Solitary Basaloid Follicular Hamartoma: A Report of Two Cases.

Basaloid follicular hamartoma (BFH) is rare benign follicular malformation that is often clinically misdiagnosed. Patients with BFH demonstrate a variety of clinical manifestations and associated abnormalities. BFH may be a familial, congenital, or acquired condition with localized or generalized distribution. Several clinical variants of generalized BFH are known, and they can be associated with a diverse spectrum of abnormalities. Herein, we report two cases of solitary BFH in pediatric patients, both documented dermoscopically.

Advances in polymeric nano-delivery systems targeting hair follicles for the treatment of acne.

Acne is a common chronic inflammatory disorder of the sebaceous gland in the hair follicle. Commonly used external medications cause skin irritation, and the transdermal capacity is weak, making it difficult to penetrate the cuticle skin barrier. Hair follicles can aid in the breakdown of this barrier. As nanomaterials progress, polymer-based nanocarriers are routinely used for hair follicle drug delivery to treat acne and other skin issues. Based on the physiological and anatomical characteristics of hair follicles, this paper discusses factors affecting hair follicle delivery by polymer nanocarriers, summarizes the common combination technology to improve the targeting of hair follicles by carriers, and finally reviews the most recent research progress of different polymer nanodrug-delivery systems for the treatment of acne by targeting hair follicles.

Efficient Generation of Pancreatic Progenitor Cells from Induced Pluripotent Stem Cells Derived from a Non-Invasive and Accessible Tissue Source-The Plucked Hair Follicle.

The advent of induced pluripotent stem cell (iPSC) technology has brought about transformative advancements in regenerative medicine, offering novel avenues for disease modeling, drug testing, and cell-based therapies. Patient-specific iPSC-based treatments hold the promise of mitigating immune rejection risks. However, the intricacies and costs of producing autologous therapies present commercial challenges. The hair follicle is a multi-germ layered versatile cell source that can be harvested at any age. It is a rich source of keratinocytes, fibroblasts, multipotent stromal cells, and the newly defined Hair Follicle-Associated Pluripotent Stem Cells (HAP). It can also be obtained non-invasively and transported via regular mail channels, making it the ideal starting material for an autologous biobank. In this study, cryopreserved hair follicle-derived iPSC lines (HF-iPS) were established through integration-free vectors, encompassing a diverse cohort. These genetically stable lines exhibited robust expression of pluripotency markers, and showcased tri-lineage differentiation potential. The HF-iPSCs effectively differentiated into double-positive cKIT+/CXCR4+ definitive endoderm cells and NKX6.1+/PDX1+ pancreatic progenitor cells, affirming their pluripotent attributes. We anticipate that the use of plucked hair follicles as an accessible, non-invasive cell source to obtain patient cells, in conjunction with the use of episomal vectors for reprogramming, will improve the future generation of clinically applicable pancreatic progenitor cells for the treatment of Type I Diabetes.

Upregulation of caveolae-associated structural proteins in the hair follicle bulge of lichen planopilaris and frontal fibrosing alopecia.

Lichen planopilaris (LPP) and frontal fibrosing alopecia (FFA) are primary cicatricial alopecia that cause a major impact on quality of life due to irreversible hair loss and symptoms as itching, burning and pain. They are characterized by permanent loss of hair follicle stem cells (HFSCs) by pathomechanisms still poorly understood, resulting in poor efficacy of currently available treatments. Caveolae are flask-shaped lipid rafts invaginated within the plasma membrane of multiple cell types. Although their role in the HF physiology and pathophysiology is relatively unknown, we have previously demonstrated that the primary structural component of caveolae (caveolin-1 or Cav1) is upregulated in FFA. Thus, we propose to investigate the expression and localization of caveolae-associated structural proteins (Cav1, Cav2, and Cavin-1) and HFSCs (identified by K15) in both LPP and FFA. We analyzed 4 patients with LPP biopsied in affected and non-affected (NA) scalp, 4 patients with FFA biopsied in affected scalp and 4 healthy controls. Affected scalp of LPP and FFA demonstrated increased levels of Cav1 and Cavin-1 compared with HC and LPP-NA. Moreover, Cav1, Cav2 and Cavin1 all exhibit high colocalization with K15 and their expression appears to be negatively correlated, supporting the hypothesis that these proteins are important players in LPP/FFA and may serve as therapeutic targets in future treatments.

