Table 1 presents in vivo studies of inner ear hearing loss based on stem cell source and year of study. Several types of stem cells, including BMSCs, AdSCs, umbilical cord stem cells (UCSCs), tongue-derived stem cells (TSCs), olfactory epithelium neural stem cells (oeMSCs), nasal tissue-derived stem cells (NSCs), and hematopoietic stem cells (HSCs) have been utilized in the treatment of inner ear hearing loss in animal models of mouse, rat, gerbil, and Guinea pig. There are many studies regarding the use of BMSCs in treatment of inner ear hearing loss. Li et al. used BMSCs in rats and observed cell transformation into neuron-like cells and positive expression of neurofilament (NF-200), microassociate protein-2 (MAP-2), neuron-specific nuclear protein (NeuN), nestin, gial fibrillary acidic protein (GFAP), GAD, and ChAT by immunohisitochemistry [66]. Intravenous transplantation of BMSCs and HSCs in mice resulted in integration of cells in the cochlea, and their differentiation, to inner ear fibrocyte-like cells without any adverse effects on auditory function [67]. Perilymphatic transplantation of green fluorescent protein (GFP) transgenic mouse BMSCs in the gerbil model of auditory neuropathy (deafened with ouabain) via scala tympani or modiolar injection demonstrated survival of MSCs within the modiolus that participated in the regeneration of damaged SGNs without any evidence of hyperacute rejection [68]. Transplantation of BMSCs into lateral semicircular canals in the rat model of acute SNHL, secondary to fibrocyte dysfunction (mitochondrial toxin), resulted in detection of MSCs in injured lateral cochlear wall, expression of connexin 26 and connexin 30, reactivation in gap junction between neighboring cells, an increase in cell survival, and acceleration of hearing recovery through the repair of injured cochlear fibrocytes [69]. Ratajczac et al. utilized BMSCs in mice and noted very small, embryonic-like cells with the potential to develop into neural and other cells for tissue repair [70]. GFP transgenic mouse BMSCs transplantation into the perilymphatic space of normal cochleae in mice displayed that transplanted cells could settle within the cochlear tissues, especially in the SLs and the spiral limbus, although most transplants were located in the perilymphatic space. Some of the transplanted cells expressed the cochlear gap-junction protein connexin 26, indicating their potential for restoration of cochlear cells [71].

Tan et al. in autoimmune deafened adult Guinea pig by use of IL-4-expressing BMSCs into scala tympani, via lateral wall, reported homing and survival capability of cells to the deafened cochlea, and transdifferentiation of them to any cochlea cell types [72]. Cho et al. utilized human BMSCs in vitro neural differentiated cells into the scala tympani to treat a Guinea pig animal model with ouabain-induced auditory neuropathy, leading to an increase in SGNs number and improvement of hearing function [73]. Intravenous injection of human BMSCs in a rat model of noise-induced or ototoxic SNHL resulted in survival of a small number of MSCs within the spiral ganglion area while most MSCs were trapped in the lungs [74]. In the Guinea pig model, the potential of AdSCs and BMSCs differentiation into neuron-like cells was demonstrated [75]. In mice, hearing recovery, after transplantation of BMSCs into the inner ear, was shown to happen, due to transdifferentiation of BMSCs into sensory fibrocyte-like (SFL) cells and stimulation of host SFLs regeneration [76].

Mouse MSCs transplanted into ampulla of superior semicircular canal in young and old healthy mice could show migration of BMSCs to cochlea, and their differentiation into SFL cells, in young mice without any adverse effects on auditory brainstem response (ABR) [77]. Human BMSCs transplanted into neomycin-deafened Guinea pig cochlea led to an increase in number of SGNs in organ of Corti and spiral ganglion, and their differentiation into neuronal progenitor cells and neuronal cells, to be used in treatment of SNHL, based on the induction of stem cell homing factors in the host cochlear tissue [78]. Magnetic labeled MSCs have been investigated in otorhinolaryngology too. To track the condition of cells transplanted in the inner ear, superparamagnetic iron oxide nanoparticles (SPIONs) have been incorporated into BMSCs, and the cells were injected to the inner ear of Guinea pig and monitored by a 1.5 Tesla MRI (Siemens, Munich, Germany) to confirm that the stem cells were successfully engrafted in the inner ear [79]. Mahmoudian-Sani et al. suggested that BMSCs, in comparison to AdSCs and UCSCs, had more efficacy to migrate and survive in the cochlear tissues, regenerating inner ear, and treating SNHL [80].

