Hair Regeneration Research Articles

One-step generation of core–shell biomimetic microspheres encapsulating double-layer cells using microfluidics for hair regeneration

Tissue engineering of hair follicles (HFs) has enormous potential in the treatment of hair loss. HF morphogenesis is triggered by reciprocal interactions between HF germ epithelial and mesenchymal layers. Here, a microfluidic-assisted technology is developed for the preparation of double aqueous microdroplets that entrap double-layer cells and growth factors to ultimately be used for hair regeneration. Mouse mesenchymal cells (MSCs) and epidermal cells (EPCs) are encapsulated in gelatin methacrylate (GelMA) cores and photo-curable catechol-grafted hyaluronic acid (HAD) shells to fabricate GelMA-MSC/HAD-EPC (G/HAD) microspheres. The findings show that the G/HAD microspheres exhibit ultrafast gelation, aqueous phase separation, superior biocompatibility, and favorable wet adhesion properties. G/HAD microspheres can also support cell proliferation and sustain growth factor release. These composite cell microspheres are capable of efficient HF generation upon transplantation into the dorsal dermis of nude mice. This finding facilitates the large-scale preparation of approximately 80 double-layer cell spheres per min. This simple double-layer cell sphere preparation approach is a promising strategy for improving current hair-regenerative medicine techniques and can potentially be applied along with other organoid techniques for extended applications.

Keratin 79 is a PPARA target that is highly expressed by liver damage

Keratins are key structural proteins found in skin and other epithelial tissues. Keratins also protect epithelial cells from damage or stress. Fifty-four human keratins were identified and classified into two families, type I and type II. Accumulating studies showed that keratin expression is highly tissue-specific and used as a diagnostic marker for human diseases. Notably, keratin 79 (KRT79) is type II cytokeratin that was identified as regulator of hair canal morphogenesis and regeneration in skin, but its role in liver remains unclear. KRT79 is undetectable in normal mouse but its expression is significantly increased by the PPARA agonist WY-14643 and fenofibrate, and completely abolished in Ppara-null mice. The Krt79 gene has functional PPARA binding element between exon 1 and exon 2. Hepatic Krt79 is regulated by HNF4A and HER2. Moreover, hepatic KRT79 is also significantly elevated by fasting- and high-fat diet-induced stress, and these increases are completely abolished in Ppara-null mice. These findings suggest that hepatic KRT79 is controlled by PPARA and is highly associated with liver damage. Thus, KRT79 may be considered as a diagnostic marker for human liver diseases.

Hordenine Activated Dermal Papilla Cells and Promoted Hair Regrowth by Activating Wnt Signaling Pathway.

Hordenine is effective in treating hyperpigmentation, fighting diabetes and resisting fibrosis and acute inflammation. However, the role of Hordenine on hair growth has not been elucidated. Here, we found that Hordenine treatments significantly enhance proliferation of primary mouse dermal-papilla cells (DPCs) and increase the activity of DPCs in a dose-dependent manner. Additionally, Hordenine markedly promoted the elongation of the hair shaft in the model of in vitro-cultured mouse vibrissa follicle and accelerated hair regrowth in a mouse model of depilation-induced hair regeneration. Real-time PCR, Western Blot and immunofluorescent assays showed that nuclear β-catenin and its downstream gene expression such as Lef1, Axin2, Cyclin D1 and ALP were greatly upregulated in DPCs and mouse hair follicles after Hordenine treatments. Moreover, the increased DPCs' proliferation and hair shaft elongation of cultured mouse vibrissa follicles induced by Hordenine treatments were rescued by a Wnt/β-catenin signaling inhibitor, FH535. These data indicate that Hordenine can effectively enhance DPCs' activity and accelerate hair regrowth through activating the Wnt/β-catenin signaling pathway. Therefore, these findings suggest Hordenine/its derivatives may be potentially used for preventing and treating alopecia in the future.

