@phdthesis{Koschitzki2020,
  author    = {Koschitzki, Kim Christine Cornelia},
  title     = {Evaluation of preclinical animal models in bone tissue engineering and their success in clinical translation},
  doi       = {10.25972/OPUS-20759},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207593},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Autologous bone still represents today's gold standard for the treatment of critical size bone defects and fracture non-unions despite associated disadvantages regarding limitations in availability, donor site morbidity, costs and efficacy. Bone tissue engineered constructs would present a promising alternative to currently available treatments. However, research on preclinical animal studies still fails to provide clinical applicable results able to allow the replacement of currently applied methods. It seems that the idea of bone tissue engineering, which has now been integral part of academic studies for over 30 years, got somehow stuck at an intermediate level, in between intense preclinical research and striven stages of initial clinical trial phases. A clear discrepancy exists between the number of studies with preclinical animal models for bone tissue engineering and the number of clinically approved bone tissue engineered constructs available to patients. The aim of this thesis was hence to evaluate preclinical animal models for bone tissue engineering as well as the perception of scientists and clinicians towards these models. Moreover, the general role of bone tissue engineering and its clinical need assessed by scientists and surgeons was investigated. A survey was conducted questioning both scientific and clinical opinions on currently available study designs and researchers' satisfaction with preclinical animal models. Additionally, a literature research was conducted, resulting in 167 papers from the last 10 years that report current designs of preclinical orthotopic animal studies in bone tissue engineering. Thereby, the focus lied on the description of the models regarding animal species, strain, age, gender and defect design. The outcome of the literature search was evaluated and compared to the outcome obtained from the survey. The survey data revealed that both scientists and surgeons generally remain positive about the future role of bone tissue engineering and its step to clinical translation, at least in the distant future, where it then might replace the current gold standard, autologous bone. Moreover, most of the participants considered preclinical animal models as relevant and well developed but the results as not yet realizable in the clinics. Surgeons thereby demonstrated a slightly more optimistic perception of currently conducted research with animal models compared to scientists. However, a rather inconsistent description of present preclinical study designs could be discerned when evaluating the reported study designs in the survey and the papers of the literature search. Indeed, defining an appropriate animal species, strain, age, gender, observation time, observation method and surgical design often depends on different indications and research questions and represents a highly challenging task for the establishment of a preclinical animal model. The existing lack of valid guidelines for preclinical testing of bone tissue engineering leads hence to a lack of well standardized preclinical animal models. Moreover, still existing knowledge gaps regarding aspects that affect the process of fracture healing, such as vascularization or immunological aspects, were found to hinder clinical translation of bone tissue engineered constructs. Using literature review and survey, this thesis points out critical issues that need to be addressed to allow clinical translation of bone tissue engineered constructs. It can be concluded that currently existing study designs with preclinical animal models cannot live up to the claim of providing suitable results for clinical implementation. The here presented comprehensive summary of currently used preclinical animal models for bone tissue engineering reveals a missing consensus on the usage of models such as an apparent lack of reporting and standardization regarding the study designs described in both papers from the literature review and the survey. It thereby indicates a crucial need to improve preclinical animal models in order to allow clinical translation. Despite the fact that participants of the survey generally revealed a positive perception towards the use of bone tissue engineered constructs and affirmed the clinical need for such novel designs, the missing standardization constitutes a main weak point for the provision of reliable study outcome and the translational success of the models. The optimization of reproducibility and reliability, as well as the further understanding of ongoing mechanisms in bone healing in order to develop effective tissue engineered constructs, need to form the basis of all study designs. The study outcomes might then fulfill the requirements of maybe today's and hopefully tomorrow's aging population.},
