Innate lymphoid cell-based immunomodulatory hydrogel microspheres containing Cutibacterium acnes extracellular vesicles for the treatment of psoriasis.

Psoriasis is a chronic skin inflammation influenced by dysregulated skin microbiota, with the role of microbiota in psoriasis gaining increasing prominence. Bacterial extracellular vesicles (bEVs) serve as crucial regulators in the interaction between hosts and microbiota. However, the mechanism underlying the therapeutic potential of bEVs from commensal bacteria in psoriasis remains unclear. Here, we investigated the therapeutic role of Cutibacterium acnes (C. acnes)-derived extracellular vesicles (CA-EVs) in psoriasis treatment. To prolong the active duration of CA-EVs, we encapsulated them in gelatin methacrylate (GelMA) to fabricate hydrogel microspheres (CA-EVs@GHM) with sustained release properties. As GelMA degraded, CA-EVs were gradually released, maintaining a high concentration in mouse skin even 96 h post-treatment. In human keratinocyte cells (HaCaT), CA-EVs@GHM enhanced resistance to Staphylococcus aureus (S. aureus), promoted proliferation and migration of HaCaT cells exposed to S. aureus, and significantly reduced the expression of inflammatory genes such as interleukin (IL)-6 and C-X-C motif chemokine ligand 8 (CXCL8). In vivo, CA-EVs@GHM, more potent than CA-EVs alone, markedly attenuated proinflammatory gene expression, including tumor necrosis factor (TNF), Il6, Il17a, Il22 and Il23a in imiquimod (IMQ)-induced psoriasis-like mice, and restored skin barrier function. 16S rRNA sequencing revealed that CA-EVs@GHM might provide therapeutic effects against psoriasis by restoring microbiota diversity on the back skin of mice, reducing Staphylococcus colonization, and augmenting lipid metabolism. Furthermore, flow cytometry analysis showed that CA-EVs@GHM prevented the conversion of type 2 innate lymphoid cells (ILC2) to type 3 innate lymphoid cells (ILC3) in psoriasis-like mouse skin, reducing the pathogenic ILC3 population and suppressing the secretion of IL-17 and IL-22. In summary, our findings demonstrate that the long-term sustained release of CA-EVs alleviated psoriasis symptoms by controlling the transformation of innate lymphoid cells (ILCs) subgroups and restoring skin microbiota homeostasis, thus offering a promising therapy for psoriasis treatment. STATEMENT OF SIGNIFICANCE: Cutibacterium acnes, which is reduced in psoriasis skin, has been reported to promote skin homeostasis by regulating immune balance. Compared to live bacteria, bacterial extracellular vesicles (bEVs) are less prone to toxicity and safety concerns. bEVs play a pivotal role in maintaining bacterial homeostasis and modulating the immune system. However,bEVs without sustained release materials are unable to function continuously in chronic diseases. Therefore, we utilized hydrogel microspheres to encapsulate Cutibacterium acnes (C. acnes)-derived extracellular vesicles (CA-EVs), enabling long term sustained release. Our findings indicate that, CA-EVs loaded gelatin methacrylate hydrogel microspheres (CA-EVs@GHM) showed superior therapeutic effects in treating psoriasis compared to CA-EVs. CA-EVs@GHM exhibited a more significant regulation of pathological type 3 innate lymphoid cells (ILC3) and skin microbiota, providing a promising approach for microbiota-derived extracellular vesicle therapy in the treatment of skin inflammation.

Autophagy induces hair follicle stem cell activation and hair follicle regeneration by regulating glycolysis.