Hypoxia inducible factor-1α promotes trichogenic gene expression in human dermal papilla cells

Dermal papilla cells (DPCs) play critical roles in hair follicle development, but the underlying mechanisms that contribute to hair regeneration have yet to be fully elucidated, particularly in terms of alterations in androgenetic alopecia patients. In this study, we demonstrated that hypoxia-inducible factor-1α (HIF-1α) is suppressed in scalp tissues of androgenetic alopecia patients and potentially associated with hair follicle development. Using RT-qPCR and western blot, we found that mRNA and protein levels of trichogenic genes, LEF1 and versican (VCAN), were attenuated in HIF-1α knockdown DPCs. Under an in vivo mimicked environment in a three-dimensional spheroid culture, HIF-1α-suppressed DPCs downregulated the expression of hair induction-related genes. Finally, treatment with a HIF-1α activator resulted in the elevated expression of trichogenic genes in DPCs. This study highlights the importance of dermal HIF-1α expression in regulating trichogenic genes and provides a promising therapeutic target and a fundamental tissue engineering approach for hair loss treatment.

Microinjury-Induced Tumor Necrosis Factor-α Surge Stimulates Hair Regeneration in Mice

Introduction: Tumor necrosis factor (TNF)-α released after follicular injury such as that caused by plucking plays a role in the activation of hair regeneration. Microneedle (MN) treatment is applied to the scalp to increase permeability and facilitate the delivery of any number of compounds. Because the MN treatment causes injury to the epidermis, albeit minor, we reasoned that this treatment would lead to a temporary TNF-α surge and thereby promote hair regeneration. Methods: To investigate the effects of MN-treatment-induced microinjury and TNF-α on hair growth, we used C57BL/6N mice which were divided into six experimental groups: three groups of 1) negative control (NC), 2) plucking positive control (PK), and 3) MN therapy system (MTS) mice; and three groups identical to above were treated with a TNF-α blocker for 3 weeks: 4) NCB, 5) PKB, and 6) MTSB group. Results: After injury, TNF-α surge occurred on day 3 in the PK group and on day 6 in the MTS group. Wnt proteins and VEGF expression were markedly increased in the PK group on day 3 and on day 6 in the MTS group compared to the NC group. Following wound healing, only MTS and PK groups displayed thickened epidermis and longer HF length. Within the 2 weeks following treatment, the rate of hair growth was much slower in the injured mice treated with the TNF-α blocker. Conclusion: Our findings indicate that microinjury stimulates the wound-healing mechanism via TNF-α/Wnt/VEGF surge to induce hair growth, and that blocking TNF-α inhibits this growth process.

3D printing of microneedle arrays for hair regeneration in a controllable region

Hair loss is a common skin disease that causes intense emotional suffering. Hair regeneration in a personalized area is highly desirable for patients with different balding conditions. However, the existing pharmaceutical treatments have difficulty precisely regenerating hair in a desired area. Here, we show a method to precisely control the hair regeneration using customized microneedle arrays (MNAs). The MNA with a customized shape is fast fabricated by a static optical projection lithography process in seconds, which is a 3D printing technology developed by our group. In the mouse model, MNA treatment could induce hair regrowth in a defined area corresponding to the customized shape of MNA. And the regenerated hair promoted by MNAs had improved quality. Cellular and molecular analysis indicated that MNA treatment could recruit macrophages in situ and then initiate the proliferation of hair follicle stem cells, thereby improving hair regeneration. Meanwhile, the activation of the Wnt/β-catenin signaling pathway was observed in hair follicles. The expressions of Hgf, Igf 1 and Tnf-α were also upregulated in the treated skin, which may also be beneficial for the MNA-induced hair regeneration. This study provides a strategy to precisely control hair regeneration using customized microneedle arrays by recruiting macrophages in situ, which holds the promise for the personalized treatment of hair loss.

Ganoderma lucidum ethanolic extract for the treatment of androgenic alopecia in rats with testosterone-induced Baldness.