  language  = {en}
}
@phdthesis{Wiesner2020,
  author    = {Wiesner, Miriam},
  title     = {Stem Cell-based Adipose Tissue Engineering - Engineering of Prevascularized Adipose Tissue Constructs In Vitro \& Investigation on Gap Junctional Intercellular Communication in Adipose-derived Stem Cells},
  doi       = {10.25972/OPUS-18500},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-185005},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {In reconstructive and plastic surgery, there exists a growing demand of adequate tissue implants, since currently available strategies for autologous transplantation are limited by complications including transplant failure and donor site morbidity. By developing in vitro and in vivo autologous substitutes for defective tissue sites, adipose tissue engineering can address these challenges, although there are several obstacles to overcome. One of the major limitations is the sufficient vascularization of in vitro engineered large constructs that remains crucial and demanding for functional tissues. Decellularized jejunal segments may represent a suitable scaffolding system with preexisting capillary structures that can be repopulated with human microvascular endothelial cells (hMVECs), and a luminal matrix applicable for the adipogenic differentiation of human adipose-derived stem cells (hASCs). Hence, co-culture of these cells in jejunal segments, utilizing a custom-made bioreactor system, was characterized in terms of vascularization and adipose tissue development. Substantial adipogenesis of hASCs was demonstrated within the jejunal lumen in contrast to non-induced controls, and the increase of key adipogenic markers was verified over time upon induction. The development of major extracellular matrix components of mature adipose tissue, such as laminin and collagen IV, was shown within the scaffold in induced samples. Successful reseeding of the vascular network with hMVECs was demonstrated in long-term culture and co-localization of vascular structures and adipogenically differentiated hASCs was observed. Therefore, these results represent a novel approach for in vitro engineering of vascularized adipose tissue constructs that warrants further investigations in preclinical studies. Another still existing obstacle in adipose tissue engineering is the insufficient knowledge about the applied cells, for instance the understanding of how cells can be optimally expanded and differentiated for successful engineering of tissue transplants. Even though hASCs can be easily isolated from liposuction of abdominal fat depots, yielding low donor site morbidity, huge numbers of cells are required to entirely seed complex and large 3D matrices or scaffolds. Thus, cells need to be large-scale expanded in vitro on the premise of not losing their differentiation capacity caused by replicative aging. Accordingly, an improved differentiation of hASCs in adipose tissue engineering approaches remains still desirable since most engineered constructs exhibit an inhomogeneous differentiation pattern. For mesenchymal stem cells (MSCs), it has been shown that growth factor application can lead to a significant improvement of both proliferation and differentiation capacity. Especially basic fibroblast growth factor (bFGF) represents a potent mitogen for MSCs, while maintaining or even promoting their osteogenic, chondrogenic and adipogenic differentiation potential. As there are currently different contradictory information present in literature about the applied bFGF concentration and the explicit effect of bFGF on ASC differentiation, here, the effect of bFGF on hASC proliferation and differentiation capacity was investigated at different concentrations and time points in 2D culture. Preculture of hASCs with bFGF prior to adipogenic induction showed a remarkable effect, whereas administration of bFGF during culture did not improve adipogenic differentiation capacity. Furthermore, the observations indicated as mode of action an impact of this preculture on cell proliferation capacity, resulting in increased cellular density at the time of adipogenic induction. The difference in cell density at this time point appeared to be pivotal for increased adipogenic capacity of the cells, which was confirmed in a further experiment employing different seeding densities. Interestingly, furthermore, the obtained results suggested a cell-cell contact-mediated mechanism positively influencing adipogenic differentiation. As a consequence, subsequently, studies were conducted focusing on intercellular communication of these cells, which has hardly been investigated to date. Despite the multitude of literature on