Single-cell profiling reveals a potent role of quercetin in promoting hair regeneration

Hair loss affects millions of people at some time in their life,and safe and efficient treatments for hair loss are a significant unmet medical need.We report that topical delivery of quercetin(Que)stimulates resting hair follicles to grow with rapid follicular keratinocyte proliferation and replenishes perifollicular microvasculature in mice.We construct dynamic single-cell transcriptome landscape over the course of hair regrowth and find that Que treatment stimulates the differentiation trajectory in the hair follicles and induces an angiogenic signature in dermal endothelial cells by activating HIF-1αin endothelial cells.Skin administration of a HIF-1αagonist partially recapitulates the pro-angiogenesis and hair-growing effects of Que.Together,these findings provide a molecular understanding for the efficacy of Que in hair regrowth,which underscores the translational potential of targeting the hair follicle niche as a strategy for regenerative medicine,and suggest a route of pharmacological intervention that may promote hair regrowth.

Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae

Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.

Retrovirus-Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possible utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through mor-phology observation, immunnocytochemical staining and RT-PCR analysis. The Hath1 gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1(a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differentiate into hair cell-like cells when forced to express Hath1. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.

ULTRASTRUCTURAL OBSERVATIONS ON REGENERATING HAIR CELLS IN THE CHICK BASILAR PAPILLA FOLLOWING AMINOGLYCOSIDE OTOTOXICITY

Newly hatched chicks were injected with so mg/(kg' d) of gentamycin sulfate (GM )for 10 days. After 18st injection, animals were killed on survival days 3,6 .9 and 12. The chick basilar papillae (BP) were observed by transmission electron microscopy (TEM ). The results sho've'Ithat regenerated hair cells were erupted to the hasal membrane surrace in the early phase. The cytoplasm of these regenerated hair cells was heavily stained an contained numerous mitochondria. Afterseveral days, stereocilia appeared. When stereocilis bundles were rormed and cuticular layer was integrated, hasal nerve terminals were round. The results suggested that regenerating hair cells 'veredirectly produced on the hasal membrane surface following gentamycin induced ototoxlcity and thenI,roliferated and developed Into mature normal hair cells.

Stem Cell-Based Hair Cell Regeneration and Therapy in the Inner Ear

Hearing loss has become increasingly prevalent and causes considerable disability,thus gravely burdening the global economy.Irreversible loss of hair cells is a main cause of sensorineural hearing loss,and currently,the only relatively effective clinical treatments are limited to digital hearing equipment like cochlear implants and hearing aids,but these are of limited benefit in patients.It is therefore urgent to understand the mechanisms of damage repair in order to develop new neuroprotective strategies.At present,how to promote the regeneration of functional hair cells is a key scientific question in the field of hearing research.Multi-ple signaling pathways and transcriptional factors trigger the activation of hair cell progenitors and ensure the maturation of newborn hair cells,and in this article,we first review the principal mechanisms underlying hair cell reproduction.We then further discuss therapeutic strategies involving the co-regulation of multiple signaling pathways in order to induce effective functional hair cell regeneration after degeneration,and we summarize current achievements in hair cell regeneration.Lastly,we discuss potential future approaches,such as small molecule drugs and gene therapy,which might be applied for regenerating functional hair cells in the clinic.

Design of a biofluid-absorbing bioactive sandwich-structured Zn-Si bioceramic composite wound dressing for hair follicle regeneration and skin burn wound healing

The deep burn skin injures usually severely damage the dermis with the loss of hair follicle loss,which are difficult to regenerate.Furthermore,severe burns often accompanied with large amount of wound exudates making the wound moist,easily infected,and difficult to heal.Therefore,it is of great clinical significance to develop wound dressings to remove wound exudates and promote hair follicle regeneration.In this study,a sandwich-structured wound dressing(SWD)with Janus membrane property was fabricated by hot compression molding using hydrophilic zinc silicate bioceramics(Hardystonite,ZnCS)and hydrophobic polylactic acid(PLA).This unique organic/inorganic Janus membrane structure revealed excellent exudate absorption property and effectively created a dry wound environment.Meanwhile,the incorporation of ZnCS bioceramic particles endowed the dressing with the bioactivity to promote hair follicle regeneration and wound healing through the release of Zn^(2+)and SiO^(2-)_(3)ions,and this bioactivity of the wound dressing is mainly attributed to the synergistic effect of Zn^(2+)and SiO^(2-)_(3)to promote the recruitment,viability,and differentiation of hair follicle cells.Our study demonstrates that the utilization of the Janus membrane and synergistic effect of different type bioactive ions are effective approaches for the design of wound dressings for burn wound healing.

