This study demonstrated that injection of hASC spheroids with HA-based hydrogel into the aged larynx improved the morphological characteristics in terms of increased intrinsic laryngeal muscles and HA in the lamina propria. Functional analysis showed that the phase difference in the mucosal wave was minimal with hASC spheroid injection, while other injections, including single hASCs, showed substantial phase differences in vibration cycles.
Three layers of intracartilaginous laryngeal structure undergo changes with age. Loss of muscle mass with age in the human thyroarytenoid muscle has been reported in addition to decreased innervation and metabolic activity of mitochondria . The thickness of the epithelium usually decreases, and the lamina propria shows a variety of changes, including thickening/edema of the superficial layer and degeneration/atrophy of elastic fibers . As dysfunction of the larynx due to aging can lead to problems with speech and social communication, several studies have been performed that used tissue-engineering techniques to address these issues. Many studies focused on restoration of volume at the area of muscle using various biomaterials . In our previous study, we examined the use of injectable basic fibroblast growth factor (bFGF)-loaded alginate (ALG)/HA hydrogel for rejuvenation of geriatric laryngeal muscles, which showed increased expression of myogenic regulatory factor-related genes, hypertrophy of muscle fibers, proliferation of muscle satellite cells, and angiogenesis and decreased interstitial fibrosis .
As MSCs have self-renewal capability and tissue regeneration potential by differentiation and maturation into specific phenotypes, they have remarkable potential for clinical applications [29, 30]. The research library of the National Institutes of Health (NIH) includes reports of 993 clinical studies in which MSCs have been used to treat different diseases. The trials with MSCs frequently failed in controlled studies with large cohorts, probably because of lack of comprehension of the regenerative and immunomodulatory mechanisms. MSC spheroids showed considerable changes in gene expression pattern , and 3D cell culture has emerged as a new therapeutic alternative [32, 33]. There is evidence that MSC spheroids show better stemness, angiogenesis, differential potential, paracrine, and immunomodulatory effects than single MSCs [34–36].
In this study, we found that muscle volume increased to a significantly greater extent in the SP group than in the other groups, including the MONO group. In addition, the interstitial space decreased, although we did not evaluate whether this was due to decreased fibrosis or passive compression of the space by increased muscle volume. According to in vivo studies evaluating the therapeutic effects of hASC spheroids, wound healing and regeneration are possible with various cytokines and growth factors, including fibroblast growth factor, vascular endothelial growth factor, hepatocyte growth factor, etc., while in vitro studies usually evaluated stemness based on expression of the transcription factors Nanog, Oct-3/4, Sox-2, Klf4, c-Myc, and STAT3, in addition to surface markers, such as CD105, CD90, CD73, and CD34 [35, 37, 38]. As impaired muscle repair characterized by uncontrolled ECM remodeling results in the formation of fibrotic tissue in skeletal muscle , restoration of muscle volume may result in decreased interstitial fibrosis.
In addition, this study showed that the composition of HA in the lamina propria was increased significantly in the SP group compared to the other groups. HA, as one of the main components of the lamina propria in the larynx, plays a significant role in phonation via its vibratory potential. As most cases of presbylaryngis show decreased thickness of the epithelium and lamina propria and lack of vibratory tissue, increased HA in the lamina propria may result in improvements in the voice. Several studies have shown that the differentiation and activation of stem cells were facilitated by ECM of various organs, such as the kidney, liver, heart, and lung [40–42]. In terms of vocal-fold injury, Lungova et al. showed that applying human embryonic stem cells on gels composed of collagen and vocal-fold fibroblasts increased the vocal-fold lamina propria . A study using bone-marrow-derived mesenchymal stromal cells revealed increased HA distribution and decreased dense collagen networks, resulting in improved biomechanical properties . As spheroids of stem cells showed better regenerative potential, our study showed a greater increase in HA in the SP group than in the MONO group.
Most laryngeal injection procedures and injected materials can influence the mucosal wave. As vocal dysphonia is the main reason for treatment of geriatric larynx, impairment of the mucosal wave by injected material should be avoided because abnormalities in the mucosal wave are correlated with voice dysfunction, particularly hoarseness. Restoration of a normal mucosal wave is indicated by symmetric and periodic sinusoidal functions with sustained inter-vocal fold contact between cycles . Symmetric mucosal wave was observed in the SP group, which showed the minimal phase difference in bilateral vocal fold vibration. In comparison to the sham group, the MONO group showed a negative impact on mucosal wave. As the injection itself and injection material induce inflammatory responses in the injected area, changes in the viscoelasticity of the overall laryngeal inner structure may have influenced the mucosal wave. As discussed above, hASC spheroids were superior to single cells in terms of immunomodulatory function and thereby may evoke minimal surrounding inflammation with a better mucosal wave.
Vocal-fold bowing and glottal gap are typical findings of presbylarynx. For a clear voice, glottal contact should be maintained during vocal-fold adduction. The SP group in this study had a much smaller vocal gap area compared to the other groups. As with the microscopy findings, appropriate muscle volume in the SP group may have resulted in a reduced vocal gap area.