@phdthesis{Hafen2015, author = {Hafen, Bettina}, title = {Physical contact between mesenchymal stem cells and endothelial precursors induces distinct signatures with relevance to tissue regeneration and engineering}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119417}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2015}, abstract = {The goal of the project VascuBone is to develop a tool box for bone regeneration, which on one hand fulfills basic requirements (e.g. biocompatibility, properties of the surface, strength of the biomaterials) and on the other hand is freely combinable with what is needed in the respective patient's situation. The tool box will include a variation of biocompatible biomaterials and cell types, FDA-approved growth factors, material modification technologies, simulation and analytical tools like molecular imaging-based in vivo diagnostics, which can be combined for the specific medical need. This tool box will be used to develop translational approaches for regenerative therapies of different types of bone defects. This project receives funding from the European Union's Seventh Framework Program (VascuBone 2010). The present study is embedded into this EU project. The intention of this study is to assess the changes of the global gene expression patterns of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) after direct cell-cell contact as well as the influence of conditioned medium gained from MSCs on EPCs and vice versa. EPCs play an important role in postnatal vasculogenesis. An intact blood vessel system is crucial for all tissues, including bone. Latest findings in the field of bone fracture healing and repair by the use of tissue engineering constructs seeded with MSCs raised the idea of combining MSCs and EPCs to enhance vascularization and therefore support survival of the newly built bone tissue. RNA samples from both experimental set ups were hybridized on Affymetrix GeneChips® HG-U133 Plus 2.0 and analyzed by microarray technology. Bioinformatic analysis was applied to the microarray data and verified by RT-PCR. This study gives detailed information on how EPCs and MSCs communicate with each other and therefore gives insights into the signaling pathways of the musculoskeletal system. These insights will be the base for further functional studies on protein level for the purpose of tissue regeneration. A better understanding of the cell communication of MSCs and EPCs and subsequently the targeting of relevant factors opens a variety of new opportunities, especially in the field of tissue engineering. The second part of the present work was to develop an ELISA (enzyme-linked immunosorbent assay) for a target protein from the lists of differentially expressed genes revealed by the microarray analysis. This project was in cooperation with Immundiagnostik AG, Bensheim, Germany. The development of the ELISA aimed to have an in vitro diagnostic tool to monitor e.g. the quality of cell seeded tissue engineering constructs. The target protein chosen from the lists was klotho. Klotho seemed to be a very promising candidate since it is described in the literature as anti-aging protein. Furthermore, studies with klotho knock-out mice showed that these animals suffered from several age-related diseases e.g. osteoporosis and atherosclerosis. As a co-receptor for FGF23, klotho plays an important role in bone metabolism. The present study will be the first one to show that klotho is up-regulated in EPCs after direct cell-cell contact with MSCs. The development of an assay with a high sensitivity on one hand and the capacity to differentiate between secreted and shedded klotho on the other hand will allow further functional studies of this protein and offers a new opportunity in medical diagnostics especially in the field of metabolic bone disease.}, subject = {Vorl{\"a}uferzelle}, language = {en} } @article{KauffmannHoehneAssafetal.2020, author = {Kauffmann, Frederic and H{\"o}hne, Christian and Assaf, Alexandre Thomas and Vollkommer, Tobias and Semmusch, Jan and Reitmeier, Aline and Stein, Jamal Michel and Heiland, Max and Smeets, Ralf and Rutkowski, Rico}, title = {The influence of local pamidronate application on alveolar dimensional preservation after tooth extraction — an animal experimental study}, series = {International Journal of Molecular Sciences}, volume = {21}, journal = {International Journal of Molecular Sciences}, number = {10}, issn = {1422-0067}, doi = {10.3390/ijms21103616}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-285173}, year = {2020}, abstract = {The aim of this randomized, controlled animal exploratory trial was to investigate the influence of local application of aminobisphosphonate pamidronate during the socket preservation procedure. Mandibular premolars were extracted in five G{\"o}ttingen minipigs. Two animals underwent socket preservation using BEGO OSS (n = 8 sockets) and three animals using BEGO OSS + Pamifos (15 mg) (n = 12 sockets). After jaw impression, cast models (baseline, eight weeks postoperative) were digitized using an inLab X5 scanner (Dentsply Sirona) and the generated STL data were superimposed and analyzed with GOM Inspect 2018 (GOM, Braunschweig). After 16 weeks, the lower jaws were prepared and examined using standard histological methods. In the test group (BEGO OSS + pamidronate), buccooral dimensional loss was significantly lower, both vestibulary (-0.80 ± 0.57 mm vs. -1.92 ± 0.63 mm; p = 0.00298) and lingually (-1.36 ± 0.58 mm vs. -2.56 ± 0.65 mm; p = 0.00104) compared with the control group (BEGO OSS). The test group showed a significant difference between vestibular and lingual dimensional loss (p = 0.04036). Histology showed cortical and cancellous bone in the alveolar sockets without signs of local inflammation. Adjuvant application of pamidronate during socket preservation reduces alveolar dimensional loss significantly. Further investigations with regard to dose-response relationships, volume effects, side effects, and a verification of the suitability in combination with other bone substitute materials (BSMs) are necessary.}, language = {en} } @article{RighessoTerekhovGoetzetal.2021, author = {Righesso, L. A. R. and Terekhov, M. and G{\"o}tz, H. and Ackermann, M. and Emrich, T. and Schreiber, L. M. and M{\"u}ller, W. E. G. and Jung, J. and Rojas, J. P. and Al-Nawas, B.}, title = {Dynamic contrast-enhanced magnetic resonance imaging for monitoring neovascularization during bone regeneration — a randomized in vivo study in rabbits}, series = {Clinical Oral Investigations}, volume = {25}, journal = {Clinical Oral Investigations}, number = {10}, issn = {1432-6981}, doi = {10.1007/s00784-021-03889-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-307614}, pages = {5843-5854}, year = {2021}, abstract = {Objectives Micro-computed tomography (μ-CT) and histology, the current gold standard methods for assessing the formation of new bone and blood vessels, are invasive and/or destructive. With that in mind, a more conservative tool, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), was tested for its accuracy and reproducibility in monitoring neovascularization during bone regeneration. Additionally, the suitability of blood perfusion as a surrogate of the efficacy of osteoplastic materials was evaluated. Materials and methods Sixteen rabbits were used and equally divided into four groups, according to the time of euthanasia (2, 3, 4, and 6 weeks after surgery). The animals were submitted to two 8-mm craniotomies that were filled with blood or autogenous bone. Neovascularization was assessed in vivo through DCE-MRI, and bone regeneration, ex vivo, through μ-CT and histology. Results The defects could be consistently identified, and their blood perfusion measured through DCE-MRI, there being statistically significant differences within the blood clot group between 3 and 6 weeks (p = 0.029), and between the former and autogenous bone at six weeks (p = 0.017). Nonetheless, no significant correlations between DCE-MRI findings on neovascularization and μ-CT (r =-0.101, 95\% CI [-0.445; 0.268]) or histology (r = 0.305, 95\% CI [-0.133; 0.644]) findings on bone regeneration were observed. Conclusions These results support the hypothesis that DCE-MRI can be used to monitor neovascularization but contradict the premise that it could predict bone regeneration as well.}, language = {en} } @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} } @article{SiverinoFahmyGarciaMumcuogluetal.2022, author = {Siverino, Claudia and Fahmy-Garcia, Shorouk and Mumcuoglu, Didem and Oberwinkler, Heike and Muehlemann, Markus and Mueller, Thomas and Farrell, Eric and van Osch, Gerjo J. V. M. and Nickel, Joachim}, title = {Site-directed immobilization of an engineered bone morphogenetic protein 2 (BMP2) variant to collagen-based microspheres induces bone formation in vivo}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, number = {7}, issn = {1422-0067}, doi = {10.3390/ijms23073928}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-284572}, year = {2022}, abstract = {For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2′s bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.