Combination of Autophagy and Stem Cell Enhancing Properties of Natural Product Extracts in Human Dermal Papilla Stem Cells.

BACKGROUND/AIM: Dermal papilla (DP) stem cells are known for their remarkable regenerative capacity, making them a valuable model for assessing the effects of natural products on cellular processes, including stemness, and autophagy. MATERIALS AND METHODS: Autophagy and stemness characteristics were assessed using real-time RT-PCR to analyze mRNA levels, along with immunofluorescence and western blot techniques for protein level evaluation. RESULTS: Butterfly Pea, Emblica Fruits, Kaffir Lime, and Thunbergia Laurifolia extracts induced autophagy in DP cells. Kaffir Lime-treated cells exhibited increase in the OCT4, NANOG, and SOX2 mRNA (6-, 5, and 5.5-fold, respectively), and protein levels (4-, 3-, and 1.5-fold, respectively). All extracts activated the survival protein kinase B (Akt) in DP cells. CONCLUSION: Natural products are a promising source for promoting hair growth by rejuvenating hair stem cells.

Antibacterial toner exhibits bactericidal effect against Cutibacterium acnes via keratin and sebum plug penetration.

Cutibacterium acnes is an opportunistic pathogen recognized as a contributing factor to acne vulgaris. The accumulation of keratin and sebum plugs in hair follicles facilitates C. acnes proliferation, leading to inflammatory acne. Although numerous antimicrobial cosmetic products for acne-prone skin are available, their efficacy is commonly evaluated against planktonic cells of C. acnes. Limited research has assessed the antimicrobial effects on microorganisms within keratin and sebum plugs. This study investigates whether an antibacterial toner can penetrate keratin and sebum plugs, exhibiting bactericidal effects against C. acnes. Scanning electron microscopy and next-generation sequencing analysis of the keratin and sebum plug suggest that C. acnes proliferate within the plug, predominantly in a biofilm-like morphology. To clarify the potential bactericidal effect of the antibacterial toner against C. acnes inside keratin and sebum plugs, we immersed the plugs in the toner, stained them with LIVE/DEAD BacLight Bacterial Viability Kit to visualize microorganism viability, and observed them using confocal laser scanning microscopy. Results indicate that most microorganisms in the plugs were killed by the antibacterial toner. To quantitatively evaluate the bactericidal efficacy of the toner against C. acnes within keratin and sebum, we immersed an artificial plug with inoculated C. acnes type strain and an isolate collected from acne-prone skin into the toner and obtained viable cell counts. The number of the type strain and the isolate inside the artificial plug decreased by over 2.2 log and 1.2 log, respectively, showing that the antibacterial toner exhibits bactericidal effects against C. acnes via keratin and sebum plug penetration.

Possible role of ß-hydroxybutyrate in inducing inflammation in alopecia areata.

Alopecia areata (AA) is an autoimmune inflammatory disease characterized by non-scarring hair loss due to an immune response that targets hair follicles. The current treatment approach for AA involves the use of immunosuppressants and immunomodulators to reduce cytokine levels around affected hair follicles. Sodium-glucose cotransporter 2 (SGLT2) inhibitors have emerged as potential anti-inflammatory agents with diverse beneficial effects in various medical conditions. This study investigates the role of beta-hydroxybutyrate (BHB), a ketone body produced during SGLT2 inhibition, in the pathogenesis of AA. Serum BHB levels were found to be significantly elevated in patients with AA compared with healthy controls, with higher levels correlating with severity of hair loss. BHB treatment increased inflammatory cytokine production in outer root sheath (ORS) cells, mimicking the inflammatory conditions seen in AA. The results suggest that elevated BHB levels may exacerbate the inflammatory immune response in AA patients and may be associated with chronic hair loss and resistance to treatment. Serum BHB levels may serve as a potential marker of poor prognosis in patients with severe AA. Further research is needed to elucidate the precise role of BHB in the pathogenesis of AA and its implications for disease management.

Deciphering the Complex Immunopathogenesis of Alopecia Areata.