the differentiation capacity of ASCs, little is reported about the physiological properties contributing to and controlling the process of lineage differentiation. Direct intercellular communication between adjacent cells via gap junctions has been shown to modulate differentiation processes in other cell types, with connexin 43 (Cx43) being the most abundant isoform of the gap junction-forming connexins. Thus, in the present study we focused on the expression of Cx43 and gap junctional intercellular communication (GJIC) in hASCs, and its significance for adipogenic differentiation of these cells. Cx43 expression in hASCs was demonstrated histologically and on the gene and protein expression level and was shown to be greatly positively influenced by cell seeding density. Functionality of gap junctions was proven by dye transfer analysis in growth medium. Adipogenic differentiation of hASCs was shown to be also distinctly elevated at higher cell seeding densities. Inhibition of GJIC by 18α-glycyrrhetinic acid significantly compromised adipogenic differentiation, as demonstrated by histology, triglyceride quantification, and adipogenic marker gene expression. Flow cytometry analysis showed a lower proportion of cells undergoing adipogenesis when GJIC was inhibited, further indicating the importance of GJIC in the differentiation process. Altogether, these results demonstrate the impact of direct cell-cell communication via gap junctions on the adipogenic differentiation process of hASCs and may contribute to further integrate direct intercellular crosstalk in rationales for tissue engineering approaches.},
  subject      = {Tissue Engineering},
  language  = {en}
}
@phdthesis{Tylek2021,
  author    = {Tylek, Tina},
  title     = {Establishment of a Co-culture System of human Macrophages and hMSCs to Evaluate the Immunomodulatory Properties of Biomaterials},
  doi       = {10.25972/OPUS-20357},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203570},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The outcome of the innate immune response to biomaterials mainly determines whether the material will be incorporated in the body to fulfill its desired function or, when it gets encapsulated, will be rejected in the worst case. Macrophages are key players in this process, and their polarization state with either pro- (M1), anti-inflammatory (M2), or intermediate characteristics is crucial for deciding on the biomaterial's fate. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates the proper healing and subsequent regeneration. Therefore, biomaterial-based polarization may aid in driving macrophages in the desired direction. However, the in vivo process is highly complex, and a mono-culture of macrophages in vitro displays only one part of the cellular system, but, to this date, there is a lack of established co-cultures to assess the immune response to biomaterials. Thus, this thesis aimed to establish a functional co-culture system of human macrophages and human mesenchymal stromal cells (hMSCs) to improve the assessment of the immune response to biomaterials in vitro. Together with macrophages, hMSCs are involved in tissue regeneration and inflammatory reactions and can modulate the immune response. In particular, endogenously derived hMSCs considerably contribute to the successful engrafting of biomaterials. This thesis focused on poly(ε-caprolactone) (PCL) fiber-based scaffolds produced by the technique of melt electrowriting (MEW) as biomaterial constructs. Via this fabrication technique, uniform, precisely ordered scaffolds varying in geometry and pore size have been created in-house. To determine the impact of scaffold geometries and pore sizes on macrophages, mono-cultures incubated on scaffolds were conducted. As a pre-requisite to achieve a functional co-culture system on scaffolds, setups for direct and indirect systems in 2D have initially been established. These setups were analyzed for the capability of cell-cell communication. In parallel, a co-culture medium suitable for both cell types was defined, prior to the establishment of a step-by-step procedure for the co-cultivation of human macrophages and hMSCs on fiber-based scaffolds. Regarding the scaffold morphologies tested within this thesis to improve M2-like polarization, box-shaped scaffolds outperformed triangular-, round- or disordered-shaped ones. Upon further investigation of scaffolds with box-shaped pores and precise inter-fiber spacing from 100 µm down to only 40 µm, decreasing pore sizes facilitated primary human macrophage elongation accompanied by their differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 µm. To the best of my knowledge, this was the first time that the elongation of human macrophages in a 3D environment has been correlated to their M2-like polarization. Thus, these results may set the stage for the design, the assessment, and the