Single-dose of integrated bilayer microneedles for enhanced hypertrophic scar therapy with rapid anti-inflammatory and sustained inhibition of myofibroblasts.

Hypertrophic scar (HS) tends to raised above skin level with high inflammatory microenvironment and excessive proliferation of myofibroblasts. The HS therapy remains challenging due to dense scar tissue which makes it hard to penetrate, and the side effects resulting from intralesional corticosteroid injection which is the mainstay treatment in clinic. Herein, bilayer microneedle patches combined with dexamethasone and colchicine (DC-MNs) with differential dual-release pattern is designed. Two drugs loaded in commercially available materials HA and PLGA, respectively. Specifically, after administration, outer layer rapidly releases the anti-inflammatory drug dexamethasone, which inhibits macrophage polarization to pro-inflammatory phenotype in scar tissue. Subsequently, inner layer degrades sustainedly, releasing antimicrotubular agent colchicine, which suppresses the overproliferation of myofibroblasts with extremely narrow therapeutic window, and inhibits the overexpression of collagen, as well as promotes the regular arrangement of collagen. Only applied once, DC-MNs directly delivered drugs to the scar tissue. Compared to traditional treatment regimen, DC-MNs significantly suppressed HS at lower dosage and frequency by differential dual-release design. Therefore, this study put forward the idea of integrated DC-MNs accompany the development of HS, providing a non-invasive, self-applicable, more efficient and secure strategy for treatment of HS.

CircRNA_SLC8A1 alleviates hypertrophic scar progression by mediating the Nrf2-ARE pathway.

BACKGROUND: Hypertrophic scar (HS) is associated with cosmetic defects, mobility, and functional impairments, pruritus, and pain. Previous circRNA microarray analysis identified reduced expression of circRNA_SLC8A1 in HS tissues. Therefore, this study aims to investigate the role of circRNA_SLC8A1 in modulating the abnormal behavior of HS-derived fibroblasts (HSFs) in vitro. METHODS: RT-qPCR and FISH assays were used to assess the differential expression and localization of circRNA_SLC8A1 in normal and HS tissues. Following modulation of circRNA_SLC8A1 expression, CCK-8, flow cytometry, Transwell, and wound healing assays were employed to evaluate the effects of circRNA_SLC8A1 on the biological behaviors of HSFs. The Starbase database, dual-luciferase reporter assays, and Ago2-RIP assays were utilized to predict and validate the interaction between circRNA_SLC8A1 and downstream miRNAs. RESULTS: CircRNA_SLC8A1 was found to be downregulated in HS tissues and was primarily localized in the cytoplasm. Overexpression of circRNA_SLC8A1 reduced cell viability, cell invasion, wound healing, and the expression of Vimentin, N-cadherin, Col I, and Col III, while enhancing apoptosis and E-cadherin expression in HSFs. CircRNA_SLC8A1 activates the Nrf2-ARE pathway by competitively binding to miRNA-27a-3p. miRNA-27a-3p and Nrf2 exhibited high and low expression, respectively in HS tissues, with an inverse correlation between their levels. Overexpression of miRNA-27a-3p counteracted the effects of circRNA_SLC8A1 in HSF proliferation, apoptosis, migration, EMT, collagen deposition, and Nrf2-ARE pathway activity. CONCLUSION: CircRNA_SLC8A1 inhibits the proliferation, migration, EMT, and collagen deposition of HSF through competitive binding with miRNA-27a-3p, thereby activating the Nrf2-ARE pathway. The circRNA_SLC8A1/miRNA-27a-3p/Nrf2-ARE axis may offer a promising molecular target for HS therapy.

NIR-II light based combinatorial management of hypertrophic scar by inducing autophagy in fibroblasts.

The hypertrophic scar (HS) is a prevalent cutaneous fibrotic disorder that impacts both the aesthetic and functional aspects of the skin, there is an urgent need for a highly safe and effective approach to address the challenge of HS with thick and deep types. Inspired by the superior deep tissue penetrative ability of near-infrared-II (NIR-II) light and potential mitochondria ROS inducing effect of Chinese medicine lycorine (LYC), we fabricated a Cu2Se@LYC (CL) composite by encapsulating LYC on polyvinyl pyrrolidone (PVP) modified Cu2Se nanoparticles. After NIR-II irradiation, CL could induce the generation of reactive oxygen species (ROS) and mitochondrial damage in hypertrophic scar fibroblasts (HSFs). The subsequent release of cytochrome C (cyt-c) from mitochondria into the cytoplasm and upregulation of beclin1 leads to the activation of endogenous apoptosis and autophagy-mediated cell death. The CL + NIR-II treatment exhibited a pronounced anti-scarring effect in both in vitro and in vivo rabbit ear scar models, leading to a significant reduction in the fibrotic markers including Collagen I/III and α-smooth muscle actin (α-SMA). This study comprehensively investigated the crucial role of HSFs' autophagy in scar management and proposed a safe and effective therapy based on NIR-II laser for clinical application.

