, Coaxial and Spray electrospinning pointing out similarities anddifferences between the three. The methods used to characterize the materials include scalemeasurements and SEM image analysis to determine morphology, fibre diameter, layerthickness and distance between particles. Glass particles were used as substitutes for livingcells for the sake of proof of concept which showed that these can successfully be integratedsimultaneously in an electrospun material. However porosity and the number of particles haveto be further optimized for the material to be ready for use in tissue engineering.

Poly(methacrylic acid -co- methyl methacrylate) beads were previously shown to have a therapeutic effect on wound closure through the promotion of angiogenesis. However, it was unclear how this polymer elicited its beneficial properties. The goal of this thesis was to characterize the host response to MAA beads by identifying molecules of interest involved in MAA-mediated angiogenesis (in comparison to poly(methyl methacrylate) beads, PMMA). Using a model of diabetic wound healing and a macrophage-like cell line (dTHP-1), eight molecules of interest were identified in the host response to MAA beads. Gene and/or protein expression analysis showed that MAA beads increased the expression of Shh, IL-1β, IL-6, TNF-α and Spry2, but decreased the expression of CXCL10 and CXCL12, compared to PMMA and no beads. MAA beads also appeared to modulate the expression of OPN. In vivo, the global gene expression of OPN was increased in wounds treated with MAA beads, compared to PMMA and no beads. In contrast, dTHP-1 decreased OPN gene expression compared to PMMA and no beads, but expressed the same amount of secreted OPN, suggesting that the cells decreased the expression of the intracellular isoform of OPN. Interestingly, MAA beads had no effect on the expression of pro-angiogenic growth factors VEGF, bFGF and PDGF-B in vivo or in vitro, suggesting that MAA beads do not induce angiogenesis by simply increasing the expression of pro-angiogenic factors, but use more subtle mechanisms. It was hypothesized that these mechanisms may involve modulation of toll-like receptor signaling in macrophages interacting with the protein layer adsorbed on to MAA beads, in a manner distinct from PMMA beads and no beads. Taken together, the results suggest that MAA beads promote angiogenesis through increased expression of Shh, decreased expression of CXCL10 and modulation of the expression of OPN, but not through increased expression of typical pro-angiogenic growth factors. The resulting vessel-rich “alternative foreign body reaction” has exciting clinical implications as the polymer itself was found to exert a therapeutic effect in the absence of bioactive components or transplanted cells. Understanding the mechanism could lead to new applications for this material and others designed on similar principles.

Poly(methacrylic acid -co- methyl methacrylate) beads were previously shown to have a therapeutic effect on wound closure through the promotion of angiogenesis. However, it was unclear how this polymer elicited its beneficial properties. The goal of this thesis was to characterize the host response to MAA beads by identifying molecules of interest involved in MAA-mediated angiogenesis (in comparison to poly(methyl methacrylate) beads, PMMA). Using a model of diabetic wound healing and a macrophage-like cell line (dTHP-1), eight molecules of interest were identified in the host response to MAA beads. Gene and/or protein expression analysis showed that MAA beads increased the expression of Shh, IL-1β, IL-6, TNF-α and Spry2, but decreased the expression of CXCL10 and CXCL12, compared to PMMA and no beads. MAA beads also appeared to modulate the expression of OPN. In vivo, the global gene expression of OPN was increased in wounds treated with MAA beads, compared to PMMA and no beads. In contrast, dTHP-1 decreased OPN gene expression compared to PMMA and no beads, but expressed the same amount of secreted OPN, suggesting that the cells decreased the expression of the intracellular isoform of OPN. Interestingly, MAA beads had no effect on the expression of pro-angiogenic growth factors VEGF, bFGF and PDGF-B in vivo or in vitro, suggesting that MAA beads do not induce angiogenesis by simply increasing the expression of pro-angiogenic factors, but use more subtle mechanisms. It was hypothesized that these mechanisms may involve modulation of toll-like receptor signaling in macrophages interacting with the protein layer adsorbed on to MAA beads, in a manner distinct from PMMA beads and no beads. Taken together, the results suggest that MAA beads promote angiogenesis through increased expression of Shh, decreased expression of CXCL10 and modulation of the expression of OPN, but not through increased expression of typical pro-angiogenic growth factors. The resulting vessel-rich “alternative foreign body reaction” has exciting clinical implications as the polymer itself was found to exert a therapeutic effect in the absence of bioactive components or transplanted cells. Understanding the mechanism could lead to new applications for this material and others designed on similar principles.

Research on the interactions between microbes and polymeric materials constitutes an important part in antimicrobial identification and provides an insight into microbial response on the polymer surfaces. Herein, a high-content screening method with polymer microarray technology was developed to investigate microbe-polymer interactions, especially in studying adhesion/repellence of microbes (bacteria and parasites). Firstly, the polymer microarray approach was used to successfully identify polymers which either selectively captured or prevented the binding of major food-borne pathogen, Salmonella Typhimurium. A parallel study with a lab strain of Escherichia coli was also carried out, revealing polymers which either displayed a common binding activity or which exhibited species discrimination. Likewise, this polymer microarray technology was applied to more bacterial strains, such as Campylobacter, Clostridium, Streptococcus, Klebsiella and their cocktails to discover families of substrates that displayed strong broad-spectrum bacterial non-binding activity. These synthetic polymers represented a novel class of coating materials which can be used to prevent surface colonisation and subsequent formation of bacterial biofilms. The study of protozoan-polymer interactions was also explored in this thesis. Polymers were identified which either bound or prevented parasites (Crysporidium parvum and Giardia lamblia) binding. Material properties, including wettability, surface roughness and polymer composition were analysed to study correlation of parasite binding and the generation of polymer structure function relationships.

Zugl.: Mannheim, Universiẗat Diss., 2007.