selection of new biomaterials, which can positively affect the tissue regeneration. The cell communication of both cell types, detected via mitochondria exchange in direct and indirect co-cultures systems, took place in both directions, i.e., from hMSCs to macrophages and vice versa. Thereby, in direct co-culture, tunneling nanotubes enabled the transfer from one cell type to the respective other, while in indirect co-culture, a non-directional transfer through extracellular vesicles (EVs) released into the medium seemed likely. Moreover, the phagocytic activity of macrophages after 2D co-cultivation and hence immunomodulation by hMSCs increased with the highest phagocytic rate after 48 h being most pronounced in direct co-cultivation. As the commonly used serum supplements for macrophages and hMSCs, i.e., human serum (hS) and fetal calf serum (FCS), respectively, failed to support the respective other cell type during prolonged cultivation, these sera were replaced by human platelet lysate (hPL), which has been proven to be the optimal supplement for the co-cultivation of human macrophages with hMSCs within this thesis. Thereby, the phenotype of both cell types, the distribution of both cell populations, the phagocytic activity of macrophages, and the gene expression profiles were maintained and comparable to the respective standard mono-culture conditions. This was even true when hPL was applied without the anticoagulant heparin in all cultures with macrophages, and therefore, heparin was omitted for further experiments comprising hPL and macrophages. Accordingly, a step-by-step operating procedure for the co-cultivation on fiber-based scaffolds has been established comprising the setup for 3D cultivation as well as the description of methods for the analysis of phenotypical and molecular changes upon contact with the biomaterial. The evaluation of the macrophage response depending on the cultivation with or without hMSCs and either on scaffolds or on plastic surfaces has been successfully achieved and confirmed the functionality of the suggested procedures. In conclusion, the functional co-culture system of human macrophages and hMSCs established here can now be employed to assess biomaterials in terms of the immune response in a more in vivo-related way. Moreover, specifically designed scaffolds used within the present thesis showed auspicious design criteria positively influencing the macrophage polarization towards the anti-inflammatory, pro-healing type and might be adaptable to other biomaterials in future approaches. Hence, follow-up experiments should focus on the evaluation of the co-culture outcome on promising scaffolds, and the suggested operating procedures should be adjusted to further kinds of biomaterials, such as cements or hydrogels.},
  subject      = {Makrophage},
  language  = {en}
}
@phdthesis{Simann2015,
  author    = {Simann, Meike},
  title     = {Aufkl{\"a}rung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von prim{\"a}ren humanen Knochenmark-Stroma-Zellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119322},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Regulating and reverting the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) represents a promising approach for osteoporosis therapy and prevention. Fibroblast growth factor 1 (FGF1) and its subfamily member FGF2 were scored as lead candidates to exercise control over lineage switching processes (conversion) in favor of osteogenesis previously. However, their impact on differentiation events is controversially discussed in literature. Hence, the present study aimed to investigate the effects of these FGFs on the adipogenic and osteogenic differentiation and conversion of primary hBMSCs. Moreover, involved downstream signaling mechanisms should be elucidated and, finally, the results should be evaluated with regard to the possible therapeutic approach. This study clearly revealed that culture in the presence of FGF1 strongly prevented the adipogenic differentiation of hBMSCs as well as the adipogenic conversion of pre-differentiated osteoblastic cells. Lipid droplet formation was completely inhibited by a concentration of 25 ng/µL. Meanwhile, the expression of genetic markers for adipogenic initiation, peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAAT/enhancer binding protein alpha (C/EBPa), as well as subsequent adipocyte maturation, fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL), were significantly downregulated. Yet, the genetic markers of osteogenic commitment and differentiation were not upregulated during adipogenic differentiation and conversion under FGF supplementation, not supporting an event of osteogenic lineage switching. Moreover, when examining the effects on the osteogenic differentiation of hBMSCs and the osteogenic conversion of pre-differentiated adipocytic cells, culture in the presence of FGF1 