An efficient strategy for establishing a model of sensorineural deafness in rats

Ototoxic drugs can be used to produce a loss of cochlear hair cells to create animal models of deafness. However, to the best of our knowledge, there is no report on the establishment of a rat deafness model through the combined application of aminoglycosides and loop diuretics. The aim of this study was to use single or combined administration of furosemide and kanamycin sulfate to establish rat models of deafness. The rats received intravenous injections of different doses of furosemide and/or intramuscular injections of kanamycin sulfate. The auditory brainstem response was measured to determine the hearing threshold after drug application. Immunocytochemistry and confocal microscopy were performed to evaluate inner ear morphology. In the group receiving combined administration of furosemide and kanamycin, the auditory brainstem response threshold showed significant elevation 3 days after administration, higher than that produced by furosemide or kanamycin alone. The hair cells showed varying degrees of injury, from the apical turn to the basal turn of the cochlea and from the outer hair cells to the inner hair cells. The spiral ganglion cells maintained a normal morphology during the first week after the hair cells completely disappeared, and then gradually degenerated. After 2 months, the majority of spiral ganglion cells disappeared, but a few remained. These findings demonstrate that the combined administration of furosemide and kanamycin has a synergistic ototoxic effect, and that these drugs can produce hair cell loss and hearing loss in rats. These findings suggest that even in patients with severe deafness, electronic cochlear implants may partially restore hearing.

Thermodynamically stable ionic liquid microemulsions pioneer pathways for topical delivery and peptide application

Copper peptides(GHK-Cu)are a powerful hair growth promoter with minimal side effects when compared with minoxidil and finasteride;however,challenges in delivering GHK-Cu topically limits their non-invasive applications.Using theoretical calculations and pseudo-ternary phase diagrams,we designed and constructed a thermodynamically stable ionic liquid(IL)-based microemulsion(IL-M),which integrates the high drug solubility of ILs and high skin permeability of microemulsions,thus improving the local delivery of copper peptides by approximately three-fold while retaining their biological function.Experiments in mice validated the effectiveness of our proposed IL-M system.Furthermore,the exact effects of the IL-M system on the expression of growth factors,such as vascular endothelial growth factor,were revealed,and it was found that microemulsion increased the activation of the Wnt/β-catenin signaling pathway,which includes factors involved in hair growth regulation.Overall,the safe and non-invasive IL microemulsion system developed in this study has great potential for the clinical treatment of hair loss.

Rassf2 overexpression mediated by AAV promotes the supporting cell-to-hair cell transformation in the cochlea

Sensory hair cells are responsible for detecting and transmitting sound in the inner ear,and damage to HCs leads to hearing loss.HCs do not regenerate spontaneously in adult mammals,which makes the hearing loss permanent.However,hair cells and supporting cells have the same precursors in the inner ear,and in newborn mice,the adjacent SCs can be activated by gene manipulation to differentiate into newly regenerated hair cells.Here,we demonstrate the role of the Ras association domain family member 2(Rassf2)in supporting cell to hair cell trans-differentiation in the inner ear.Using the AAV vector(AAV-ie)to upregulate Rassf2 expression promoted supporting cell division and hair cell production in cultured cochlear organoids.Also,AAV-Rassf2 enhanced the regenerative ability of Lgr5+SCs in the postnatal cochlea without impairing hearing,and this might due to the modulation of the Wnt,Hedgehog and Notch signaling pathways.Furthermore,AAV-Rassf2 enhances cochlear supporting cell division and hair cell production in the neomycin injury model.In summary,our results suggest that Rassf2 is a key component in HC regenerative repair,and gene modulation mediated by adeno-associated virus may be a promising gene therapy for hearing repair.

Composite double-layer microneedle loaded with traditional Chinese medicine for the treatment of androgenic alopecia

Androgenetic alopecia(AGA)is an androgen-mediated alopecia affected by both genes and hormones.Medication is a relatively common treatment.As a new drug delivery method,microneedles(MNs)can effectively break through the stratum corneum barrier,deliver drugs more efficiently,and achieve better therapeutic effects.In this study,we develop a composite double-layer MN through multi-step casting fabrication using a polydimethylsiloxane mold.The needle tip was fabricated by mixed solution of chitosan and polyvinylpyrrolidone which was loaded with Polygonum multiflorum extract,and the base layer was prepared by mixed solution of polyvinyl alcohol and polyvinylpyrrolidone.In vitro mechanical tests showed that the maximum load of a single tip of the drug-loaded MN was about 3.5 N,which met the mechanical requirements of skin puncture(>1 N).The drug release experiment showed that the MN could achieve gradual drug release.In the animal experiment,pigmentation and hair regrowth occurred earlier in the Polygonum multiflorum-MN(Pm-MN)group than in the other groups,and hair growth finally appeared in almost the entire area.Compared with the AGA model mice,mice in the Pm-MN group achieved an increase in the number and diameter of hair follicles.In conclusion,the Pm-MN is scientific and feasible for treating AGA.