Angiogene Auswirkung der retardierten Freisetzung von rh-BMP-2 und rh-VEGF im Muskel der Ratte. Immunhistochemische Auswertung von CD31 und CD34
Angiogenic effect of sustained release of rh-BMP-2 and rh-VEGF in the rat muscle. Immunohistochemical evaluation of CD31 and CD34
by Florian Bernd Obermeyer
Date of Examination:2018-02-05
Date of issue:2018-01-23
Advisor:Prof. Dr. Dr. Henning Schliephake
Referee:Prof. Dr. Nicolai Miosge
Referee:Prof. Dr. Rainer Mausberg
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Abstract
English
Bone defects of the facial skull are associated with a extensive amount of treatment. In therapy, the focus is not only on the functional reconstruction, but also on a satisfactory aesthetic rehabilitation for the patient. Current gold standard of clinical therapy is the autogenous bone graft. The second and third surgery required to remove the graft and the osteosynthesis material, results in increased patient morbidity. There is a particular interest in alternative treatment options by implantation of replacement materials with tissue-inducing effects. In addition to osteogenesis, angiogenesis plays a key role. In the present work, polylactide implants served as carrier material for the growth factors VEGF and BMP. These carriers were implanted in the gluteal musculature of male Wistar rats. The growth factor carrier was loaded by foaming the polylactide with the respective growth factor, or the growth factor was dropped onto the polylactide carriers. The test animals were sacrificed after a period of 4 or 13 weeks. The muscles were taken together with the carrier and processed in thin sections. After immunohistochemical staining for CD31 and CD34, the newly created vascular areas were measured. CD31 staining showed that higher levels of VEGF lead to more vascular growth in the incorporation-loaded vehicles. Even after an implantation period of 13 weeks, this remained constant. In the group with dripped carriers, this was not observed, although after 4 weeks they were superior to the carriers prepared by incorporation of the VEGF. Also, the combination of VEGF with BMP is not superior to VEGF as a single dose. Evaluation of CD34 did not significantly increase the results of either VEGF dose or prolongation of implantation time in the incorporation slides or in the dripped slides. The used combinations of VEGF with BMP showed significantly higher values in the foamed growth factors by a prolongation of the implantation time from 4 to 13 weeks. After 13 weeks, only the combinations of 1.5 μg VEGF with 48 μg BMP were significantly superior to the single administration of 1.5 μg VEGF. It was also unexpected that the single administration of BMP in two cases led to a significantly higher result than the combination of BMP with VEGF. In the case of carriers loaded with drops, the BMP was also superior in one case of the combination of VEGF with BMP. It is possible that the BMP will produce more antigens that can be stained by the anti-CD34 antibody. Not all functions of the surface marker CD34 are still researched. In comparison of the two production methods, the values of the carriers prepared by incorporation were again significantly superior after 13 weeks. Conclusions: 1. Incorporated VEGF can be delivered from preformed polylactide carriers with calcium carbonate supplement over a period of 13 weeks and results in a significant increase in vascularization. 2. After 4 weeks runtime, the foam load is superior to dripping. 3. There is no evidence of vaso-inducing effects of BMP in established CD31 staining. 4. The staining with CD34 antibodies leads in some cases to higher values for BMP in comparison to VEGF and implies further research needs for the elucidation of all functions of CD34. 5. For future studies, based on the results obtained here, a combined loading method of the polylactide carriers by incorporating a part of the growth factor and an additional dripping of the other part of the growth factor is recommended.
Keywords: Growth factor; VEGF; BMP-2; Tissue engineering; Bone augmentation; Polylactide