markedly decreased extracellular matrix (ECM) mineralization. Additionally, the gene expression of the osteogenic marker alkaline phosphatase (ALP) was significantly reduced and ALP enzyme activity was decreased. Furthermore, genetic markers of osteogenic commitment, like the master regulator runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 4 (BMP4), as well as markers of osteogenic differentiation and ECM formation, like collagen 1 A1 (COL1A1) and integrin-binding sialoprotein (IBSP), were downregulated. In contrast, genes known to inhibit ECM mineralization, like ANKH inorganic pyrophosphate transport regulator (ANKH) and osteopontin (OPN), were upregulated. ANKH inhibition revealed that its transcriptional elevation was not crucial for the reduced matrix mineralization, perhaps due to decreased expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) that likely annulled ANKH upregulation. Like FGF1, also the culture in the presence of FGF2 displayed a marked anti-adipogenic and anti-osteogenic effect. The FGF receptor 1 (FGFR1) was found to be crucial for mediating the described FGF effects in adipogenic and osteogenic differentiation and conversion. Yet, adipogenic conversion displayed a lower involvement of the FGFR1. For adipogenic differentiation and osteogenic differentiation/conversion, downstream signal transduction involved the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)/ERK kinases 1 and 2 (MEK1/2), probably via the phosphorylation of FGFR docking protein FGFR substrate 2a (FRS2a) and its effector Ras/MAPK. The c-Jun N-terminal kinase (JNK), p38-MAPK, and protein kinase C (PKC) were not crucial for the signal transduction, yet were in part responsible for the rate of adipogenic and/or osteogenic differentiation itself, in line with current literature. Taken together, to the best of our knowledge, our study was the first to describe the strong impact of FGF1 and FGF2 on both the adipogenic and osteogenic differentiation and conversion processes of primary hBMSCs in parallel. It clearly revealed that although both FGFs were not able to promote the differentiation and lineage switching towards the osteogenic fate, they strongly prevented adipogenic differentiation and lineage switching, which seem to be elevated during osteoporosis. Our findings indicate that FGF1 and FGF2 entrapped hBMSCs in a pre-committed state. In conclusion, these agents could be applied to potently prevent unwanted adipogenesis in vitro. Moreover, our results might aid in unraveling a pharmacological control point to eliminate the increased adipogenic differentiation and conversion as potential cause of adipose tissue accumulation and decreased osteoblastogenesis in bone marrow during aging and especially in osteoporosis.},
  subject      = {Mesenchymzelle},
  language  = {en}
}
@phdthesis{RamaniMohan2021,
  author    = {Ramani Mohan, Ramkumar},
  title     = {Effect of Mechanical Stress On Stem Cells to Improve Better Bone Regeneration},
  doi       = {10.25972/OPUS-24013},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240134},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Critical size bone defects and nonunion fractures remain difficult to treat. Although cell-loaded bone substitutes have improved bone ingrowth and formation, the lack of methods for achieving viability and the uniform distribution of cells in the scaffold limits their use as bone grafts. In addition, the predominant mechanical stimulus that drives early osteogenic cell maturation has not been clearly identified. Further, it is challenging to evaluate mechanical stimuli (i.e., deformation and fluid-flow-induced shear stress) because they are interdependent. This thesis compares different mechanical stimuli applied to cell-seeded scaffolds to develop bone grafts efficiently for the treatment of critical size bone defects. It also seeks to understand how deformation strain and interstitial fluid-flow-induced shear stress promote osteogenic lineage commitment. In this thesis, different scaffolds were seeded with primary human bone marrow mesenchymal stem cells (BM-MSCs) from different donors and subjected to static and dynamic culture conditions. In contrast with the static culture conditions, homogenous cell distributions were accomplished under dynamic culture conditions. Additionally, the induction of osteogenic lineage commitment without the addition of soluble factors was observed in the bioreactor system after one week of cell culture. To determine the role of mechanical stimuli, a bioreactor was developed to apply mechanical deformation force to a mesenchymal stem sell (MSC) line (telomerase reverse transcriptase (TERT)) expressing a strain-responsive AP-1 luciferase reporter construct on porous scaffolds. Increased luciferase expression was observed in the deformation strain compared with the shear stress strain. Furthermore, the expression of osteogenic