Background: Alopecia is a widespread hair loss condition affecting numerous individuals worldwide. This study aims to investigate the potential hair growth-promoting properties of a preparation containing an ethanolic extract of Ganoderma lucidum and its primary components in a model of testosterone-induced alopecia. Aim: The objective of this research is to assess the effects of Ganoderma lucidum extract on hair regeneration using a testosterone-induced alopecia model. Materials and Methods: The study was conducted using five groups of rats: negative control group (n = 6), positive control group (n = 6), testosterone plus minoxidil 2% (n = 6), testosterone and ethanolic extract of Ganoderma lucidum extract (10 mg/kg) (n = 6), and testosterone and ethanolic extract of Ganoderma lucidum extract (15 mg/kg). To induce alopecia, subcutaneous testosterone (1 mg/kg SC) was administered daily to all groups except the negative control group for 21 consecutive days. After 21 days, Anagen/telogen (A/T) ratio and the number of follicles were measured and recorded. Results: The standard group, treated with minoxidil 2%, showed a higher anagen/telogen (A/T) ratio, follicular density, and hair length compared to the positive control group. The groups treated with ethanolic extract of Ganoderma lucidum at 10mg/kg and 15mg/kg displayed increased anagen/telogen ratio and improved hair follicle quantity and morphology compared to the positive control group. Furthermore, the group treated with 15mg/kg of the extract showed more favorable outcomes compared to the 10mg/kg group. These results were comparable to the effects of the commonly prescribed drug, Minoxidil 2%, used to promote hair growth in individuals with androgenetic alopecia. Conclusion: The preparation containing an ethanolic extract of Ganoderma lucidum, especially at a concentration of 15mg/kg, demonstrated significant hair growth-promoting effects in the testosterone-induced alopecia model. Our findings suggest the potential of Ganoderma lucidum extract as a possible treatment for alopecia and merit further exploration for therapeutic applications.

Therapeutic Approach for Hair Growth and Regeneration Using Bioactive Formulation Containing Mesenchymal Stromal Cell-Derived Conditioned Medium

Therapeutic Approach for Hair Growth and Regeneration Using Bioactive Formulation Containing Mesenchymal Stromal Cell-Derived Conditioned Medium

3D multicellular micropatterning biomaterials for hair regeneration and vascularization.

Hair loss caused by the abnormal functions of hair follicles in skin can seriously impact the quality of an individual's life. The development of sophisticated skin tissue-engineered constructs is required to enable the function recovery of hair follicles. However, effective hair regrowth in skin substitutes still remains a great challenge. In this study, a 3D multicellular micropattern was successfully fabricated by arranging the hair follicle-related cells orderly distributed in the interval of vascular-cell networks via bioprinting technology. By combining the stable biomimetic micropattern structure and the bio-inducing substrate incorporated with magnesium silicate (MS) nanomaterials, the 3D multicellular micropattern possessed significant follicular potential and angiogenic capacity in vitro. Furthermore, the 3D multicellular micropattern with MS incorporation contributed to efficient hair regrowth during skin tissue regeneration in both immunodeficient mice and androgenetic alopecia (AGA) mice models. Thus, this study proposes a novel 3D micropatterned multicellular system assembling a biomimetic micro-structure and modulating the cell-cell interaction for hair regeneration during skin reconstruction.

Mechanical force drives the initial mesenchymal-epithelial interaction during skin organoid development.