BACKGROUND: Hair follicle stem cells (HFSCs) typically remain quiescent and are activated only during the transition from telogen to anagen to ensure that the hair follicle enters a new cycle. The metabolic behavior of stem cells in tissues is regulated by macroautophagy/autophagy, and changes in HFSC metabolism directly affect their activation and maintenance. However, the role of autophagy in the regulation of HFSC metabolism and function remains unclear. METHODS: Back skin samples were obtained from mice at different hair follicle cycle stages, and immunofluorescence staining was used to monitor autophagy in HFSCs. Mouse and human hair follicles were treated with rapamycin (Rapa, an autophagy activator) or 3-methyladenine (3-MA, an autophagy inhibitor). The effects of autophagy on the hair follicle cycle and HFSC were investigated by imaging, cell proliferation staining, and HFSC-specific marker staining. The influence and mechanism of autophagy on HFSC metabolism were explored using RNA sequencing, real-time polymerase chain reaction, immunohistochemical staining, and detection of lactate and glucose concentrations. Finally, the influence of autophagy-induced glycolysis on HFSC and the hair follicle cycle was verified by stem cell characteristics and in vivo functional experiments. RESULTS: Autophagy in HFSC was highest during the transition from telogen to anagen. Inhibiting autophagy with 3-MA led to early entry into catagen and prolonged telogen, whereas Rapa promoted autophagy and hair growth. Autophagy activated HFSC by increasing the expression and activity of HFSC lactate dehydrogenase (Ldha), thereby transforming HFSC metabolism into glycolysis. Inhibition of Ldha expression counteracted the effects of autophagy. CONCLUSIONS: Autophagy activated HFSC by promoting the transition from HFSC metabolism to glycolysis, ultimately initiating the hair follicle cycle and promoting hair growth.

Lonicerin promotes wound healing in diabetic rats by enhancing blood vessel regeneration through Sirt1-mediated autophagy.

Among the numerous complications of diabetes mellitus, diabetic wounds seriously affect patients' quality of life and result in considerable psychological distress. Promoting blood vessel regeneration in wounds is a crucial step in wound healing. Lonicerin (LCR), a bioactive compound found in plants of the Lonicera japonica species and other honeysuckle plants, exhibits anti-inflammatory and antioxidant activities, and it recently has been found to alleviate ulcerative colitis by enhancing autophagy. In this study we investigated the efficacy of LCR in treatment of diabetic wounds and the underlying mechanisms. By comparing the single-cell transcriptomic data from healing and non-healing states in diabetic foot ulcers (DFU) of 5 patients, we found that autophagy and SIRT signaling activation played a crucial role in mitigating inflammation and oxidative stress, and promoting cell survival in wound healing processes. In TBHP-treated human umbilical vein endothelial cells (HUVECs), we showed that LCR alleviated cell apoptosis, and enhanced the cell viability, migration and angiogenesis. Furthermore, we demonstrated that LCR treatment dose-dependently promoted autophagy in TBHP-treated HUVECs by upregulating Sirt1 expression, and exerted its anti-apoptotic effect through the Sirt1-autophagy axis. Knockdown of Sirt1 significantly decreased the level of autophagy, and mitigated the anti-apoptotic effect of LCR. In a STZ-induced diabetic rat model, administration of LCR significantly promoted wound healing, which was significantly attenuated by Sirt1 knockdown. This study highlights the potential of LCR as a therapeutic agent for the treatment of diabetic wounds and provides insights into the molecular mechanisms underlying its effects.

Sorting Technology for Mesenchymal Stem Cells from a Single Tissue Source.

Mesenchymal stem cells (MSCs) are adult stem cells that can be obtained, enriched and proliferated in vitro. They owned enormous potential in fields like regenerative medicine, tissue engineering and immunomodulation. However, though isolated from the same origin, MSCs are still essentially heterogeneous cell populations with different phenotypes and functions. This heterogeneity of MSCs significantly affects their therapeutic efficacy and brings obstacles to scientific research. Thus, reliable sorting technology which can isolate or purify MSC subpopulations with various potential and differentiation pathways is urgently needed. This review summarized principles, application status and clinical implications for these sorting methods, aiming at improving the understanding of MSC heterogeneity as well as providing fresh perspectives for subsequent clinical applications.

The homing of exogenous hair follicle mesenchymal stem cells into hair follicle niches.

