Cellulose nanocrystal-stabilized Pickering emulsion gels as vehicles for follicular delivery of minoxidil.

Minoxidil (MXD) is the only topical over-the-counter medicine approved by the United States Food and Drug Administration for the treatment of androgenetic alopecia. For the purpose of targeting the delivery of MXD to dermal papilla in the hair follicle, MXD Pickering emulsion gels were fabricated based on the designability of deep eutectic solvent (DES) and the versatility of cellulose nanocrystal (CNC) and sodium carboxymethyl cellulose (CMC-Na). Structural studies and theoretical calculations results suggest that CNC can stabilize the interface between the MXD-DES and water, leading to the formation of Pickering emulsions. The rheological properties and stabilities of MXD Pickering emulsions were enhanced through gelation using CMC-Na, which highlights the good compatibility and effectiveness of natural polysaccharides in emulsion gels. Due to the particle size of emulsion droplets (679 nm) and the rheological properties of emulsion gel, the fabricated MXD formulations show in vivo hair regrowth promotion and hair follicle targeting capabilities. Interestingly, the MXD Pickering emulsion-based formulations exert therapeutic effects by upregulating the expression of hair growth factors. The proposed nanodrug strategy based on supramolecular strategies of CNC and CMC-Na provides an interesting avenue for androgenetic alopecia treatment.

Stimulation by exosomes from hypoxia-preconditioned hair follicle mesenchymal stem cells facilitates mitophagy by inhibiting the PI3K/AKT/mTOR signaling pathway to alleviate ulcerative colitis.

Background: Ulcerative colitis (UC) is an intestinal inflammatory disease that is strongly associated with mitochondrial damage and dysfunction as well as mitophagy and lacks of satisfactory treatments. Hair follicle mesenchymal stem cell (HF-MSC)-derived exosomes owe benefit effectiveness on inflammatory therapies. Hypoxia-preconditioned HF-MSCs exhibit enhanced proliferation and migration abilities, and their exosomes exert stronger effects than normal exosomes. However, the therapeutic function of Hy-Exos in UC is unknown. Methods: The inflammation model was established with LPS-treated MODE-K cells, and the mouse UC model was established by dextran sulfate sodium (DSS) administration. The therapeutic effects of HF-MSC-derived exosomes (Exos) and hypoxia-preconditioned HF-MSC-derived exosomes (Hy-Exos) were compared in vitro and in vivo. Immunofluorescence staining and western blotting were used to explore the effects of Hy-Exos on mitochondrial function, mitochondrial fission and fusion and mitophagy. MiRNA sequencing analysis was applied to investigate the differences in components between Exos and Hy-Exos. Results: Hy-Exos had a better therapeutic effect on LPS-treated MODE-K cells and DSS-induced UC mice. Hy-Exos promoted colonic tight junction proteins expression, suppressed the oxidative stress response, and reduced UC-related inflammatory injury. Hy-Exos may exert these effects via miR-214-3p-mediated inhibition of the PI3K/AKT/mTOR signaling pathway, maintenance of mitochondrial dynamic stability, alleviation of mitochondrial dysfunction and enhancement of mitophagy. Conclusion: This study revealed a vital role for Hy-Exos in suppressing inflammatory progression in UC and suggested that miR-214-3p is a potential critical target for Hy-Exos in alleviating UC.

Ontogeny of Skin Stem Cells and Molecular Underpinnings.

Skin stem cells (SCs) play a pivotal role in supporting tissue homeostasis. Several types of SCs are responsible for maintaining and regenerating skin tissue. These include bulge SCs and others residing in the interfollicular epidermis, infundibulum, isthmus, sebaceous glands, and sweat glands. The emergence of skin SCs commences during embryogenesis, where multipotent SCs arise from various precursor populations. These early events set the foundation for the diverse pool of SCs that will reside in the adult skin, ready to respond to tissue repair and regeneration demands. A network of molecular cues regulates skin SC behavior, balancing quiescence, self-renewal, and differentiation. The disruption of this delicate equilibrium can lead to SC exhaustion, impaired wound healing, and pathological conditions such as skin cancer. The present review explores the intricate mechanisms governing the development, activation, and differentiation of skin SCs, shedding light on the molecular signaling pathways that drive their fate decisions and skin homeostasis. Unraveling the complexities of these molecular drivers not only enhances our fundamental knowledge of skin biology but also holds promise for developing novel strategies to modulate skin SC fate for regenerative medicine applications, ultimately benefiting patients with skin disorders and injuries.