Alopecia areata (AA) is an autoimmune-mediated disorder in which the proximal hair follicle (HF) attack results in non-scarring partial to total scalp or body hair loss. Despite the growing knowledge about AA, its exact cause still needs to be understood. However, immunity and genetic factors are affirmed to be critical in AA development. While the genome-wide association studies proved the innate and acquired immunity involvement, AA mouse models implicated the IFN-γ- and cytotoxic CD8+ T-cell-mediated immune response as the main drivers of disease pathogenesis. The AA hair loss is caused by T-cell-mediated inflammation in the HF area, disturbing its function and disrupting the hair growth cycle without destroying the follicle. Thus, the loss of HF immune privilege, autoimmune HF destruction mediated by cytotoxic mechanisms, and the upregulation of inflammatory pathways play a crucial role. AA is associated with concurrent systemic and autoimmune disorders such as atopic dermatitis, vitiligo, psoriasis, and thyroiditis. Likewise, the patient's quality of life (QoL) is significantly impaired by morphologic disfigurement caused by the illness. The patients experience a negative impact on psychological well-being and self-esteem and may be more likely to suffer from psychiatric comorbidities. This manuscript aims to present the latest knowledge on the pathogenesis of AA, which involves genetic, epigenetic, immunological, and environmental factors, with a particular emphasis on immunopathogenesis.

Morphology of sweat glands in the skin of Saimaa (Pusa hispida saimensis) and Baltic ringed (Pusa hispida botnica) seals.

The endangered Saimaa ringed seal (Pusa hispida saimensis) is an endemic freshwater subspecies inhabiting Lake Saimaa in Finland. The Baltic ringed seal (Pusa hispida botnica) inhabits the brackish Baltic Sea, which is almost entirely landlocked. Recent research shows that Saimaa and Baltic ringed seals may be genetically even further apart from each other than from other ringed seal subspecies. We documented histologically the integument microstructure of Saimaa and Baltic ringed seals to determine whether the geographic and genetic isolation was manifested as variation in the integument microstructure of these subspecies adapted to icy aquatic environments. The skin structures of these subspecies were similar to those of other phocids. The association of the sweat glands with hair follicles in both subspecies suggested that they were small apocrine sweat glands described previously in terrestrial or aquatic mammals. None of the apocrine glands had large lumina, and some of the ducts were relatively straight and short. Further studies analysing the mode of secretion, for example, apocrine versus eccrine, in the sweat glands are necessary to confirm the types of sweat glands in seals.

Low-level laser activates Wnt/ß-catenin signaling pathway-promoting hair follicle stem cell regeneration and wound healing: Upregulate the expression of key downstream gene Lef 1.

BACKGROUND: The objective of this study is to investigate the mechanism by which low-level laser stimulation promotes the proliferation of intraepithelial hair follicle stem cells (HFSCs) in wounds. This research aims to expand the applications of laser treatment, enhance wound repair methods, and establish a theoretical and experimental foundation for achieving accelerated wound healing. METHODS: The experimental approach involved irradiating a cell model with low-level laser to assess the proliferation of HFSCs and examine alterations in the expression of proteins related to the Wnt/ß-catenin signaling pathway. A mouse back wound model was established to investigate the effects of low-level laser irradiation on wound healing rate, wound microenvironment, and the proliferation of HFSCs in relation to the Wnt/ß-catenin signaling pathway. RESULTS: The research findings indicate that low-level laser light effectively activates the Wnt signaling pathway, leading to the increased accumulation of core protein ß-catenin and the upregulation of key downstream gene Lef 1. Consequently, this regulatory mechanism facilitates various downstream biological effects, including the notable promotion of HFSC proliferation and differentiation into skin appendages and epithelial tissues. As a result, the process of wound healing is significantly accelerated. CONCLUSION: Low levels of laser activates the Wnt signalling pathway, promotes the regeneration of hair follicle stem cells and accelerates wound healing.

Mesenchymal Stem Cells from Mouse Hair Follicles Inhibit the Development of Type 1 Diabetes.

Mesenchymal stem cells (MSCs) are known for their immunosuppressive properties. Based on the demonstrated anti-inflammatory effect of mouse MSCs from hair follicles (moMSCORS) in a murine wound closure model, this study evaluates their potential for preventing type 1 diabetes (T1D) in C57BL/6 mice. T1D was induced in C57BL/6 mice by repeated low doses of streptozotocin. moMSCORS were injected intravenously on weekly basis. moMSCORS reduced T1D incidence, the insulitis stage, and preserved insulin production in treated animals. moMSCORS primarily exerted immunomodulatory effects by inhibiting CD4+ T cell proliferation and activation. Ex vivo analysis indicated that moMSCORS modified the cellular immune profile within pancreatic lymph nodes and pancreatic infiltrates by reducing the numbers of M1 pro-inflammatory macrophages and T helper 17 cells and upscaling the immunosuppressive T regulatory cells. The proportion of pathogenic insulin-specific CD4+ T cells was down-scaled in the lymph nodes, likely via soluble factors. The moMSCORS detected in the pancreatic infiltrates of treated mice presumably exerted the observed suppressive effect on CD4+ through direct contact. moMSCORS alleviated T1D symptoms in the mouse, qualifying as a candidate for therapeutic products by multiple advantages: non-invasive sampling by epilation, easy access, permanent availability, scalability, and benefits of auto-transplantation.