In mice, BMSCs were shown to enhance the regeneration and maintenance of fibrocytes in damaged SLs, leading to partial restoration of cochlear function [81]. Transtympanic transplantation of rodent BMSCs in a non-immunocompromised rat model to assess cochlear function by ABR, distortion product otoacoustic emissions (DPOAE), and histopathology did not reveal the recruitment of inflammatory leukocytes and edema in the cochlea of MSCs administered rats [82]. Fetoni et al., in a noise deafened adult Guinea pig, found that transplanted AdSCs into scala tympani, via round window, were able to survive and migrate, at the site of tissue damage, and express trophic factors to pave the way for further treatment of deafness [83]. Intraperitoneal injection of human AdSCs, two weeks after the onset of hearing loss, in mouse model of experimental autoimmune hearing loss (EAHL) was treated with β-tubulin, demonstrated immunomodulatory properties, absence of atrophy in stria vascularis or organ of Corti, and improvement in hearing function, presented as decreased thresholds of ABR, and protected HCs in established EAHL [84].

Injection of magnetically labeled AdSCs into the cochlea of kanamycin deafened rats revealed elevations of BDNF, the presence of significant number of MSCs in the cochlea, improved survival of SGNs, and improved hearing threshold levels denoting to their protective effects against loss of auditory function [85]. Intravenous transplantation of intact human UCSCs in a deaf Guinea pig model revealed an improvement in hearing thresholds via relocation and a rise in the number of SGNs [86]. Sullivan et al., in an adult mouse deafened by noise, demonstrated that administration of mouse TSCs into scala tympani/scala vestibulivia lateral wall led to an increase in cell survival and could attenuate the ototoxic effects of noise trauma [87]. The administration of oeMSCs into the mice cochleae, with lateral wall cochleostomy hearing loss, exhibited survival of implanted stem cells within the perilymphatic spaces of the scala tympani and conservation of hearing with otoprotective activity of oeMSCs, via stimulation by native spiral ligament fibrocytes and transdifferentiation into SFLs and stimulation of regeneration in situ of host spiral ligament fibrocytes, from a resident stem cell population by paracrine nature of MSCs [88].

Direct injection of rat oeMSCs, into the cochlear of noise-induced hearing loss model of rats, resulted in migration of stem cells around the spiral ganglion neurons and restoration of hearing loss after cell implantation, as assessed by ABR [89]. Injection of human olfactory stem cells (OSCs) into cochleae of mice, via lateral wall cochleostomy, with early onset progressive SNHL resulted in a reduction in inflammation, oxidative stress, cell apoptosis, a significant lower hearing threshold level, and amelioration of hearing loss [90]. The human MSCs, derived from nasal tissue, were evidenced to repair spiral ganglion loss in experimentally injured cochlear of neonatal rats via direct neuronal differentiation and secondary effects on endogenous cells [91] that can be tracked and verified, in a non-invasive manner, after cell transplantation by using MRI contrast agents [79,85,92].

Chronic otitis media is the primary cause of conductive hearing loss that involves perforation of the tympanic membrane (TM) and erosion of the ossicles. The TM, or eardrum, is a thin, protective layer of middle-ear tissue that forms a boundary between the external and middle ear. It is consisted of three main parts of larger pars tensa, the smaller pars flaccida, and umbo that are also other components of the hearing process in the auditory system. The anatomical structure of the TM has three layers of ectoderm, mesoderm, and endoderm [46].

TM is responsible for amplifying and transmission of sound vibrations through a chain of mobile ossicles and its perforations. External sound pressure, middle ear infection, severe trauma, and insertion of sharp objects into the ear can lead to deficient hearing function. Three primary issues in the repair of TM perforation have been reported including absence of structural assistance, absence of extracellular matrix, leading to weak neomembrane adhesion of cells, and limited angiogenesis and growth factors [93]. It is necessary to mention that wound healing in TM is slightly different from wound healing in other cutaneous tissues [94], so an exudate is released around the edges of the perforated TM, after injury, which can protect the tissue from dehydration and facilitate cell migration and proliferation of stratified squamous epithelial layer to the perforation center [95].

