@phdthesis{Siverino2020,
  author    = {Siverino, Claudia},
  title     = {Induction of ectopic bone formation by site directed immobilized BMP2 variants \(in\) \(vivo\)},
  doi       = {10.25972/OPUS-16935},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169359},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {In contrast to common bone fractures, critical size bone defects are unable to self-regenerate and therefore external sources for bone replacement are needed. Currently, the gold standard to treat critical size bone fractures, resulting from diseases, trauma or surgical interventions, is the use of autologous bone transplantation that is associated with several drawbacks such as postoperative pain, increased loss of blood during surgery and extended operative time. The field of bone tissue engineering focuses on the combination of biomaterials and growth factors to circumvent these adverse events and thereby to improve critical size bone defects treatment. To this aim, a promising approach is represented by using a collagen sponge soaked with one of the most powerful osteoinductive proteins, the bone morphogenetic protein 2 (BMP2). After the approval by the Food and Drug Administration (FDA), BMP2 was used to successfully treat several severe bone defects. However, the use of BMP2 delivery systems is associated with severe side effects such as inflammation, swelling, ectopic bone formation outside of the site of implantation and breathing problems if implanted in the area of the cervical spine. The occurrence of severe side effects is related to the supraphysiological amounts of the applied protein at the implantation site. The BMP2 is typically adsorbed into the scaffold and diffuses rapidly after implantation. Therefore, intensive research has been conducted to improve the protein's retention ability, since a prolonged entrapment of the BMP2 at the implantation site would induce superior bone formation in vivo due to a minimized protein release. By controlling the release from newly designed materials or changing the protein immobilization methods, it seems possible to improve the osteoinductive properties of the resulting BMP2-functionalized scaffolds. The combination of biocompatible and biodegradable scaffolds functionalized with a covalently immobilized protein such as BMP2 would constitute a new alternative in bone tissue engineering by eliminating the aforementioned severe side effects. One of the most common immobilization techniques is represented by the so-called EDC/NHS chemistry. This coupling technique allows covalent biding of the growth factor but in a non-site direct manner, thus producing an implant with uncontrollable and unpredictable osteogenic activities. Therefore, the generation of BMP2 variants harboring functional groups that allow a site-directed immobilization to the scaffold, would enable the production of implants with reproducible osteogenic activity. The new BMP2 variants harbor an artificial amino acid at a specific position of the mature polypeptide sequence. The presence of the unnatural amino acid allows to use particular covalent immobilization techniques in a highly specific and site directed manner. The two selected BMP2 variants, BMP2 E83Plk and BMP2 E83Azide, were expressed in E. coli, renatured and purified by cation exchange chromatography. The final products were intensively analyzed in terms of purity and biological activity in vitro. The two BMP2 variants enabled the application of different coupling techniques and verify the possible options for site directed immobilization to the scaffold. Intensive analyses on the possible side effects caused by the coupling reactions and on the quantification of the coupled protein were performed. Both click chemistry reactions showed high reaction efficacies when the BMP2 variants were coupled to functionalized fluorophores. Quantification by ELISA and scintillation counting of radioactively labeled protein revealed different outcomes. Moreover, the amounts of protein detected for the BMP2 variants coupled to microspheres were similar to that of the wild type protein. Therefore, it was not possible to conclude whether the BMP2 variants were covalently coupled or just adsorbed. BMP2 variants being immobilized to various microspheres induced osteogenic differentiation of C2C12 cells in vitro, but only in those cells that were located in close proximity to the functionalized beads. This selectivity strongly indicates that the protein is for a great portion covalently coupled and not just adsorbed. Moreover, the difference between the covalently coupled BMP2 variants and the adsorbed BMP2 WT was confirmed in vivo. Injection of the BMP2-functionalized microspheres in a rat model induced subcutaneous bone formation. The main aim of the animal experiment was to prove whether covalently coupled BMP2 induces bone formation at significant lower doses if compared to the amount being required if the protein is simply adsorbed. To this aim, several BMP2 concentrations were tested in this animal experiment. The BMP2 variants, being covalently immobilized, were hypothesized to be retained and therefore bio-available