Skin Development and Disease: A Molecular Perspective.

Skin, the largest organ in the human body, is a crucial protective barrier that plays essential roles in thermoregulation, sensation, and immune defence. This complex organ undergoes intricate processes of development. Skin development initiates during the embryonic stage, orchestrated by molecular cues that control epidermal specification, commitment, stratification, terminal differentiation, and appendage growth. Key signalling pathways are integral in coordinating the development of the epidermis, hair follicles, and sweat glands. The complex interplay among these pathways is vital for the appropriate formation and functionality of the skin. Disruptions in multiple molecular pathways can give rise to a spectrum of skin diseases, from congenital skin disorders to cancers. By delving into the molecular mechanisms implicated in developmental processes, as well as in the pathogenesis of diseases, this narrative review aims to present a comprehensive understanding of these aspects. Such knowledge paves the way for developing innovative targeted therapies and personalised treatment approaches for various skin conditions.

Pilomatrixoma of the Ankle: A Case Report.

Pilomatrixoma, also known as calcifying epithelioma of Malherbe, is a rare benign skin tumor originating from hair follicle matrix cells. It typically presents as a firm, painless subcutaneous nodule, most commonly found in the head, neck, and upper extremities. Pilomatrixoma can occasionally appear in atypical locations, posing a diagnostic challenge due to its nonspecific clinical presentation. A 43-year-old female presented with a painless, slowly enlarging mass on the lateral side of her left ankle, which had been present for approximately one year. Physical examination revealed a firm, well-circumscribed subcutaneous nodule measuring about 2 cm in diameter with normal overlying skin. An MRI of the left ankle demonstrated a well-circumscribed, subcutaneous mass with heterogeneous signal intensity, consistent with calcifications, suggesting pilomatrixoma. A fine-needle aspiration biopsy confirmed the presence of basaloid cells, shadow cells, and areas of calcification. The lesion was surgically excised, and histopathological examination validated the diagnosis of pilomatrixoma. The patient had an uneventful postoperative course, with no recurrence at the six-month follow-up. This case underscores the importance of considering pilomatrixoma in the differential diagnosis of subcutaneous nodules, even in unusual locations. A comprehensive diagnostic approach, including clinical evaluation, imaging, and histopathological examination, is essential for an accurate diagnosis. Surgical excision with clear margins is the treatment of choice, ensuring low recurrence rates and excellent patient outcomes. This report enhances the understanding of pilomatrixoma and highlights the necessity for a multimodal diagnostic strategy in managing this rare condition effectively.

Differentiation of cultured hair follicle neural crest stem cells into functional melanocytes.

Many autologous melanocytes are required for surgical treatment of depigmentation diseases such as vitiligo. However, primary cultured melanocytes have a limited number of in vitro passages. The production of functional epidermal melanocytes from stem cells provides an unprecedented source of cell therapy for vitiligo. This study explores the clinical application of melanocytes induced by hair follicle neural crest stem cells (HFNCSCs). This study established an in vitro differentiation model of HFNCSCs into melanocytes. Results demonstrate that most differentiated melanocytes expressed the proteins C-KIT, MITF, S-100B, TYRP1, TYRP2, and tyrosinase. The HFNCSC-derived melanocytes were successfully transplanted onto the dorsal skin of mice and survived in the local tissues, expressing marker protein of melanocytes. In conclusion, HFNCSCs in mice can be induced to differentiate into melanocytes under specific conditions. These induced melanocytes exhibit the potential to facilitate repigmentation in the lesion areas of vitiligo-affected mice, suggesting a promising avenue for therapeutic intervention.

A case of congenital pili multigemini.

Pili multigemini is an uncommon hair follicle disorder, where multiple hair shafts emerge from the same follicular opening. While it is commonly observed in men's facial hair, it has also been reported, albeit infrequently, in other areas of the body. Here we describe an unusual case of congenital pili multigemini presenting on the eyebrow of a female infant.

MicroRNA expression profiles reveal wool development and fineness regulation in Gansu alpine fine-wool sheep.