Rationale: Stem cells self-organize to form organoids that generate mini-organs that resemble the physiologically-developed ones. The mechanism by which the stem cells acquire the initial potential for generating mini-organs remains elusive. Here we used skin organoids as an example to study how mechanical force drives initial epidermal-dermal interaction which potentiates skin organoids to regenerate hair follicles. Methods: Live imaging analysis, single-cell RNA-sequencing analysis, and immunofluorescence were used to analyze the contractile force of dermal cells in skin organoids. Bulk RNA-sequencing analysis, calcium probe detection, and functional perturbations were used to verify that calcium signaling pathways respond to the contractile force of dermal cells. In vitro mechanical loading experiment was used to prove that the stretching force triggers the epidermal Piezo1 expression which negatively regulates dermal cell attachment. Transplantation assay was used to test the regenerative ability of skin organoids. Results: We found that dermal cell-derived contraction force drives the movement of dermal cells surrounding the epidermal aggregates to trigger initial mesenchymal-epithelial interaction (MEI). In response to dermal cell contraction force, the arrangement of the dermal cytoskeleton was negatively regulated by the calcium signaling pathway which further influences dermal-epidermal attachment. The native contraction force generated from the dermal cell movement exerts a stretching force on the adjacent epidermal cells, activating the stretching force sensor Piezo1 in the epidermal basal cells during organoid culture. Epidermal Piezo1 in turn drives strong MEI to negatively regulate dermal cell attachment. Proper initial MEI by mechanical-chemical coupling during organoid culture is required for hair regeneration upon transplantation of the skin organoids into the back of the nude mice. Conclusion: Our study demonstrated that mechanical-chemical cascade drives the initial event of MEI during skin organoid development, which is fundamental to the organoid, developmental, and regenerative biology fields.

Recombinant Oil-Body-Expressed Oleosin-hFGF5 in Arabidopsis thaliana Regulates Hair Growth.

FGF5 (Fibroblast Growth Factor) is a member of the fibroblast growth factor family, which not only regulates growth and development but also inhibits hair regeneration. The oil-body expression vector pOTB-hFGF5 was constructed by the genetic engineering method and it was transformed into Arabidopsis by flora dip. T3 homozygous transgenic Arabidopsis was obtained after screening and propagation by the PCR and Western blot methods. The recombinant oil-body-expressed oleosin-hFGF5 can inhibit the proliferation of hair follicle epithelial cells and it exhibits the pharmacological activity of inhibiting hair regeneration in vivo by protein hybridization and imunohistochemistry. At the same time, the potential mechanism of recombinant oil-body-expressed oleosin-hFGF5 inhibiting hair growth was also revealed by RNA-Seq. This implies that the recombinant oil-body-expressed oleosin-hFGF5 has a good effect on inhibiting hair growth.

PROTAC Degraders of Androgen Receptor-Integrated Dissolving Microneedles for Androgenetic Alopecia and Recrudescence Treatment via Single Topical Administration.

Androgenetic alopecia (AGA) is a transracial and cross-gender disease worldwide with a youth-oriented tendency, but it lacks effective treatment. The binding of androgen receptor (AR) and androgen plays an essential role in the occurrence and progression of AGA. Herein, novel proteolysis targeting chimera degrader of AR (AR-PROTAC) is synthesized and integrated with dissolving microneedles (PROTAC-MNs) to achieve AR destruction in hair follicles for AGA treatment. The PROTAC-MNs possess adequate mechanical capabilities for precise AR-PROTAC delivery into the hair follicle-residing regions for AR degradation. After applying only once topically, the PROTAC-MNs achieve an accelerated onset of hair regeneration as compared to the daily application of the first-line topical drug minoxidil. Intriguingly, PROTAC-MNs via single administration still realize superior hair regeneration in AGA recrudescence, which is the major drawback of minoxidil in clinical practice. With the degradation of AR, the PROTAC-MNs successfully regulate the signaling cascade related to hair growth and activate hair follicle stem cells. Furthermore, the PROTAC-MNs do not cause systemic toxicity or androgen deficiency-related chaos in vivo. Collectively, these AR-degrading dissolving microneedles with long-lasting efficacy, one-step administration, and high biocompatibility provide a great therapeutic potential for AGA treatment.

