ABSTRACT
OBJECTIVES/HYPOTHESIS: Injection laryngoplasty of materials for unilateral vocal-fold paralysis has shown various results regarding the long-term stability of the injected material. We evaluated a fibrin-gel based cell suspension with autologous chondrocytes in-vitro and in-vivo as long-term-stable vocal-fold augmentation material in an animal model. STUDY DESIGN: This study compises an in vitro cell-culture part as well as an in vivo animal study with New Zealand White Rabbits. METHODS: In in-vitro experiments, auricular chondrocytes harvested from 24 New Zealand White Rabbits cadavers were cultivated in pellet cultures to evaluate cartilage formation for 4 weeks using long-term-stable fibrin gel as carrier. Injectability and injection volume for the laryngoplasty was determined in-vitro using harvested cadaveric larynxes. In-vivo 24 Rabbits were biopsied for elastic cartilage of the ear and autologous P1 cells were injected lateral of one vocal cord into the paraglottic space suspended in a long-term-stable fibrin gel. Histologic evaluation was performed after 2, 4, 12, and 24 weeks. RESULTS: During 12-week pellet culture, we found extracellular matrix formation and weight-stable cartilage of mature appearance. In-vivo, mature cartilage was found in two larynxes (n = 6) at 4 weeks, in four (n = 6) at 12 weeks, and in five (n = 6) at 24 weeks mostly located in the paraglottic space and sometimes with spurs into the vocalis muscle. Surrounding tissue was often infiltrated with inflammatory cells. Material tended to dislocate through the cricothyroid space into the extraglottic surrounding tissue. CONCLUSIONS: A cell-based approach with chondrocytes for permanent vocal-fold augmentation has not previously been reported. We have achieved the formation of structurally mature cartilage in the paraglottic space, but this is accompanied by difficulties with dislocated material, deformation of the augmentation, and inflammation. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:E1624-E1632, 2021.
Subject(s)
Chondrocytes/transplantation , Fibrin/chemistry , Laryngoplasty/methods , Vocal Cord Paralysis/therapy , Animals , Cell Culture Techniques/methods , Chondrocytes/chemistry , Chondrogenesis/physiology , Disease Models, Animal , Ear Cartilage/cytology , Female , Gels , Humans , Injections, Intralesional , Male , Primary Cell Culture , Rabbits , Transplantation, Autologous , Vocal Cord Paralysis/pathology , Vocal Cords/innervation , Vocal Cords/pathologyABSTRACT
Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-ß-lactamases (MBLs) target the most widely used antibiotic class, the ß-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-ß-lactamase inhibitors, essential in the fight against antibiotic resistance.
