Poster AA18 - A New Bioengineered Decellularized Meniscus Scaffold For Tympanic Membrane Repair

Introduction: Chronic tympanic membrane (TM) perforations if left untreated have the potential to cause further harm to the TM and middle ear by means of impaired hearing, recurrent infections, and pain during daily activities like bathing and swimming. While most TM perforations heal naturally, in some cases this does not occur, and a surgical intervention is required via tympanoplasty to patch the perforated eardrum using either autologous fascia or cartilage harvested from the patient. Bioengineered grafts for TM repair have the potential to avoid the need for a second surgical site to harvest the autologous tissue, saving operating room (OR) time and also decreasing discomfort for the patient and the risk of donor site morbidity. A key parameter for a successful bioengineered graft is being able to withstand the mechanical properties of the native TM. To this end, we have designed a porcine derived cartilaginous scaffold based on meniscus decellularization (MEND), and we have shown that MEND is laden with microchannels to enable recellularization. In this work we explore the influence of fabrication parameters on MEND’s structure, and its potential to heal a tympanic membrane defect in chinchillas, an accepted human preclinical model.

Materials and

Methods: The MEND fabrication process for the TM repair. Yorkshire, Landrace, and Duroc porcine meniscus were sourced from a commercial provider, Collagen Solutions (MN). Three (n=3) porcine menisci were characterized by histology and ImageJ analysis to define a consistent region for the harvest of the MEND grafts. Each meniscus was then devitalized with four freeze-thaw cycles, one of which in an isotonic buffered solution, and then sliced using a freezing-stage microtome consistently achieving 300 micrometers thickness. The meniscus slices were then subjected to pepsin and elastase enzymatic treatment to enzymatically remove the elastin bundles and to create microchannels. Mechanical properties of the scaffold were examined before and after each fabrication step, as well as histology and histochemistry such as H&E, Collagen I, DAPI, Alcian Blue, Van Gieson’s stain and modified Hart’s stain to assess structure, decellularization, and the removal of elastin. Total DNA and GAG content were further quantified via Quan-iT PicoGreen assay and DMMB dye assay. The bioengineered scaffolds were then lyophilized prior to sterilization by ethylene oxide. MEND constructs were then used to repair the TM in an in vivo chinchilla animal study of N=5. A defect in the TM was made. Repair conditions included: no graft, auricular cartilage graft, fascia, or MEND. TM closure and graft integration was evaluated endoscopically for 6 weeks until sacrifice. The TM were then evaluated via histology and immunohistochemistry and second harmonics generation was used to characterize the distribution and orientation of the collagen fibers before and after implant.

Results, Conclusions, and Discussions: The red zone of the meniscus was determined to be the ideal anatomically relevant region to utilize for MEND fabrication. After enzymatically removing elastase from the native tissue, the MEND constructs showed well-defined micro channels approximately 8 microns in average diameter spanning across the scaffold parallel to the sliced graft surface. Modified Hart’s stain showed successful removal of elastin content. Mechanics testing revealed that after sterilization, MEND became stiffer. Second harmonic generation on native tissue shows that the collagen fibers in the meniscus, align with the circumferential and radial fibers of the TM, this alignment is not affected by the decellularization process and is partially remodelled after implantation. Our initial in vivo study shows that MEND grafts adhered well to the native TM for the duration of 6 weeks post-op, while autologous grafts were more unstable. MEND integrated with the native tissue and was easily infiltrated by native TM cells. Our findings support the potential of MEND to improve tympanoplasty.

Acknowledgements (Optional): The Children’s Hospital of Philadelphia Research Institute, McKay Orthopedic Research Laboratory, Penn Health-Tech NEMO Prize,