Isolation, characterization and differentiation of dermal papilla cells from Small-tail Han sheep

Dermal papilla cells (DPCs) are the key dermal component of the hair follicle that directly regulates hair follicle development, growth and regeneration. Successfully isolated and cultured DPCs from Small-tail Han sheep could provide a good model for the study of hair follicle development mechanism in vitro. DPCs were isolated using enzyme digestion and dissecting microscope from Small-tail Han sheep. Adherent cells were identified by cell characteristics, particular gene expression, differentiation capability to adipocyte and osteoblast using specific differentiation mediums. Additionally, flow cytometry was used to detect the cell cycle of DPCs. Cells originating from the dermal papilla showed the morphological appearance of mesenchymal cells (fibroblast-like cells). Purified DPCs were positive for α-SMA (α smooth muscle actin) and vimentin; in addition to their strong proliferation abilities in vitro, these DPCs can be differentiated into adipocyte and osteoblasts lineage under appropriate culture condition. DPCs were successfully isolated and subcultured from Small-tail Han sheep, which exhibited progenitor cell features and multiple differentiation potency. It provides a material for studying the molecular mechanism of hair follicle development and hair cycle, which will promote wool production in the future.

Engineered Nanovesicles from Fibroblasts Modulate Dermal Papillae Cells In Vitro and Promote Human Hair Follicle Growth Ex Vivo.

Alopecia is a common medical condition affecting both sexes. Dermal papilla (DP) cells are the primary source of hair regeneration in alopecia patients. Therapeutic applications of extracellular vesicles (EVs) are restricted by low yields, high costs, and their time-consuming collection process. Thus, engineered nanovesicles (eNVs) have emerged as suitable therapeutic biomaterials in translational medicine. We isolated eNVs by the serial extrusion of fibroblasts (FBs) using polycarbonate membrane filters and serial and ultracentrifugation. We studied the internalization, proliferation, and migration of human DP cells in the presence and absence of FB-eNVs. The therapeutic potential of FB-eNVs was studied on ex vivo organ cultures of human hair follicles (HFs) from three human participants. FB-eNVs (2.5, 5, 7.5, and 10 µg/mL) significantly enhanced DP cell proliferation, with the maximum effect observed at 7.5 µg/mL. FB-eNVs (5 and 10 µg/mL) significantly enhanced the migration of DP cells at 36 h. Western blotting results suggested that FB-eNVs contain vascular endothelial growth factor (VEGF)-a. FB-eNV treatment increased the levels of PCNA, pAKT, pERK, and VEGF-receptor-2 (VEGFR2) in DP cells. Moreover, FB-eNVs increased the human HF shaft size in a short duration ex vivo. Altogether, FB-eNVs are promising therapeutic candidates for alopecia.

Pyruvate Kinase M2 Promotes Hair Regeneration by Connecting Metabolic and Wnt/β-Catenin Signaling.

Hair follicle stem cells (HFSCs) utilize glycolytic metabolism during their activation and anagen induction. However, the role of pyruvate kinase M2 (PKM2), which catalyzes the final step of glycolysis, in hair regeneration has not been elucidated. In this study, we investigated the expression pattern and activity of PKM2 during the depilation-induced anagen progression in mice. We found that TEPP-46, a selective activator of PKM2, enhanced hair re-growth and proliferation of HFSCs. PKM2 expression was increased via up-regulation of Wnt/β-catenin signaling, which is involved in hair regeneration. Moreover, a combined treatment with KY19382, a small molecule that activates Wnt/β-catenin signaling, and TEPP-46 significantly enhanced hair re-growth and wound-induced hair follicle neogenesis (WIHN). These results indicate that simultaneous activation of the PKM2 and Wnt/β-catenin signaling could be a potential strategy for treating alopecia patients.

Regenerative medicine strategies for hair growth and regeneration: A narrative review of literature.