Combination of autologous FCS and MPG versus each technique alone in the treatment of stable vitiligo.

Follicular cell suspension (FCS) transplantation is a novel surgical method for treating resistant stable vitiligo, whereas mini punch grafting is an established effective method for treating stable vitiligo. The combination of FCS and mini punch grafting is a better strategy for the treatment of resistant stable vitiligo. The aim of the study was to evaluate the efficacy of follicular cell suspension, mini punch grafting, and a combination of both techniques in the treatment of stable vitiligo. This prospective comparative study was conducted on 48 patients with stable vitiligo. They were divided into three equal groups, including group A (treated with follicular cell suspension), group B (treated with mini punch grafting), and group C (treated with the combination of both techniques). All patients were followed-up for six months for the assessment of their therapeutic response regarding clinical outcomes. By comparing the data of the three studied groups, we found that the difference in the degree of re-pigmentation after one and three months of treatment was not significant. However, the progress of re-pigmentation was significantly different after six months of treatment among the three studied groups (P = 0.027). Specifically, re-pigmentation was significantly better in group C than in groups A and B (P = 0.037 and 0.017, respectively), but it was not significantly different between groups A and B.

Comprehensive transcriptome profiling between balding and non-balding scalp of female pattern hair loss in Asian.

While many gene expression studies have focused on male pattern baldness (MPB), few studies have investigated the genetic differences between bald and non-bald hair follicles in female pattern hair loss (FPHL). This study aimed to identify molecular biomarkers associated with FPHL through genetic analysis of paired bald and non-bald hair follicles from 18 FPHL patients, using next-generation sequencing (NGS) techniques. RNA transcriptome analysis was performed to identify differentially expressed genes (DEGs) between bald and non-bald hair follicles in FPHL. The DEGs were validated using real-time PCR, and protein expression was confirmed through immunohistochemistry and western blot analysis. Our findings suggest that HOXB13, SFRP2, PTGDS, CXCR3, SFRP4, SOD3, and DCN are significantly upregulated in bald hair follicles compared to non-bald hair follicles in FPHL. SFRP2 and PTGDS were found to be consistently highly expressed in bald hair follicles in all 18 samples. Additionally, elevated protein levels of SFRP2 and PTGDS were confirmed through western blot and immunohistochemical analysis. Our study identified SFRP2 and PTGDS as potential biomarkers for FPHL and suggests that they may play a role in inducing hair loss in this condition. These findings provide a foundation for further research on the pathogenesis of FPHL and potential therapeutic targets.

Hair Follicle-Targeted Delivery of Azelaic Acid Micro/Nanocrystals Promote the Treatment of Acne Vulgaris.

Purpose: Acne vulgaris is a chronic inflammatory skin disorder centered on hair follicles, making hair follicle-targeted delivery of anti-acne drugs a promising option for acne treatment. However, current researches have only focused on the delivering to healthy hair follicles, which are intrinsically different from pathologically clogged hair follicles in acne vulgaris. Patients and Methods: Azelaic acid (AZA) micro/nanocrystals with different particle sizes were prepared by wet media milling or high-pressure homogenization. An experiment on AZA micro/nanocrystals delivering to healthy hair follicles was carried out, with and without the use of physical enhancement techniques. More importantly, it innovatively designed an experiment, which could reveal the ability of AZA micro/nanocrystals to penetrate the constructed clogged hair follicles. The anti-inflammatory and antibacterial effects of AZA micro/nanocrystals were evaluated in vitro using a RAW264.7 cell model stimulated by lipopolysaccharide and a Cutibacterium acnes model. Finally, both the anti-acne effects and skin safety of AZA micro/nanocrystals and commercial products were compared in vivo. Results: In comparison to commercial products, 200 nm and 500 nm AZA micro/nanocrystals exhibited an increased capacity to target hair follicles. In the combination group of AZA micro/nanocrystals and ultrasound, the ability to penetrate hair follicles was further remarkably enhanced (ER value up to 9.6). However, toward the clogged hair follicles, AZA micro/nanocrystals cannot easily penetrate into by themselves. Only with the help of 1% salicylic acid, AZA micro/nanocrystals had a great potential to penetrate clogged hair follicle. It was also shown that AZA micro/nanocrystals had anti-inflammatory and antibacterial effects by inhibiting pro-inflammatory factors and Cutibacterium acnes. Compared with commercial products, the combination of AZA micro/nanocrystals and ultrasound exhibited an obvious advantage in both skin safety and in vivo anti-acne therapeutic efficacy. Conclusion: Hair follicle-targeted delivery of AZA micro/nanocrystals provided a satisfactory alternative in promoting the treatment of acne vulgaris.