lineage commitment markers such as osteonectin, osteocalcin (OC), osteopontin, runt-related transcription factor 2 (RUNX2), alkaline phosphate (AP), and collagen type 1 was significantly downregulated in the shear stress strain compared with the deformation strain. These findings establish that the deformation strain was the predominant stimulus causing skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation. Finally, these findings were used to develop a bioreactor in vitro test system in which the effect of medication on osteoporosis could be tested. Primary human BM-MSCs from osteoporotic donors were subjected to strontium ranelate (an osteoporotic drug marketed as Protelos®). Increased expression of collagen type 1 and calcification was seen in the drugtreated osteoporotic stem cells compared with the nondrug-treated osteoporotic stem cells. Thus, this bioreactor technology can easily be adapted into an in vitro osteoporotic drug testing system.},
  language  = {en}
}
@phdthesis{OliveiraAlvesPereira2022,
  author    = {Oliveira Alves Pereira, Ana Rita},
  title     = {Modelling of Mesenchymal Stromal Cells Interactions within the Skeletal Niche},
  doi       = {10.25972/OPUS-26660},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-266603},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Mesenchymal stem/stromal cells (MSCs) are a rare subpopulation of cells first identified in bone marrow with the potential to proliferate in plastic-adherent colonies and to generate de novo bone marrow stroma and its environment upon serial transplantation to heterotopic anatomical sites. Given their multipotency and self renewal competence, MSCs are prime prospective candidates for most modern musculoskeletal-tissue engineering and regenerative medicine approaches. Still, their envisioned therapeutic use is being questioned with concerns regarding their definition, characterization and integrative functions in vivo. It is well established that microenvironmental cues such as the extracellular matrix (ECM)-chemistry, the mechanical environment and local cellular and/or paracrine interactions critically control MSCs behavior. Yet, most of the scientific knowledge regarding the biology and therapeutic effect of MSCs originates from mechanistic in vitro studies where microenvironmental cues are hardly addressed. Therefore, manifestable changes in cell proliferation behavior and multilineage differentiation potential might be triggered that eventually compromise the translation of results to clinics. This thesis aims to address the complexity of MSCs interactions within the skeletal niche microenvironment in order to provide alternative methods to bypass the current MSCs in vitro culture limitations. Firstly, the influence of ECM-chemistry on MSCs behavior in vitro was explored by means of decellularized human bone models here established. Basal or osteogenic tailored cell-derived decellularized 2D matrices (dECM), proved to be suitable culture substrates for MSCs expansion by providing close-to-native cell-ECM interactions. Moreover, quantified morphological shape changes suggested a material osteo supportive potential, further functionally validated by observable spontaneous mineralization of MSCs. Aiming to identify novel intrinsic ECM regulatory features specific to the skeletal niche, 3D decellularized human trabecular bone scaffolds (dBone) were additionally developed and comprehensively characterized. Remarkably, the MSCs cultured on dBone scaffolds exhibit upregulation of genes associated with stemness as well as niche-related protein expression advocating for the conservation of the na{\"i}ve MSCs phenotype. vi On the other hand, the effect of biomimetic mineralization on MSCs osteogenic lineage differentiation potential was further addressed by hydroxyapatite functionalization of type-I collagen in presence of magnesium. Mineralized scaffolds exhibited higher cell viability and a clear trend of osteogenic genes upregulation comparing with non-mineralized scaffolds. Lastly, in order to mimic the complexity of the native MSCs environment, a dynamic culture system was applied to the 3D decellularized bone constructs, previously studied in single static conditions. Mechanical stimuli generated by (1) continuous perfusion of cell culture medium at 1.7 mL/min and (2) compressive stress from 10\% uniaxial load at 1 Hz, resulted in an improved cell repopulation within the scaffold and boosting of de novo ECM production. The stress-induced gene expression pattern suggested early MSCs commitment towards the osteogenic lineage mediated by integrin matrix adhesion, therefore further corroborating the recapitulation of a reliable in vitro bone niche model in dBone scaffolds. To conclude, the here developed in vitro models provide a progressive increased biomimicking complexity through which significant insights regarding MSC interactions with microenvironmental features in the skeletal niche can be obtained, thus surely paving the way for a better understanding of the role of MSCs in bone homeostasis and regeneration.},
