Autor: Pia Katharina Sophia Ostach

Model for the implantation of implants stimulating the body’s own mechanisms for repair and regeneration of ovine annulus fibrosus defects. The surgical management of herniated discs often involves removal of the prolapsed tissue and subsequent replacement of the nucleus pulposus. Invasive surgical access via the annulus fibrosus frequently leads to further tissue defects and consequently to additional damage to the existing damage of the annulus fibrosus. Numerous experimental studies on the treatment of degenerative and traumatic changes in intervertebral discs report results of surgical treatment with the aim of repair and regeneration of the nucleus pulposus. The success of these therapeutic methods largely depends on the functional restoration of the annulus fibrosus, since only an intact annulus fibrosus can resist the pressure from inside the intervertebral disc and thus prevent recurrent herniation. Therapeutic methods taking advantage of the regenerative potential of the annulus fibrosus are referred to as Annulus Fibrosus Tissue Engineering. They are based on the implantation of various different scaffolds and cells fostering repair and regeneration. The aim of this study was to assess the suitability of the model under review to test the suitability of the absorbable implants used for the treatment of annulus fibrosus defects. In the present study, for the first time, an implant in combination with a human recombinant chemokine TECK (CCL25) and in combination with PRP was tested for its bio-compatibility and stability in an in vivo test for annulus fibrosus defect healing in a sheep model. Since the chemokine TECK is a human recombinant chemokine, the effect of this specific chemokine on ovine annulus fibrosus cells was studied in vitro in a chemotaxis assay and also in a 3D cell culture before testing in vivo. In vitro, the human recombinant chemokine TECK was able to dose-dependently induce migration of ovine annulus fibrosus cells, which was significant at concentrations of 750nM and 1000nM. Hence, the receptor ligand of the human recombinant chemokine TECK not only binds to human cells, but also to the chemokine receptor of ovine annulus fibrosus cells. In addition, in 3D cell culture, the ovine annulus fibrosus cells were able to generate proteoglycans in the presence of human recombinant chemokine TECK. In combination with PRP, the ovine annulus fibrosus cells generated almost no proteoglycans. For the first time, the suitability of the human recombinant chemokine TECK for use in animal experiments on sheep could be demonstrated with these in vitro experiments. In the following animal experiment, 3.5mm x 3.5mm defects were set via a retroperitoneal approach to the annulus fibrosus of the lumbar intervertebral discs of 30 experimental ovine animals. The experimental animals were randomized into 5 groups of 6 (groups A to E). Group A (empty defect), where a defect was set in the annulus fibrosus and subsequently not covered with an implant, was the control group to study the body's own repair and regeneration mechanisms. In groups B to E, the defect was covered with various combinations of implants and therapeutic substances. A macroscopic and histological evaluation was carried out 3 months after surgery. The assessed model is suitable for testing absorbable implants as this thesis shows, that: • the sheep was an acceptable model for the study of new surgical therapies with implants for the repair and regeneration of annulus fibrosus defects, since the proportional anatomical conditions are comparable with humans and the same surgical techniques (instruments and implants) were used as would be under clinical conditions for humans. In summary, the model and surgical techniques are transferable to humans. The assessed model is suitable to demonstrate this. • the retroperitoneal access as well as the defect setting with scalpel and tweezers was reproducible in all animals with increasing efficiency. • the attachment method with sutures at the four corners of the implant reliably prevented dislocation of the absorbable implants. • the explantation, anatomical preparation, and preparation of specimens were reproducible. • histological hematoxylin / eosin staining proved to be a reliable diagnostic tool to study implant bio-degeneration, recurrent herniation rates, and, thus, defect stabilization. For future use of the assessed model in new studies however, the implant attachment method should be modified to allow for clinically safe, reproducible application and easy integration of the method into existing surgical protocols. A surgical application aid should be used for the challenging application of sutures, since in each instance several attempts to correctly place the implant were required. Also, hematoxylin / eosin staining as used in the assessed model did not allow conclusions as to the quality of the regenerated tissue, and, thus, not as to the effectiveness of the applied therapeutic substances on tissue repair and regeneration. Only quantitative effects could be assessed. The hypothesis, whether human recombinant chemokine TECK (CCL25), in addition to the demonstrated in vitro effect, also has an in vivo effect on defect healing of ovine annulus fibrosus cells, could not be assessed with the methods under review. Hence, for future use of the model, defects of equal size should be set in all animals, and the study of the defect content should also be in the focus of further research. Since the model is suitable in principle, with defects set of equal size, a classification system for quantitative analysis of tissue repair and regeneration between groups of animals as well as relative to the chemokine TECK used can be developed.