}, language = {en} } @article{SiverinoFahmyGarciaNiklausetal.2023, author = {Siverino, Claudia and Fahmy-Garcia, Shorouk and Niklaus, Viktoria and Kops, Nicole and Dolcini, Laura and Misciagna, Massimiliano Maraglino and Ridwan, Yanto and Farrell, Eric and van Osch, Gerjo J. V. M. and Nickel, Joachim}, title = {Addition of heparin binding sites strongly increases the bone forming capabilities of BMP9 in vivo}, series = {Bioactive Materials}, volume = {29}, journal = {Bioactive Materials}, doi = {10.1016/j.bioactmat.2023.07.010}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350470}, pages = {241-250}, year = {2023}, abstract = {Highlights • Despite not being crucial for bone development BMP9 can induce bone growth in vivo. • BMP9 induced bone formation is strongly enhanced by introduced heparin binding sites. • BMP9s bone forming capabilities are triggered by extracellular matrix binding. • Heparin binding BMP9 (BMP9 HB) can improve the current therapies in treating bone fractures. Abstract Bone Morphogenetic proteins (BMPs) like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects. In recent in vitro studies, BMP9 revealed the highest osteogenic potential compared to other BMPs, possibly due to its unique signaling pathways that differs from other osteogenic BMPs. However, in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7. In silico analysis of the BMP9 protein sequence revealed that BMP9, in contrast to other osteogenic BMPs such as BMP2, completely lacks so-called heparin binding motifs that enable extracellular matrix (ECM) interactions which in general might be essential for the BMPs' osteogenic function. Therefore, we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9′s mature part. The resulting protein (BMP9 HB) showed higher heparin binding affinity than BMP2, similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9. However, remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats, showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9. Even at 10-fold lower BMP9 HB doses bone tissue formation was observed. This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs, could constitute a valuable replacement to the commonly used BMPs. The possibility to use lower protein doses demonstrates BMP9 HB's high translational potential.}, language = {en} } @article{WangStoecklLietal.2022, author = {Wang, Chenglong and St{\"o}ckl, Sabine and Li, Shushan and Herrmann, Marietta and Lukas, Christoph and Reinders, Yvonne and Sickmann, Albert and Gr{\"a}ssel, Susanne}, title = {Effects of extracellular vesicles from osteogenic differentiated human BMSCs on osteogenic and adipogenic differentiation capacity of na{\"i}ve human BMSCs}, series = {Cells}, volume = {11}, journal = {Cells}, number = {16}, issn = {2073-4409}, doi = {10.3390/cells11162491}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-286112}, year = {2022}, abstract = {Osteoporosis, or steroid-induced osteonecrosis of the hip, is accompanied by increased bone marrow adipogenesis. Such a disorder of adipogenic/osteogenic differentiation, affecting bone-marrow-derived mesenchymal stem cells (BMSCs), contributes to bone loss during aging. Here, we investigated the effects of extracellular vesicles (EVs) isolated from human (h)BMSCs during different stages of osteogenic differentiation on the osteogenic and adipogenic differentiation capacity of na{\"i}ve (undifferentiated) hBMSCs. We observed that all EV groups increased viability and proliferation capacity and suppressed the apoptosis of na{\"i}ve hBMSCs. In particular, EVs derived from hBMSCs at late-stage osteogenic differentiation promoted the osteogenic potential of na{\"i}ve hBMSCs more effectively than EVs derived from na{\"i}ve hBMSCs (na{\"i}ve EVs), as indicated by the increased gene expression of COL1A1 and OPN. In contrast, the adipogenic differentiation capacity of na{\"i}ve hBMSCs was inhibited by treatment with EVs from osteogenic differentiated hBMSCs. Proteomic analysis revealed that osteogenic EVs and na{\"i}ve EVs contained distinct protein profiles, with pro-osteogenic and anti-adipogenic proteins encapsulated in osteogenic EVs. We speculate that osteogenic EVs could serve as an intercellular communication system between bone- and bone-marrow adipose tissue, for transporting osteogenic factors and thus favoring pro-osteogenic processes. Our data may support the theory of an endocrine circuit with the skeleton functioning as a ductless gland.}, language = {en} }