Comparison of Normal Human Skin and Hypertrophic Scar Tissue Samples of Different Ages, Locations, and Stages of Maturity.

BACKGROUND: Scars disrupt the normal structure and function of the skin. The primary goal of plastic surgery is to prevent and reduce scarring. Therefore, we aimed to establish a comparison scheme between normal skin (NS) tissues of different ages and locations; hypertrophic scars (HTS) of different ages, locations, and maturities; and NS and HTS tissues to provide evidence on scar severity for improving treatment evaluation. METHODS: Various methods including histology, immunohistochemistry, and immunofluorescence were employed to compare the general appearance, macrophage infiltration, fibroblast activity, degree of angiogenesis, and collagen fiber type and arrangement in human-sourced NS and HTS tissues of different ages, locations, and maturities in seven patients (three with NS and four with HTS) from the Department of Burn and Plastic Surgery of the Shandong Provincial Hospital from January 2019 to December 2020. RESULTS: The thicknesses of the epidermis and dermis of NS tissues varied with age and location. The epidermis of the upper arms, face, and upper eyelids of NS tissues sequentially thickened, whereas the dermis was sequentially thinner. Several glandular structures were identified in the upper eyelids but rarely in the face and upper arms. Histological changes in HTS tissue of different ages, locations, and maturity occur as scar formation time is prolonged, accompanied by increased CD86 levels and fibrosis. As the scar matured, connexin and VEGFR2 expression decreased, indicating reduced inflammation, fibroblast activity, and angiogenesis. The comparison between NS and HTS tissue also revealed significant differences; the positive expression of VEGFR2 and total collagen in HTS tissue was higher than that in NS tissue. CONCLUSIONS: We discovered significant differences among NS, HTS, and NS and HTS tissues of different ages, locations, and maturities. Further, this study may provide a basis for clarifying the treatment effect of different methods for HTS compared with those for NS, efficiently individualizing patients' treatment plans and ultimately shortening the scar treatment process.

Exosomal miR­194 from adipose­derived stem cells impedes hypertrophic scar formation through targeting TGF­ß1.

Hypertrophic scars, which result from aberrant fibrosis and disorganized collagen synthesis by skin fibroblasts, emerge due to disrupted wound healing processes. These scars present significant psychosocial and functional challenges to affected individuals. The current treatment limitations largely arise from an incomplete understanding of the underlying mechanisms of hypertrophic scar development. Recent studies, however, have shed light on the potential of exosomal non­coding RNAs interventions to mitigate hypertrophic scar proliferation. The present study assessed the impact of exosomes derived from adipose­derived stem cells (ADSCs­Exos) on hypertrophic scar formation using a rabbit ear model. It employed hematoxylin and eosin staining, Masson's trichrome staining and immunohistochemical staining techniques to track scar progression. The comprehensive analysis of the present study encompassed the differential expression of non­coding RNAs, enrichment analyses of functional pathways, protein­protein interaction studies and micro (mi)RNA­mRNA interaction investigations. The results revealed a marked alteration in the expression levels of long non­coding RNAs and miRNAs following ADSCs­Exos treatment, with little changes observed in circular RNAs. Notably, miRNA (miR)­194 emerged as a critical regulator within the signaling pathways that govern hypertrophic scar formation. Dual­luciferase assays indicated a significant reduction in the promoter activity of TGF­ß1 following miR­194 overexpression. Reverse transcription­quantitative PCR and immunoblotting assays further validated the decrease in TGF­ß1 expression in the treated samples. In addition, the treatment resulted in diminished levels of inflammatory markers IL­1ß, TNF­α and IL­10. In vivo evidence strongly supported the role of miR­194 in attenuating hypertrophic scar formation through the suppression of TGF­ß1. The present study endorsed the strategic use of ADSCs­Exos, particularly through miR­194 modulation, as an effective strategy for reducing scar formation and lowering pro­inflammatory and fibrotic indicators such as TGF­ß1. Therefore, the present study advocated the targeted application of ADSCs­Exos, with an emphasis on miR­194 modulation, as a promising approach to managing proliferative scarring.