Hair loss, or alopecia, is associated with several psychosocial and medical comorbidities, and it remains an economic burden to individuals and the society. Alopecia is attributable to varied mechanisms and features a multifactorial predisposition, and the available conventional medical interventions have several limitations. Thus, several therapeutic strategies for alopecia in regenerative medicine are currently being explored, with increasing evidence suggesting that mesenchymal stem cell (MSC) implantation, MSC-derived secretome treatment, and blood-derived platelet-rich plasma therapies are potential treatment options. In this review, we searched the Cochrane Library, MEDLINE (PubMed), EMBASE, and Scopus using various combinations of terms, such as "stem cell," "alopecia," "hair loss," "Androgenetic alopecia," "male-pattern hair loss," "female-pattern hair loss," "regenerative hair growth," "cell therapy," "mesenchymal stem cells," "MSC-derived extracellular vesicles," "MSC-derived exosomes," and "platelet-rich plasma" and summarized the most promising regenerative treatments for alopecia. Moreover, further opportunities of improving efficacy and innovative strategies for promoting clinical application were discussed.

Wnt/β-catenin signaling activator restores hair regeneration suppressed by diabetes mellitus

Diabetes mellitus is one of the most prevalent diseases in modern society. Many complicationssuch as hepatic cirrhosis, neuropathy, cardiac infarction, and so on are associated with diabetes. Although a relationship between diabetes and hair loss has been recently reported, the treatment of diabetic hair loss by Wnt/β-catenin activators has not been achieved yet. In this study, we found that the depilation-induced anagen phase was delayed in both db/db mice and high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic mice. In diabetic mice, both hair regrowth and wound-induced hair follicle neogenesis (WIHN) were reduced because of suppression of Wnt/β-catenin signaling and decreased proliferation of hair follicle cells. We identified that KY19382, a small molecule that activates Wnt/β-catenin signaling, restored the capabilities of regrowth and WIHN in diabetic mice. The Wnt/β-catenin signaling activator also increased the length of the human hair follicle which was decreased under high glucose culture conditions. Overall, the diabetic condition reduced both hair regrowth and regeneration with suppression of the Wnt/β-catenin signaling pathway. Consequently, the usage of Wnt/β-catenin signaling activators could be a potential strategy to treat diabetes-induced alopecia patients. [BMB Reports 2022; 55(11): 559-564].

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-1α in endothelial cells. Skin administration of a HIF-1α agonist 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.

Keratin-mediated hair growth and its underlying biological mechanism

Here we show that intradermal injection of keratin promotes hair growth in mice, which results from extracellular interaction of keratin with hair forming cells. Extracellular application of keratin induces condensation of dermal papilla cells and the generation of a P-cadherin-expressing cell population (hair germ) from outer root sheath cells via keratin-mediated microenvironmental changes. Exogenous keratin-mediated hair growth is reflected by the finding that keratin exposure from transforming growth factor beta 2 (TGFβ2)-induced apoptotic outer root sheath cells appears to be critical for dermal papilla cell condensation and P-cadherin-expressing hair germ formation. Immunodepletion or downregulation of keratin released from or expressed in TGFβ2-induced apoptotic outer root sheath cells negatively influences dermal papilla cell condensation and hair germ formation. Our pilot study provides an evidence on initiating hair regeneration and insight into the biological function of keratin exposed from apoptotic epithelial cells in tissue regeneration and development.

Transcriptome Analysis Reveals Genes Contributed to Min Pig Villi Hair Follicle in Different Seasons.

The Min pig, a local pig breed in China, has a special trait which has intermittent villus and coat hair regeneration. However, the regulation and mechanism of villus in Min pigs have not yet been described. We observed and described the phenotype of Min pig dermal villi in detail and sequenced the mRNA transcriptome of Min pig hair follicles. A total of 1520 differentially expressed genes (DEG) were obtained.K-means hierarchical clustering showed that there was a significant expression pattern difference in winter compared with summer. Gene enrichment and network analysis results showed that the hair growth in Min pigs was closely related to the composition of desmosomes and regulated by an interaction network composed of eight core genes, namely DSP, DSC3, DSG4, PKP1, TGM1, KRT4, KRT15, and KRT84. Methylation analysis of promoters of target genes showed that the PKP1 gene was demethylated. Our study will help to supplement current knowledge of the growth mechanism of different types of hair.