at the site of implantation for a prolonged time. However, in the animal experiments, lower doses of either coupled or adsorbed protein were unable to induce any bone formation within the 12 weeks. In contrast, the highest doses induced bone formation that was first detected at week 4. During the 12 weeks of the experiment, an increase in bone density and a steady state bone volume was observed. These results were obtained only for the covalently coupled BMP2 E83Azide but not for BMP2 E83Plk that did not induce bone formation in any condition. The negative outcome after application of BMP2 E83Plk suggested that the coupling reaction might have provoked changes in the protein structure that extremely influenced its osteogenic capabilities in vivo. However, the histological examination of the different ossicles induced either by BMP2 WT or BMP2 E83Azide, revealed clear morphological differences. BMP2 WT induced a bone shell-like structure, while the covalently coupled protein induced uniform bone formation also throughout the inner part. The differences between the two newly formed bones can be clearly associated with the different protein delivery mechanisms. Thus, the developed functionalized microspheres constitute a new interesting strategy that needs further investigations in order to be able to be used as replacement of the currently used BMP2 WT loaded medical devices.},
  language  = {en}
}
@phdthesis{Bleier2023,
  author    = {Bleier, Michael},
  title     = {Underwater Laser Scanning - Refractive Calibration, Self-calibration and Mapping for 3D Reconstruction},
  isbn      = {978-3-945459-45-4},
  doi       = {10.25972/OPUS-32269},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-322693},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {There is great interest in affordable, precise and reliable metrology underwater: Archaeologists want to document artifacts in situ with high detail. In marine research, biologists require the tools to monitor coral growth and geologists need recordings to model sediment transport. Furthermore, for offshore construction projects, maintenance and inspection millimeter-accurate measurements of defects and offshore structures are essential. While the process of digitizing individual objects and complete sites on land is well understood and standard methods, such as Structure from Motion or terrestrial laser scanning, are regularly applied, precise underwater surveying with high resolution is still a complex and difficult task. Applying optical scanning techniques in water is challenging due to reduced visibility caused by turbidity and light absorption. However, optical underwater scanners provide significant advantages in terms of achievable resolution and accuracy compared to acoustic systems. This thesis proposes an underwater laser scanning system and the algorithms for creating dense and accurate 3D scans in water. It is based on laser triangulation and the main optical components are an underwater camera and a cross-line laser projector. The prototype is configured with a motorized yaw axis for capturing scans from a tripod. Alternatively, it is mounted to a moving platform for mobile mapping. The main focus lies on the refractive calibration of the underwater camera and laser projector, the image processing and 3D reconstruction. For highest accuracy, the refraction at the individual media interfaces must be taken into account. This is addressed by an optimization-based calibration framework using a physical-geometric camera model derived from an analytical formulation of a ray-tracing projection model. In addition to scanning underwater structures, this work presents the 3D acquisition of semi-submerged structures and the correction of refraction effects. As in-situ calibration in water is complex and time-consuming, the challenge of transferring an in-air scanner calibration to water without re-calibration is investigated, as well as self-calibration techniques for structured light. The system was successfully deployed in various configurations for both static scanning and mobile mapping. An evaluation of the calibration and 3D reconstruction using reference objects and a comparison of free-form surfaces in clear water demonstrate the high accuracy potential in the range of one millimeter to less than one centimeter, depending on the measurement distance. Mobile underwater mapping and motion compensation based on visual-inertial odometry is demonstrated using a new optical underwater scanner based on fringe projection. Continuous registration of individual scans allows the acquisition of 3D models from an underwater vehicle. RGB images captured in parallel are used to create 3D point clouds of underwater scenes in full color. 3D maps are useful to the operator during the remote control of underwater vehicles and provide the building blocks to enable offshore inspection and surveying tasks. The advancing automation of the measurement technology will allow non-experts to use it, significantly reduce acquisition time and increase accuracy, making underwater metrology more cost-effective.},
  subject      = {Selbstkalibrierung},
  language  = {en}
}