The development of wool has a complex regulatory mechanism both influenced by genetic and environmental factors. MicroRNAs (miRNA) were involved in various biological processes of animals, and may play an important role in the regulation of wool development. In this study, we comprehensively analyzed and identified the histological parameters of hair follicles, as well as the miRNAs, target genes, pathways, and Gene Ontology terms related to wool fineness regulation and wool growth and development using HE staining and RNA-Seqs methods. Both coarse (group C, mean fiber diameter (MFD) = 22.26 ± 0.69 µm, n = 6) and fine (group F, MFD = 16.91 ± 0.29 µm, n = 6) of Gansu alpine fine-wool sheep with different wool fineness were used in this study. The results showed that the primary follicle diameter and secondary wool fiber diameter in group C were significantly higher than those in group F (P < 0.05). And the number of primary and secondary hair follicles in group C was significantly lower than that in group F (P < 0.05). Furthermore, a total of 67 DE miRNAs and 290 potential DE miRNAs target genes were screened in the skin tissues of sheep from groups F and C, and some potential target genes related to wool fineness regulation were screened, such as CDH2, KRT82, FOXN1, LOC101106296, KRT20, MCOLN3, KRT71, and TERT. These genes were closely related to Glutathione metabolism, epidermal cell differentiation, keratinization, and regulation of hair cycle. Moreover, the regulatory network of miRNAs-mRNAs suggested that miRNAs (miR-129-x, novel m0079-3p, miR-2484-z, novel m0025-5P, etc.) may play a key role in the wool development and wool fineness regulation of Gansu alpine fine-wool sheep. In summary, this study expands the existing miRNAs database and provides new information for studying the regulation of wool development in Gansu alpine fine wool sheep.

Hair follicle punch grafts in hard-to-heal wounds: A monocenter study and patient survey.

Background and Aims: Chronic wounds present a growing challenge in the aging population, resulting in an extended course of treatment and an increased influx of patients with recalcitrant wounds seeking admission to hospitals. Furthermore, the general trend in patient care is toward simple and inexpensive treatment methods, feasible in an outpatient setting with little material costs. This retrospective case study aims to elucidate the operative procedure and outcomes associated with hair follicle unit transplantation utilizing punch grafts from the scalp for the management of hard-to-heal wounds. Methods: A cohort of 28 patients, comprising 20 males and eight females, with a mean age of 72.61 years (range: 48-89) and an average wound area of 82.49 cm2, underwent dissection of punch grafts containing hair follicles (2-3 mm in diameter) from the scalp. Subsequently, these grafts were transplanted into the wound bed. The retrospective evaluation of ulcer healing encompassed photo documentation and clinical records, while patient satisfaction was assessed through structured questionnaires. Results: In 78.6% (22) of the cases, a favorable impact on wound healing was observed, characterized by epithelization, and in 57.1% (16) of the patients, complete wound closure was achieved. With the exception of one donor site, all other sites healed without complications. The patient survey indicated that the majority of individuals subjected to the procedure did not perceive it as painful or time-consuming. Notably, 81% (17) of individuals expressed a willingness to undergo the treatment again. Even in challenging wound conditions, such as pyoderma gangrenosum, our method demonstrated a positive effect on wound healing. Conclusion: Within our cohort, the utilization of hair follicle units in the management of hard-to-heal wounds resulted in either complete or partial wound closure for the majority of patients, accompanied by minimal morbidity, reduced operation time, and a low incidence of complications and associated costs.

An induced pluripotent stem cell-based approach for hair follicle development and regeneration.

Because hair loss is a common concern for many individuals, potential regenerative therapies of hair follicles have been extensively researched. Induced pluripotent stem cells (iPSCs) are a promising avenue for hair follicle regeneration. This review explores current iPSC-based approaches and highlights their potential applications and challenges in hair restoration. The principles of iPSC technology, iPSC differentiation into hair follicle precursor cells, and potential clinical implications for hair follicle regeneration are also discussed. This overview of iPSCs and their applications aims to contribute to our understanding of their role in hair restoration and potential future therapeutic applications.

Role of Csdc2 in Regulating Secondary Hair Follicle Growth in Cashmere Goats.