Hair loss is a debilitating condition associated with the depletion of dermal papilla cells (DPCs), which can be replenished by dermal sheath cells (DSCs). Hence, strategies aimed at increasing the populations of DPCs and DSCs hold promise for the treatment of hair loss. In this study, we demonstrated in mice that introduced exogenous DPCs and DSCs (hair follicle mesenchymal stem cells) could effectively migrate and integrate into the dermal papilla and dermal sheath niches, leading to enhanced hair growth and prolonged anagen phases. However, the homing rates of DPCs and DSCs were influenced by various factors, including recipient mouse depilation, cell passage number, cell dose, and immune rejection. Through in vitro and in vivo experiments, we also discovered that the CXCL13/CXCR5 pathway mediated the homing of DPCs and DSCs into hair follicle niches. This study underscores the potential of cell-based therapies for hair loss by targeted delivery of DPCs and DSCs to their respective niches and sheds light on the intriguing concept that isolated mesenchymal stem cells can home back to their original niche microenvironment.

Hair follicle extraction combined with an expanded scalp flap for facial organ reconstruction.

BACKGROUND: Use of scalp skin for facial organ reconstruction represents a mainstream procedure for organ reconstruction. In most cases, adequate amounts of skin can be obtained by using tissue expanders, but harvesting sufficient scalp tissue in patients with low hairlines is challenging. Hair follicular unit extraction (FUE) is one approach to resolve this problem. With FUE, hair follicles are removed from the scalp skin, which can then be prepared as a donor site to obtain sufficient amounts of hairless skin. OBJECTIVES: To evaluate the safety and efficacy of FUE when combined with an expanded scalp flap for facial organ reconstruction. MATERIAL AND METHODS: Patients with low hairlines requiring facial organ reconstruction were selected for this study. The area of skin extension and hair removal were determined prior to surgery, a process which was performed in three stages. Stage I consisted of hair follicle removal using the FUE technique at the donor site. Stage II involved expander implantation using water injections. In Stage III facial organ reconstruction was completed. RESULTS: With the use of the FUE technique, hair follicles from the donor scalp were thoroughly removed and the donor scalp tissue was successfully expanded. Postoperatively, no evident scar formation at the reconstruction site or contracture of the expanded flap was observed. All patients were satisfied with the outcome of their reconstruction procedure. CONCLUSION: FUE provides a means for hair follicle removal from the donor site and can be employed to achieve a safe and effective procedure for facial reconstruction in patients with low hairlines.

A clinical trial of treating androgenic alopecia with mesenchymal stem cell suspension derived from autologous hair follicle.

BACKGROUND: Androgenic alopecia (AGA) is characterized by progressive hair follicle miniaturization, and novel treatments are needed to intervene in the miniaturization process. We aimed to evaluate the efficacy, safety, effectiveness, and effective population of autologous hair follicle mesenchymal stem cell therapy for the treatment of advanced AGA in Chinese people. METHODS: 50 patients ranging from 25 to 45 years old, with an average age of 32 ± 1.24 years were included. None of them had ever used minoxidil, finasteride, or other drugs to promote hair growth. Healthy hair follicles were extracted from the occipital area and treated to obtain hair follicle mesenchymal stem cells suspensions. The recipient sites were divided into two groups. Nine points were injected in a 1 cm 2 area, and 100 µl of solution containing either 1 × 10 5 cells or normal saline was injected at each point. The follow-up duration was 9 months. Observers were blinded to patient groupings and measurements. RESULTS: An increased proportion of terminal hair and hair shaft diameter was observed in the experimental group at 1 month; the effect lasted until 3 months. The hair thickening effect of advanced miniaturized hair follicles with hair shaft diameter less than 60 µm was more notable than that above 60 µm. None of the patients experienced any obvious side effects. CONCLUSIONS: Hair follicle mesenchymal stem cells were effective in the treatment of Chinese advanced AGA, and a hair shaft diameter of 60µm can be used as a key index to predict the effectiveness of the therapy.