Comprehensive analysis of the expression profiles of mRNA, lncRNA, circRNA, and miRNA in primary hair follicles of coarse sheep fetal skin.

BACKGROUND: The Qinghai Tibetan sheep, a local breed renowned for its long hair, has experienced significant deterioration in wool characteristics due to the absence of systematic breeding practices. Therefore, it is imperative to investigate the molecular mechanisms underlying follicle development in order to genetically enhance wool-related traits and safeguard the sustainable utilization of valuable germplasm resources. However, our understanding of the regulatory roles played by coding and non-coding RNAs in hair follicle development remains largely elusive. RESULTS: A total of 20,874 mRNAs, 25,831 circRNAs, 4087 lncRNAs, and 794 miRNAs were annotated. Among them, we identified 58 DE lncRNAs, 325 DE circRNAs, 924 DE mRNAs, and 228 DE miRNAs during the development of medullary primary hair follicle development. GO and KEGG functional enrichment analyses revealed that the JAK-STAT, TGF-ß, Hedgehog, PPAR, cGMP-PKG signaling pathway play crucial roles in regulating fibroblast and epithelial development during skin and hair follicle induction. Furthermore, the interactive network analysis additionally identified several crucial mRNA, circRNA, and lncRNA molecules associated with the process of primary hair follicle development. Ultimately, by investigating DEmir's role in the ceRNA regulatory network mechanism, we identified 113 circRNA-miRNA pairs and 14 miRNA-mRNA pairs, including IGF2BP1-miR-23-x-novel-circ-01998-MSTRG.7111.3, DPT-miR-370-y-novel-circ-005802-MSTRG.14857.1 and TSPEAR-oar-miR-370-3p-novel-circ-005802- MSTRG.10527.1. CONCLUSIONS: Our study offers novel insights into the distinct expression patterns of various transcription types during hair follicle morphogenesis, establishing a solid foundation for unraveling the molecular mechanisms that drive hair development and providing a scientific basis for selectively breeding desirable wool-related traits in this specific breed.

Activation of the integrated stress response in human hair follicles.

Unravelling how energy metabolism and stress responses are regulated in human scalp hair follicles could reveal novel insights into the controls of hair growth and provide new targets to manage hair loss disorders. The Mitochondrial Pyruvate Carrier (MPC) imports pyruvate, produced via glycolysis, into the mitochondria, fuelling the TCA cycle. Previous work has shown that MPC inhibition promotes lactate generation, which activates murine epithelial hair follicle stem cells (eHFSCs). However, by pharmacologically targeting the MPC in short-term human hair follicle ex vivo organ culture experiments using UK-5099, we induced metabolic stress-responsive proliferative arrest throughout the human hair follicle epithelium, including within Keratin 15+ eHFSCs. Through transcriptomics, MPC inhibition was shown to promote a gene expression signature indicative of disrupted FGF, IGF, TGFß and WNT signalling, mitochondrial dysfunction, and activation of the integrated stress response (ISR), which can arrest cell cycle progression. The ISR, mediated by the transcription factor ATF4, is activated by stressors including amino acid deprivation and ER stress, consistent with MPC inhibition within our model. Using RNAScope, we confirmed the upregulation of both ATF4 and the highly upregulated ATF4-target gene ADM2 on human hair follicle tissue sections in situ. Moreover, treatment with the ISR inhibitor ISRIB attenuated both the upregulation of ADM2 and the proliferative block imposed via MPC inhibition. Together, this work reveals how the human hair follicle, as a complex and metabolically active human tissue system, can dynamically adapt to metabolic stress.