Cashmere goats possess two types of hair follicles, with the secondary hair follicles producing valuable cashmere fiber used for textiles. The growth of cashmere exhibits a seasonal pattern arising from photoperiod change. Transcription factors play crucial roles during this process. The transcription factor, cold-shock domain, containing C2 (Csdc2) plays a crucial role in modulating cell proliferation and differentiation. Our preceding research indicated that the expression of Csdc2 changes periodically during anagen to telogen. However, the mechanisms of Csdc2 in regulating SHF growth remain unclear. Here, we found that the knockdown of Csdc2 inhibits the proliferation of dermal papilla cells. ChIP-Seq analysis showed that Csdc2 had a unique DNA binding motif in SHFs. Through conjoint analysis of ChIP-Seq and RNA-Seq, we revealed a total of 25 candidate target genes of Csdc2. Notably, we discovered a putative Csdc2 binding site within roundabout guidance receptor 2 (Robo2) on chromosome 1 of the goat genome. Furthermore, qRT-PCR and dual-luciferase reporter assay confirmed Csdc2's positive regulatory influence on Robo2. These findings expand the research field of hair follicle transcriptional regulatory networks, offering insights into molecular breeding strategies to enhance cashmere production in goats.

Follicular unit grafting in chronic ulcers: a valuable technique for integrated management

Abstract Chronic ulcers significantly affect the quality of life of patients and impose a high cost on the healthcare system. The therapeutic management should be comprehensive, taking into consideration the etiological diagnosis of the wound and the characteristics of the wound bed when deciding on a therapeutic proposal appropriate to the healing phase, correcting factors that delay healing. During the epithelialization phase, repair techniques with grafts are recommended to shorten re-epithelialization time, improve the quality of scar tissue, and achieve adequate pain management. Currently, due to the reported benefits of skin appendages, the technique of follicular unit auto-grafting obtained with a scalp punch is among the chosen strategies for wound repair. This is a minimally invasive, outpatient practice, whose technique has advantages over the donor site, patients recovery and well-being.

Stimulation of mouse hair regrowth by exosomes derived from human umbilical cord mesenchymal stem cells.

BACKGROUND: There is an urgent need for new treatments to solve hair loss problem. As mesenchymal stem cells were proved to have effects on promoting tissue repair and regeneration, in which the exosome plays a vital role, we aim to investigate the influence of umbilical cord mesenchymal stem cells exosome (UCMSC-Exos) on hair growth and its mechanism. METHODS: The hUCMSC-Exos were extracted by ultracentrifugation. Primary fibroblasts were cultured with or without hUCMSC-Exos and cell proliferation was evaluated by CCK-8 assay. C57BL/6 mice model of depilation-induced hair regrowth was treated with either hUCMSC-Exos (200 µg/mL) or PBS on one side of the dorsal back. Real time quantitative PCR, flow cytometry analysis, immunohistochemistry and Immunofluorescent staining were used to analyze the regulative effect of hUCMSC-Exos on hair follicle stem/progenitor cells and Wnt/ß-catenin pathway. RESULTS: The proliferation of fibroblasts incubated with hUCMSC-Exos at the concentration of 200 µg/mL was greater than other groups. Treatment with hUCMSC-Exos resulted in rapid reentry into anagen. Hair follicle stem/progenitor cell markers (K15, Lgr5, Lgr6, CD34 and Lrig1) and Wnt/ß-catenin pathway related factors (Wnt5, Lef1, Lrp5 and ß-catenin) were increased in hUCMSC-Exos-injected region. CONCLUSION: hUCMSC-Exos promote fibroblasts proliferation and accelerate mouse hair regrowth by upregulating hair follicle stem/progenitor cell and Wnt/ß-catenin pathway, which suggests potential therapeutic approaches for hair loss disorders.

Combining multiomics to analyze the molecular mechanism of hair follicle cycle change in cashmere goats from Inner Mongolia.