An optimized force-triggered density gradient sedimentation method for isolation of pelage follicle dermal papilla cells from neonatal mouse skin.

BACKGROUND: The dermal papilla cells are a specialized population of mesenchymal cells located at the base of the hair follicle (HF), which possess the capacity to regulate HF morphogenesis and regeneration. However, lack of cell-type specific surface markers restricts the isolation of DP cells and application for tissue engineering purposes. METHODS: We describe a novel force-triggered density gradient sedimentation (FDGS) method to efficiently obtain purified follicular DP-spheres cells from neonatal mouse back skin, utilizing only centrifugation and optimized density gradients. RESULTS: Expression of characteristic DP cell markers, alkaline phosphatase, ß-catenin, versican, and neural cell adhesion molecules, were confirmed by immunofluorescence. Further, the patch assays demonstrated that DP cells maintained their hair regenerative capacity in vivo. Compared with current methods, including microdissection and fluorescence-activated cell sorting, the FDGS technique is simpler and more efficient for isolating DP cells from neonatal mouse skin. CONCLUSIONS: The FDGS method will improve the research potential of neonatal mouse pelage-derived DP cells for tissue engineering purposes.

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.

Cellular Heterogeneity Facilitates the Functional Differences Between Hair Follicle Dermal Sheath Cells and Dermal Papilla Cells: A New Classification System for Mesenchymal Cells within the Hair Follicle Niche.

Mesenchymal stem cells (MSCs) are known for their self-renewal and multi-lineage differentiation potential, with these cells often being evaluated in the regulation and maintenance of specific cellular niches including those of the hair follicle. Most mesenchymal stem cells in the hair follicles are housed in the dermal papilla (DP) and dermal sheath (DS), with both niches characterized by a broad variety of cellular subsets. However, while most previous studies describing the hair follicle mesenchymal niche treated all DP and DS cells as Hair Follicle Mesenchymal Stem Cells (HF-MSCs), the high number of cellular subsets would suggest that these cells are actually too heterogenous for such a broad definition. Given this we designed this study to evaluate the differentiation processes in these cells and used this data to create a new set of classifications for DP and DS cells, dividing them into "hair follicle mesenchymal stem cells (HF-MSCs)", "hair follicle mesenchymal progenitor cells (HF-MPCs)", and "hair follicle mesenchymal functional cells (HF-MFCs)". In addition, those cells that possess self-renewal and differentiation were re-named hair follicle derived mesenchymal multipotent cells (HF-MMCs). This new classification may help to further our understanding of the heterogeneity of hair follicle dermal cells and provide new insights into their evaluation.

Ficoll density gradient sedimentation isolation of pelage hair follicle mesenchymal stem cells from adult mouse back skin: a novel method for hair follicle mesenchymal stem cells isolation.

BACKGROUND: Hair follicle mesenchymal stem cells (HF-MSCs) have great potential for cell therapy. Traditional method to isolate whisker HF-MSC is time-consuming and few in cell numbers. How to quickly and conveniently obtain a large number of HF-MSC for experimental research is a problem worth exploring. METHODS: Two-step Ficoll Density Gradient Sedimentation (FDGS) was performed to isolate pelage HF-MSC from adult mice. The characteristic of the isolated cells was identified and compared with whisker HF-MSC by immunofluorescence staining, flow cytometry, three-lineage differentiation and hair follicle reconstruction. Pelage HF-MSC and exosomes were injected into the dorsal skin of mice as well as hair follicle organ culture to explore its role in promoting hair growth. The cells and exosomes distribution were located by immunofluorescence staining. RESULTS: Isolated pelage HF-MSC expressed similar markers (ALP, Versican, NCAM, Nestin), showed similar growth pattern, possessed similar mesenchymal stem cells function and hair follicle induction ability as whisker HF-MSC. A large number of cells can be obtained with fewer mice compared to traditional method. Injected pelage HF-MSC promoted hair growth by secreting exosomes. CONCLUSION: A large number of Pelage HF-MSC can be isolated by FDGS, which can promote hair growth by secreting exosomes which may target the dermal papilla and hair matrix region of host hair follicle.