  language  = {en}
}
@phdthesis{Confalonieri2018,
  author    = {Confalonieri, Davide},
  title     = {Development and characterization of a bone marrow stem cell niche model},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163128},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Kritische Knochendefekte stellen heutzutage ein ungel{\"o}stes Problem in der klinischen Praxis dar, da die verf{\"u}gbaren prothetischen Optionen oft die mechanische Anpassung an das Gewebe nicht gew{\"a}hrleisten oder zu wichtigen immunologischen und Implantat-bedingten Komplikationen f{\"u}hren. In diesem Kontext erm{\"o}glichen Tissue Engineering-Ans{\"a}tze neue Strategien, um in vitro Zell-Material Interaktionen zu untersuchen und so die Implantatmaterialien zu optimieren. In dieser Arbeit habe ich Zell-Material Interaktionen eines neuen Kollagen-basierten Scaffolds untersucht, das langfristig als Tr{\"a}gerstruktur f{\"u}r eine zellbasierte Therapie f{\"u}r kritische Knochendefekte entwickelt werden soll. Im Rahmen der Dissertation konnte ich belegen, dass die Kollagen-basierten makropor{\"o}se Mikrocarrier f{\"u}r die Zellvermehrung humaner mesenchymaler Stammzellen (MSC) und deren osteogene Differenzierung unter GMP Bedingungen verwendet werden k{\"o}nnen. Außerdem habe ich die die Kokultur von h{\"a}matopoietischen Stammzellen des Knochenmarks und multiplen Myelomzellen funktionell charakterisiert. Ich konnte erstmals Kulturbedingungen etablieren, die die Langzeitkultur ohne die Verwendung von Zytokinen erm{\"o}glicht. Mittels dieser Kokultur konnte ich ein Knochenmarknischen-Modell etablieren und die Untersuchung der Expression von zentralen Signalkaskaden der Hom{\"o}ostase dieser Nische untersuchen. Ich konnte die Expression von zwei verschiedenen Isoformen von Osteopontin nachweisen, die in Tiermodellen nicht gefunden werden. Diese Isoformen des Osteopontins habe ich kloniert und die rekombinanten Isoformen exprimiert und ihre Rollen in der Hom{\"o}ostase der Knochenmarknische untersucht. Critical size bone defects represent nowadays an unresolved problem in the clinical practice, where the available prosthetic options often lack adequate mechanical matching to the host tissue or lead to important immunological and implant-related complications. In this context, Tissue Engineering approaches promise more effective strategies to study cell-material interactions in vitro and consequently optimize implant materials. In this work, I investigated the cell-scaffold interactions of a new collagen-based scaffold for a putative cell-based therapy for critical size defects to be developed. In the context of this thesis, I could demonstrate that the collagen-based macroporous microcarriers could be employed for the expansion and osteogenic differentiation of human mesenchymal stromal cells (MSCs) under GMP-compliant conditions. Moreover, I functionally characterized the co-culture of bone marrow hematopoietic stem cells and multiple myeloma cells. I was for the first time able to establish culture conditions allowing their long-term culture in absence of externally supplemented cytokines. Using this co-culture, I was able to establish a bone marrow niche model to investigate the expression of key signaling pathways involved in the niche´s homeostasis. I was able to demonstrate the expression of two different isoforms of Osteopontin, that could not previously be detected in animal models. Finally, I cloned these Osteopontin isoforms, expressed recombinant versions of the isoforms, and investigated their roles in the homeostasis of the bone marrow niche.},
  subject      = {bone marrow niche},
  language  = {en}
}
@phdthesis{Youssef2022,
  author    = {Youssef, Almoatazbellah},
  title     = {Fabrication of Micro-Engineered Scaffolds for Biomedical Application},
  doi       = {10.25972/OPUS-23545},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-235457},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Thermoplastic polymers have a history of decades of safe and effective use in the clinic as implantable medical devices. In recent years additive manufacturing (AM) saw increased clinical interest for the fabrication of customizable and implantable medical devices and training models using the patients' own radiological data. However, approval from the various regulatory bodies remains a significant hurdle. A possible solution is to fabricate the AM scaffolds using materials and techniques with a clinical safety record, e.g. melt processing of polymers. Melt Electrowriting (MEW) is a novel, high resolution AM technique which uses thermoplastic polymers. MEW produces scaffolds with microscale fibers and precise fiber placement, allowing the control of the scaffold