To close the perforated eardrum, surgical procedures (myringoplasty or tympanoplasty) are undertaken by otorhinolaryngologists, but limitations, such as discomfort, side effects, and high cost of surgical treatment have necessitated the use of better alternatives, such as tissue engineering and MSCs transplantation, as a promising tool to overcome the limitations, the operational risks and to restore, to maintain, and to improve the TM function [46]. In an injured eardrum, cell-based therapies have opened a way to solve these limitations, because MSCs can migrate towards the site of injury and participate in cell survival, cell proliferation, and tissue angiogenesis by secretion of trophic factors such as vascular endothelial growth factor (VEGF), EGF, IGF, hepatocyte growth factor (HGF), nerve growth factor (NGF), TGF-α, and stromal derived factor-1 (SDF-1), along with chemoattractant gradients in the stromal extracellular matrix and peripheral blood [96], where local factors such as hypoxia, toll-like receptor ligands, and the cytokines activate the MSCs to foster the entry of more growth factors to boost tissue regeneration [97].

In this relation, MSCs in the TM must have an appropriate microenvironment to facilitate cell survival and proliferation. If, during introduction of MSCs in the perforated TM, the cells are dropped into the middle ear cavity, the cells would be easily susceptible to air-drying through external auditory meatus [98]. Another important point in cell transplantation is the cell delivery that uses of scaffolds as an increasingly popular technique that can provide protection and controlled spatial cues for seeded stem cells. The delivery of MSCs at the ruptured TM sites was shown to enhance the activation of epithelial stem cells for faster closure of TM perforation. So, the fibroblasts and collagen in the middle connective tissue layer produce a neomembrane framework to close the perforation [99]. Danti et al. have fabricated colonization of human MSCs on scaffolds to allow an osteoblastic maturation in vitro [100].

Rahman et al. used drops of gelatin, containing human BMSCs, on the perforated TM of rats and assessed the thickness of pars tensa region and lamina propria under otomicroscopy, mechanical stiffness of the healed TM tissue by Moiré interferometry and lamina propria, middle ear cavity, and external ear canal wall tissue via microscopy and illustrated a decrease in the stiffness of the healed tympanic membrane and a healing process with an enhanced restoration [101]. When GFP expressing BMSCs were embedded in porcine-derived (Gelita-Spon GS), hyaluronate-derived (EpiDisc ED), and polyvinyl alcohol (PVA) scaffolds and injected in an injured TM of a mouse model, the transplanted cells were deposited in the injured tissue and differentiated into epithelial-like cells and formed a thicker neotympanum that can be a promising alternative to tympanoplasty [102].

The first animal model trial of concurrent use of MSCs and a three-dimensional (3D) bioprinted scaffold (polycaprolactone/collagen/alginate) was carried out in closing of subacute TM perforations in rats undergoing otoendoscopy for acoustic measurements as per ABR thresholds. The findings denoted to the recovery of the hearing capacity at all frequencies, along with regeneration of the thick neodrum assessed by optical coherence tomography (OCT). Goncalves et al., by use of BMSCs seeded on hyaluronic acid (HA) scaffold in mice bilateral large tympanic perforations, demonstrated the repair in TM and restoration of the trilaminar structure in TM. Assessment by histology indicated formation of an intact neotympanum in the perforated areas. Neo-tympanal integrity and transparency were confirmed by otoscopy too [103]. The mechanical properties of the regenerated TM, such as membrane stiffness, membrane stability, and efficient nanovibration were evaluated by a laser Doppler vibrometer (LDV), revealing an acceleration in the healing process in the TM perforations and formation of a thickened prominent fibrous layer. The acoustic mechanical properties were recovered in the healed TM [104]. Ong et al. found that human AdSCs in mice with sub-total pars tensa perforations could lead to paracrine function, secretion of growth factors, a promoted significant keratinocyte proliferation and migration and TM wound healing [105]. Table 2 presents in vivo studies oftympanic membrane related hearing loss based on stem cell source and year of study.