Theoretical basis and clinical practice for FUE megasession hair transplantation in the treatment of large area androgenic alopecia.

BACKGROUND: Large area androgenic alopecia patients seeking hair transplantation treatment has become common. FUE Megasession has become a choice for more and more people. Long-term in vitro preservation of hair follicles during FUE Megasession has become a new challenge. OBJECTIVE: To explore optimal in vitro preservation condition according to FUE Megasession long-period surgery time and to perform clinical practice to confirm the feasibility. METHODS: Human follicles were obtained from informed patients by FUE Megasession and preserved under different conditions. Live and dead staining with DAPI was used to assess the survival rate of cells. Hair follicles were preserved in vitro for 7 days under different conditions, and the extension of the hair shaft was observed. We also performed some clinical procedures to illustrate the effectiveness of these methods. RESULTS: Under the condition of 4℃ Ringer's solution, the death rate of hair follicle cells was lower than that of the rest. 4℃ Ringer's solution supported superior growth of the hair follicle unit according to organ culture. 8-h preservation in 4℃ Ringer's solution was kept as high survival rate as the traditional hair transplantation surgery（P > .05）. Clinical procedures confirmed the feasibility of FUE Megasession hair transplantation surgery. CONCLUSION: 4℃ Ringer's solution in vitro preservation is optimal for clinical FUE Megasession surgery which ensures the hair follicle survival rate and postoperative results.

The effect of hyperbaric oxygen therapy combined with hair transplantation surgery for the treatment of alopecia.

BACKGROUND: Transplanted hair follicles suffer from various injuries, which are difficult to prevent. Hyperbaric oxygen therapy (HBOT) was reported to be an excellent procedure to promote capillary regeneration and reduce ischemia-reperfusion injury. AIM: To evaluate the clinical efficacy of HBOT as an adjuvant therapy for hair transplantation surgery. METHODS: Thirty-four patients with II-IV alopecia were divided into the control group and HBOT group randomly. The control group was treated with routine FUE procedure, while HBOT group combined with HBOT. Patients were treated with 100% oxygen under 2.0 atmospheres absolute pressure for 60 minutes through a facemask during HBOT and take the therapy daily for 7 days continuously after operation. Satisfaction and clinical improvement were evaluated at the fourth week and the sixth month postoperatively. RESULTS: Itching and folliculitis were significantly decreased in HBOT group (11.8% vs 35.3%). In addition, HBOT resulted in a lower postoperative shedding rate (27.6 ± 2.6% vs 69.1 ± 2.4%); nevertheless, the survival rate at 9 months showed no significant difference between HBOT (96.9 ± 0.5%) and control (93.8 ± 0.6%). The early postoperative satisfaction in control group was much lower than HBOT group (52.9% vs 88.2%), whereas all patients showed satisfaction with the final result. CONCLUSION: Hyperbaric oxygen therapy is able to minimize the postsurgical follicle shedding and lead to less folliculitis and itching, which provides evidence for HBOT to act as an adjuvant therapy for hair transplantation surgery.

A novel model for designing and performing parietal whorl hair transplantation.

BACKGROUND: Hair transplantation is the gold standard method for the treatment of alopecia. Recently, demand for the surgery has increased rapidly. However, high demands also require more professional training for the surgeons. With the maturing of hairline design skills, parietal whorl design has become the next challenge and no appropriate training models have yet been reported. AIMS: To present an innovative, simple, and appropriate training model for designing the scalp parietal whorl. METHODS: Outward divergent arc curves were drawn from the navel of an orange. Recipient sites were created with 23G needles or 1.0-mm knives or 1.0-mm blades according to the outward divergent arc curves, then hair shafts were transplanted using forceps. RESULTS: Different densities of hair shafts were transplanted. Transplantation of 10-20 follicular units (FU)/cm2 was too sparse, 30 FU/cm2 achieved a good appearance, but 40-50 FU/cm2 was better. Through practicing on the model, excellent results of parietal whorl transplantation on patients can be achieved. CONCLUSIONS: This new model can perfectly suit the lines and direction of the parietal whorl and also present different density transplantation practicing. We provide a training method for new surgeons as well as a rehearsal opportunity for previously trained doctors.