Formulation development of tazarotene-loaded PLGA nanoparticles for follicular delivery in the treatment of inflammatory skin diseases.

Tazarotene is a widely prescribed topical retinoid for acne vulgaris and plaque psoriasis and is associated with skin irritation, dryness, flaking, and photosensitivity. In vitro permeation of tazarotene was studied across the dermatomed human and full-thickness porcine skin. The conversion of tazarotene to the active form tazarotenic acid was studied in various skin models. Tazarotene-loaded PLGA nanoparticles were prepared using the nanoprecipitation technique to target skin and hair follicles effectively. The effect of formulation and processing variables on nanoparticle properties, such as particle size and drug loading, was investigated. The optimized nanoparticle batches with particle size <500 µm were characterized further for FT-IR analysis, which indicated no interactions between tazarotene and PLGA. Scanning electron microscopy analysis showed uniform, spherical, and non-agglomerated nanoparticles. In vitro release study using a dialysis membrane indicated a sustained release of 40-70 % for different batches over 36 h, following a diffusion-based release mechanism based on the Higuchi model. In vitro permeation testing (IVPT) in full-thickness porcine skin showed significantly enhanced follicular and skin delivery from nanoparticles compared to solution. The presence of tazarotenic acid in the skin from tazarotene nanoparticles indicated the effectiveness of nanoparticle formulations in retaining bioconversion ability and targeting follicular delivery.

Oxidative stress in hair follicle development and hair growth: Signalling pathways, intervening mechanisms and potential of natural antioxidants.

Hair follicle development and hair growth are regulated by multiple factors and multiple signalling pathways. The hair follicle, as an important skin appendage, is the basis for hair growth, and it has the functions of safeguarding the body, perceiving the environment and regulating body temperature. Hair growth undergoes a regular hair cycle, including anagen, catagen and telogen. A small amount of physiological shedding of hair occurs under normal conditions, always in a dynamic equilibrium. Hair loss occurs when the skin or hair follicles are stimulated by oxidative stress, inflammation or hormonal disorders that disrupt the homeostasis of the hair follicles. Numerous researches have indicated that oxidative stress is an important factor causing hair loss. Here, we summarize the signalling pathways and intervention mechanisms by which oxidative stress affects hair follicle development and hair growth, discuss existing treatments for hair loss via the antioxidant pathway and provide our own insights. In addition, we collate antioxidant natural products promoting hair growth in recent years and discuss the limitations and perspectives of current hair loss prevention and treatment.

Hair-follicle associated pluripotent (HAP)-cell-sheet implantation enhanced wound healing in diabetic db/db mice.

Diabetes often results in chronic ulcers that fail to heal. Effective treatment for diabetic wounds has not been achieved, although stem-cell-treatment has shown promise. Hair-follicle-associated-pluripotent (HAP)-stem-cells from bulge area of mouse hair follicle have been shown to differentiate into keratinocytes, vascular endothelial cells, smooth muscle cells, and some other types of cells. In the present study, we developed HAP-cell-sheets to determine their effects on wound healing in type-2 diabetes mellitus (db/db) C57BL/6 mouse model. Flow cytometry analysis showed cytokeratin 15 expression in 64% of cells and macrophage expression in 3.6% of cells in HAP-cell-sheets. A scratch cell migration assay in vitro showed the ability of fibroblasts to migrate and proliferate was enhanced when co-cultured with HAP-cell-sheets. To investigate in vivo effects of the HAP-cell-sheets, they were implanted into 10 mm circular full-thickness resection wounds made on the back of db/db mice. Wound closure was facilitated in the implanted group until day 16. The thickness of epithelium and granulation tissue volume at day 7 were significantly increased by the implantation. CD68 positive area and TGF-ß1 positive area were significantly increased; meanwhile, iNOS positive area was reduced at day 7 in the HAP-cell-sheets implanted group. After 21 days, CD68 positive areas in the implanted group were reduced to under the control group level, and TGF-ß1 positive area had no difference between the two groups. These observations strongly suggest that the HAP-cell-sheets implantation is efficient to facilitate early macrophage activity and to suppress inflammation level. Using immuno-double-staining against CD34 and α-SMA, we found more vigorous angiogenesis in the implanted wound tissue. The present results suggest autologous HAP-cell-sheets can be used to heal refractory diabetic ulcers and have clinical promise.