microarchitecture. Additionally, MEW can process medical-grade thermoplastic polymers, without the use of solvents paving the way for the production of medical devices for clinical applications. This pathway is investigated in this thesis, where the layout is designed to resemble the journey of a medical device produced via MEW from conception to early in vivo experiments. To do so, first, a brief history of the development of medical implants and the regenerative capability of the human body is given in Chapter 1. In Chapter 2, a review of the use of thermoplastic polymers in medicine, with a focus on poly(ε-caprolactone) (PCL), is illustrated, as this is the polymer used in the rest of the thesis. This review is followed by a comparison of the state of the art, regarding in vivo and clinical experiments, of three polymer melt AM technologies: melt-extrusion, selective laser sintering and MEW. The first two techniques already saw successful translation to the bedside, producing patient-specific, regulatory-approved AM implants. To follow in the footsteps of these two technologies, the MEW device parameters need to be optimized. The MEW process parameters and their interplay are further discussed in Chapter 3 focusing on the importance of a steady mass flow rate of the polymer during printing. MEW reaches a balance between polymer flow, the stabilizing electric field and moving collector to produce reproducible, high-resolution scaffolds. An imbalance creates phenomena like fiber pulsing or arcing which result in defective scaffolds and potential printer damage. Chapter 4 shows the use of X-ray microtomography (µCT) as a non-destructive method to characterize the pore-related features: total porosity and the pore size distribution. MEW scaffolds are three-dimensional (3D) constructs but have long been treated in the literature as two-dimensional (2D) ones and characterized mainly by microscopy, including stereo- and scanning electron microscopy, where pore size was simply reported as the distance between the fibers in a single layer. These methods, together with the trend of producing scaffolds with symmetrical pores in the 0/90° and 0/60/120° laydown patterns, disregarded the lateral connections between pores and the potential of MEW to be used for more complex 3D structures, mimicking the extracellular matrix. Here we characterized scaffolds in the aforementioned symmetrical laydown patterns, along with the more complex 0/45/90/135° and 0/30/60/90/120/150° ones. A 2D pore size estimation was done first using stereomicroscopy, followed by and compared to µCT scanning. The scaffolds with symmetrical laydown patterns resulted in the predominance of one pore size, while those with more complex patterns had a broader distribution, which could be better shown by µCT scans. Moreover, in the symmetrical scaffolds, the size of 3D pores was not able to reach the value of the fiber spacing due to a flattening effect of the scaffold, where the thickness of the scaffold was less than the fiber spacing, further restricting the pore size distribution in such scaffolds. This method could be used for quality assurance of fabricated scaffolds prior to use in in vitro or in vivo experiments and would be important for a clinical translation. Chapter 5 illustrates a proof of principle subcutaneous implantation in vivo experiment. MEW scaffolds were already featured in small animal in vivo experiments, but to date, no analysis of the foreign body reaction (FBR) to such implants was performed. FBR is an immune reaction to implanted foreign materials, including medical devices, aimed at protecting the host from potential adverse effects and can interfere with the function of some medical implants. Medical-grade PCL was used to melt electrowrite scaffolds with 50 and 60 µm fiber spacing for the 0/90° and 0/60/120° laydown patterns, respectively. These implants were implanted subcutaneously in immunocompetent, outbred mice, with appropriate controls, and explanted after 2, 4, 7 and 14 days. A thorough characterization of the scaffolds before implantation was done, followed by a full histopathological analysis of the FBR to the implants after excision. The scaffolds, irrespective of their pore geometry, induced an extensive FBR in the form of accumulation of foreign body giant cells around the fiber walls, in a manner that almost occluded available pore spaces with little to no neovascularization. This reaction was not induced by the material itself, as the same reaction failed to develop in the PCL solid film controls. A discussion of the results was given with special regard to the literature available on flat surgical meshes, as well as other hydrogel-based porous scaffolds with similar pore sizes. Finally, a general summary of the thesis in Chapter 6 recapitulates the most important points with a focus on future directions for MEW.},
  language  = {en}
}