Ameliorative effects of indomethacin at different concentrations on endothelial insulin resistance through two distinct pathways.

Indomethacin (IDMT), a non-selective inhibitor of cycloxygenase-2 (COX-2), plays important roles in anti-inflammation and analgesia and it is commonly used to treat the patients with rheumatic and rheumatoid arthritis. Besides, various literatures reported that IDMT is a synthetic ligand of peroxisome proliferator activated receptor gamma (PPARγ). Rosiglitazone (RSG), an insulin-sensitizer, is also a synthetic ligand and applied clinically to cure the patients with type 2 diabetes mellitus. However, up to date little is known about whether IDMT ameliorates endothelial insulin resistance (IR). Accordingly, the purpose of this study is to investigate the effects of IDMT on endothelial IR and its underlying mechanism. Our present results showed that IDMT improved the endothelial IR induced by high glucose and fat concentration (HG/HF) in a concentration and time-dependent manner. Intriguingly, we further identified that 0.25 mM of IDMT noticeably induced the expression levels of PPARγ, AKT and endothelial nitric oxide synthase (eNOS) but failed to notably reverse the increases in expression levels of COX-2, inhibitory κB kinase (IKK) and tumor necrosis factor alpha (TNFα) induced by HG/HF; whereas 1.0 mM of IDMT exerted opposite effects compared with 0.25 mM of IDMT. Therefore, we conclude that IDMT ameliorates the endothelial IR induced by HG/HF through two distinct pathways, i.e., a lower concentration of IDMT through a PPARγ-AKT-eNOS pathway while a higher concentration mainly via an IKK-COX-2/TNFα pathway. The findings might provide a novel clinical use for IDMT to cure IR-related disorders.

Epigallocatechin-3-gallate suppresses differentiation of adipocytes via regulating the phosphorylation of FOXO1 mediated by PI3K-AKT signaling in 3T3-L1 cells.

Epigallocatechin-3-gallate (EGCG) is a pivotal effective component of green tea. It is known that EGCG has antioxidant activity, anti-angiogenesis, anti-tumor, cardiovascular protection and blood lipid regulation functions. Forkhead box-O1 (FOXO1) is one of the downstream signals of protein kinase B (AKT) and takes part in adipogenesis. The purpose of this study is to investigate the effects of EGCG on adipose differentiation and the likely mechanisms. 3T3-L1 cells were induced by DMI for 2, 4, 6 and 8 days, respectively. During induction, the cells were treated with EGCG (5 µM, 10 µM, 50 µM and 100 µM) or DMSO for the first 2 days. In addition, another batch of 3T3-L1cells were treated with SC-3036 (PI3K activator, 10 µM), or LY294002 (PI3K inhibitor, 10 µM) alone or combined with EGCG (100 µM) for the indicated times. Medium glucose concentration, lipid accumulation, the levels of TNF-α, resistin, adiponectin and leptin and the expression of FOXO1, phosphorylated-FOXO1 (P-FOXO1), PPARγ, fatty acid synthase (FAS) were detected, respectively. The present study demonstrated that EGCG inhibited glucose uptake, lipid accumulation and adipokine secretion in a concentration-dependent manner during adipogenesis, which suggests that EGCG inhibits adipocyte's differentiation, maturation and functions. Moreover, EGCG also down-regulated the expression levels of PPARγ and P-FOXO1. Conversely, the PI3K activator reversed these changes caused by EGCG, suggesting that the inhibitory effects of EGCG may be mediated by PI3K-AKT-FOXO1 pathway to negatively regulate the expression of PPARγ. The findings will provide a solid foundation for EGCG to prevent and cure the obesity-associated diseases.