Sheep body size can directly reflect the growth rates and fattening rates of sheep and is also an important index for measuring the growth performance of meat sheep.Inner Mongolia Cashmere Goat is a local excellent breed of cashmere and meat dual-purpose, which is a typical heterogeneous indumentum. The hair follicles cycle through periods of vigorous growth (anagen), a regression caused by apoptosis (catagen), and relative rest (telogen). At present, it is not clear which genes affect the cycle transformation of hair follicles and unclear how proteins impact the creation and expansion of hair follicles.we using multi-omics joint analysis methodologies to investigated the possible pathways of transformation and apoptosis in goat hair follicles. The results showed that 917,1,187, and 716 proteins were specifically expressed in anagen, catagen andtelogen. The result of gene ontology (GO) annotation showed that differentially expressed proteins (DEPs) are in different growth cycle periods, and enriched GO items are mostly related to the transformation of cells and proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment result indicated that the apoptosis process has a great impact on hair follicle's growth cycle. The results of the protein interaction network of differential proteins showed that the ribosomal protein family (RPL4, RPL8, RPS16, RPS18, RPS2, RPS27A, RPS3) was the core protein in the network. The results of combined transcriptome and proteomics analysis showed that there were 16,34, and 26 overlapped DEGs and DEPs in the comparison of anagen VS catagen, catagen VS telogen and anagen VS telogen, of which API5 plays an important role in regulating protein and gene expression levels. We focused on API5 and Ribosomal protein and found that API5 affected the apoptosis process of hair follicles, and ribosomal protein was highly expressed in the resting stage of hair follicles. They are both useful as molecular marker candidate genes to study hair follicle growth and apoptosis,and they both have an essential function in the cycle transition process of hair follicles. The results of this study may provide a theoretical basis for further research on the growth and development of hair follicles in Inner Mongolian Cashmere goats.

Fabrication of heterocellular spheroids with controllable core-shell structure using inertial focusing effect for scaffold-free 3D cell culture models.

Three-dimensional (3D) cell culture models capable of emulating the biological functions of natural tissues are pivotal in tissue engineering and regenerative medicine. Despite progress, the fabrication ofin vitroheterocellular models that mimic the intricate structures of natural tissues remains a significant challenge. In this study, we introduce a novel, scaffold-free approach leveraging the inertial focusing effect in rotating hanging droplets for the reliable production of heterocellular spheroids with controllable core-shell structures. Our method offers precise control over the core-shell spheroid's size and geometry by adjusting the cell suspension density and droplet morphology. We successfully applied this technique to create hair follicle organoids, integrating dermal papilla cells within the core and epidermal cells in the shell, thereby achieving markedly enhanced hair inducibility compared to mixed-structure models. Furthermore, we have developed melanoma tumor spheroids that accurately mimic the dynamic interactions between tumor and stromal cells, showing increased invasion capabilities and altered expressions of cellular adhesion molecules and proteolytic enzymes. These findings underscore the critical role of cellular spatial organization in replicating tissue functionalityin vitro. Our method represents a significant advancement towards generating heterocellular spheroids with well-defined architectures, offering broad implications for biological research and applications in tissue engineering.

Hair follicle sulfotransferase activity and effectiveness of oral minoxidil in androgenetic alopecia.

BACKGROUND: Androgenetic alopecia (AGA) is common. While topical minoxidil remains the only FDA-approved therapeutic for AGA, its efficacy is limited in stimulating clinically significant hair regrowth over the longer term. Oral minoxidil, which is used off-label, is a promising alternative; however, its effectiveness and underlying mechanisms warrant further investigation. AIMS: To elucidate the site of action and infer the physiological mechanisms underlying therapeutic responses to oral minoxidil in patients with AGA. METHODS: Forty-one patients with AGA underwent 6 months of low-dose oral minoxidil treatment. Minoxidil sulfotransferase (SULT) activity was assayed in plucked scalp hair follicles. The primary outcome was hair growth after low-dose oral minoxidil treatment for a minimum of 6 months, and the secondary outcome was SULT activity in hair follicles. RESULTS: After 6 months of treatment, 26 (63.4%) patients experienced a clinical improvement in alopecia symptoms. The response rate was higher in men (19/26 [73.1%]) than in women (6/15 [40.0%]). Patients with low hair follicle SULT activity demonstrated a higher minoxidil response rate than those with high enzyme activity (85% vs. 43%, p = 0.009). CONCLUSIONS: Our findings indicate that low SULT activity within the hair follicles is associated with a favorable response to oral minoxidil therapy in patients with AGA. Further elucidation of the underlying mechanisms could significantly improve personalized therapeutic approaches through improved patient selection and the rational design of adjuvant treatments.

Pilose antler extracts promotes hair growth in androgenetic alopecia mice by activating hair follicle stem cells via the AKT and Wnt pathways.

