An Experimental Study Of Silk Fibroin Particles For Injection Laryngoplasty

Section I Preparation and biocompatibility studies of silk fibroin particlesObjectives:To prepare silk fibroin (SF) particles with different sizes and evaluate the inflammatory responses to the particles in vitro and in vivo.Methods:The regenerated silk fibroin were mechanically grinded and sieved to produce SF particles of different sizes suitable for vocal fold injection, and then the size and morphology characteristics of the particles were confirmed by scanning electron microscopy(SEM); two different sizes of SF particles were selected to investigate their role in stimulating the release of TNF-a, IL-1β and IL-6, in culture with RAW264.7macrophages in vitro, the inflammatory processes were characterized using real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR), Enzyme-Linked Immunosorbent Assay (ELISA), and light microscopy evaluations; BALB/c mouse air pouch animal models and New Zealand rabbits were used for the in vivo inflammation assessment through proinflammation cytokines detection and histological evaluation of the BALB/c mice air pouches in acute reaction and pathologic analysis of the injected sites of the New Zealand rabbits back muscles for long time observation after SF particles injection.Results:SF particles with appropriate size ranges of10-45μm (small silk fibroin particles,SFS)and45-125μm(large silk fibroin particles, FSL) were obtained by mechanically grinding and sieving, showing irregular shapes and rough surfaces under SEM; SFS and SFL in culture with RAW264.7murine macrophage cells for24h, caused up-regulation of mRNA coding for TNF-a, with statistically significant increase in this up-regulation under SFS stimulation, but without any release of IL-1β, IL-6or TNF-a relative to the control group; in the BALB/c mice air pouches, obvious secretion of IL-1β, IL-6and TNF-a could be found after48h-stimulation by SFL and SFS, among which TNF-a was the only cytokin secreted as high as the positive control, accompanied by inflammatory cell infiltration and increased thickness of the air pouches, and SFS had stronger stimulation reaction than SFL did for each evaluation indexes; injection of SFL and SFS in the dorsal paravertebral muscles of the rabbits caused obvious acute inflammatory reaction accompanied by proliferation of the fibrous connective tissue, but all gradually reduced along with time prolonged, the SF particles were wrapped with similar fibrous connective tissue and small amounts of inflammatory cells at last in both of the experimental groups, and there were no apparent degradation of the material in6-months observation.Conclusions:In vitro experiment showed that SF particles have excellent biocompatibility, but may cause inflammatory reaction mediated by TNF-α; in vivo studies indicated that SF particles could elicit obvious acute inflammatory reaction mediated by IL-1β, IL-6and mainly by TNF-a, with more severe reaction under SFS stimulation, which however gradually weakened and consequently showed good biocompatibility and immobility with time prolonged; SFL may be a promising material for injection laryngoplasty. Section Ⅱ Silk fibroin particles for injection Iaryngoplasty in animal modelsObjectives:To evaluate the suitability and efficacy of silk fibroin particles for injection laryngoplasty in animal models.Methods:New Zealand rabbits and Beagle dogs were used for the injection laryngoplasty experiments. After vocal fold paralysis by incision and ligation of the left vagus nerve(in New Zealand rabbits) or recurrent laryngeal nerve(in Beagle dogs), SFL was immediately injected into the left paraglottic space adjacent to the thyroarytenoid muscle via the thyroid cartilage. The experimental animals were sacrificed and the larynges were excised at preseted time point, for histopathological analysis of the rabbits specimen and for aerodynamic parameters measurements and high-speed digital imaging of the vocal fold vibration with excised larynx setup of the excised beagle dogs’ larynges respectively.Results:Injection of SFL for vocal fold augmentation of unilateral vocal fold paralysis in the rabbits have similar histologic changes as injection of the material into the rabbits’ dorsal paravertebral muscles.The injected SFL stayed in the paraglottic space and the thyroarytenoid muscle, did not migrate, cause slight and local inflammation, with no suppuration, necrosis and granuloma formation.The mucosa and submucosal layer of the vocal fold were not influenced. As SFL for injection laryngoplasty in the beagle dogs with unilateral vocal fold paralysis, excised larynx setup study showed distinct changes of the aerodynamical parameters and the amplitudes of the mucosa wave. At3months, phonation threshold potential (PTP), phonation threshold flow (PTF), phonation threshold power (PTW) and the mucosal wave amplitude all decreased, which indicated that the minimized glottal area and reduced work of phonation; while at6months, PTP, PTF, PTW and mucosal wave amplitude all increased, which signified the initial volume of the injected material shrinked because of the biodegradability of HA and the raised tention and viscosity caused by the proliferation of the fibrous connective tissues around the particles.Conclusions:SFL appears to be biocompatible, nonabsorbable, nonmigratory, and suitable for injection laryngoplasty. Injection laryngoplasty with SFL led to improvements in aerodynamical parameters, but caused detrimental changes to the mucosal wave. Attention must be paid to avoid the decreased efficacy caused by the degradation of the HA in the mixed material, and improvements of the viscoelasticity of the material to match the vocal fold are expected.