Background: Angrogenetic alopecia (AGA) is one of the most prevalent hair loss disorders worldwide. The hair follicle stem cell (HFSC) is closely related to the formation of hair follicle (HF) structure and HF self-renewal. The activation of HFSC in AGA is critical for hair growth. Pilose antler has been reported to have hair growth-promoting activity, but the mechanism of action on AGA and HFSC has not been reported. Methods: We previously extracted an active component from the pilose antler known as PAEs. In this study, we conducted experiments using AGA mice and HFSC. The effects of PAEs on hair growth in AGA mice were firstly detected, and then the mechanisms of PAEs for AGA were predicted by integrating network pharmacology and de novo transcriptomics data of pilose antler. Finally, biological experiments were used to validate the molecular mechanism of PAEs in treating AGA both in vivo and in vitro. Results: It was found that PAEs promoted hair regrowth by accelerating the activation of anagen, delaying the anagen-catagen transition. It also alleviated the morphological changes, such as hair shortening, thinning, miniaturization, and HF number reduction, and regulated the hair regeneration process of four subtypes of hair. We further found that PAEs could promote the proliferation of HFSC, outer root sheath (ORS) cells, and hair bulb cells in AGA mice. We then integrated network pharmacology and pilose antler transcriptomics data to predict that the mechanism of PAEs treatment in AGA mice is closely related to the PI3K-AKT/Wnt-ß-Catenin pathways. Subsequently, it was also verified that PAEs could activate both pathways in the skin of AGA mice. In addition, we found that PAEs perhaps increased the number of blood vessels around dermal papilla (DP) in experiments in vivo. Meanwhile, the PAEs stimulated the HFSC proliferation in vitro and activated the AKT and Wnt pathways. However, the proliferative activity of HFSC was inhibited after blocking the Wnt pathway and AKT activity. Conclusion: This study suggests that the hair growth-promoting effect of PAEs in AGA mice may be closely related to the stimulation of the AKT and Wnt pathways, which in turn activates the proliferation of HFSC.

Dynamic duo: Cell-extracellular matrix interactions in hair follicle development and regeneration.

Ectodermal organs, such as hair follicles, originate from simple epithelial and mesenchymal sheets through a complex developmental process driven by interactions between these cell types. This process involves dermal condensation, placode formation, bud morphogenesis, and organogenesis, and all of these processes require intricate interactions among various tissues. Recent research has emphasized the crucial role of reciprocal and dynamic interactions between cells and the extracellular matrix (ECM), referred to as the "dynamic duo", in the development of ectodermal organs. These interactions provide spatially and temporally changing biophysical and biochemical cues within tissues. Using the hair follicle as an example, this review highlights two types of cell-ECM adhesion units-focal adhesion-type and hemidesmosome-type adhesion units-that facilitate communication between epithelial and mesenchymal cells. This review further explores how these adhesion units, along with other cell-ECM interactions, evolve during hair follicle development and regeneration, underscoring their importance in guiding both developmental and regenerative processes.

The value of immunohistochemical expression of SOX9 and CD34 in alopecia areata.

BACKGROUND: Alopecia areata (AA), an immune-mediated disorder, is marked by temporary, nonscarring hair loss. The bulge area is protected from immune attacks by immune privilege; however, recent studies demonstrated immune cells infiltrating the bulge area. OBJECTIVE: This study aims to investigate the immunohistochemical expression of the sex-determining region Y-box 9 (SOX9) and cluster of differentiation 34 (CD34) in AA patients as markers of hair follicle stem cells (HFSCs) and progenitor cells, respectively. METHODS: Immunohistochemical staining of SOX9 and CD34 was applied on skin samples of 20 AA patients and 20 healthy controls. RESULTS: SOX9 and CD34 were significantly lower in lesional samples of cases compared to perilesional and control skin biopsies. Furthermore, SOX9 level was negatively correlated with the severity of alopecia tool score (SALT score) among the studied AA patients. Moreover, lowered SOX9 expression was present in patients with recurrent attacks. CONCLUSIONS: The significant reduction of stem cell markers (SOX9 and CD34) in our studied AA cases signifies the pathological affection of HFSCs and their progeny in AA. This is thought to cause a loss of competence in generating new hair in some AA cases, which needs to be validated in further research. LIMITATIONS OF THE STUDY: This study has a small sample size.