Alpha-boswellic acid accelerates acute wound healing via NF-κB signaling pathway.

BACKGROUND: Boswellic acids (BAs) showed promising effects in cancer treatment, immune response regulation, and anti-inflammatory therapy. We aimed to assess the roles of alpha-BA (α-BA) in treating acute wound healing. METHODS: In vivo wound-healing models were established to evaluate the therapeutic effects of α-BA. Cell assays were conducted to assess the impact of α-BA on cellular biological functions. Western blot analysis was employed to validate the potential mechanisms of action of α-BA. RESULTS: Animal models indicated that wound healing was notably accelerated in the α-BA group compared to the control group (P < 0.01). Hematoxylin and eosin (HE) staining and enzyme-linked immunosorbent assay (ELISA) assay preliminarily suggested that α-BA may accelerate wound healing by inhibiting excessive inflammatory reactions and increasing the protein levels of growth factors. Cell function experiments demonstrated that α-BA suppressed the proliferation and migration ability of human hypertrophic scar fibroblasts (HSFBs), thereby favoring wound healing. Additionally, α-BA exerted a significant impact on cell cycle progression. Mechanistically, the protein levels of key genes in nuclear factor kappa beta (NF-κB) signaling pathway, including cyclin D1, p65, IκBα, and p-IκBα, were downregulated by α-BA. CONCLUSIONS: α-BA demonstrated the ability to counteract the abnormal proliferation of skin scar tissues, consequently expediting wound healing. These findings suggest its potential for development as a new agent for treating acute wound healing.

Adipose-derived mesenchymal stem cells suppress fibroblast proliferation of hypertrophic scar through CCL5 and CXCL12.

BACKGROUND AND OBJECTIVE: Adipose-derived mesenchymal stem cells (ADSCs) can accelerate wound healing, reduce scar formation, and inhibit hypertrophic scar (HTS). ADSCs can secrete a large amount of CCL5, and CCL5 has been proved to be pro-inflammatory and pro-fibrotic. CXCL12 (SDF-1) is a key chemokine that promotes stem cell migration and survival. Therefore, this study selected normal skin and HTS conditioned medium to simulate different microenvironments, and analyzed the effects of different microenvironments on the expression of CCL5 and CXCL12 in human ADSCs (hADSCs). MATERIALS AND METHODS: hADSCs with silenced expression of CCL5 and CXCL12 were co-cultured with hypertrophic scar fibroblasts to verify the effects of CCL5 and CXCL12 in hADSCs on the proliferation ability of hypertrophic scar fibroblasts. A mouse model of hypertrophic scar was established to further confirm the effect of CCL5 and CXCL12 in hADSCs on hypertrophic scar formation. RESULTS: CCL5 level was found to be significantly high in hADSCs cultured in HTS conditioned medium. CXCL12 in HTS group was prominently lowly expressed compared with the normal group. Inhibition of CCL5 in hADSCs enhanced the effects of untreated hADSCs on proliferation of HTS fibroblasts while CXCL12 knockdown exerted the opposite function. Inhibition of CCL5 in hADSCs increased the percentage of HTS fibroblasts in the G0/G1 phase while down-regulation of CXCL12 decreased those. Meanwhile, the down-regulated levels of fibroblast markers including collagen I, collagen III, and α-SMA induced by CCL5 knockdown were significantly up-regulated by CXCL12 inhibition. hADSCs alleviate the HTS of mice through CCL5 and CXCL12. CONCLUSION: In summary, our results demonstrated that hADSCs efficiently cured HTS by suppressing proliferation of HTS fibroblasts, which may be related to the inhibition of CXCL12 and elevation of CCL5 in hADSCs, suggesting that hADSCs may provide an alternative therapeutic approach for the treatment of HTS.

The effect of inflammatory cytokines on the risk of hypertrophic scar: a mendelian randomization study.

Hypertrophic scar (HS) results from burns or trauma, causing aesthetic and functional issues. However, observational studies have linked inflammatory cytokines to HS, but the causal pathways involved are unclear. We aimed to determine how circulating inflammatory cytokines contribute to HS formation. Two-sample Mendelian randomization (MR) was used to identify genetic variants associated with hypertrophic scar in a comprehensive, publicly available genome-wide association study (GWAS) involving 766 patients and 207,482 controls of European descent. Additionally, data on 91 plasma proteins were drawn from a GWAS summary involving 14,824 healthy participants. Causal relationships between exposures and outcomes were investigated primarily using the inverse variance weighted (IVW) method. Furthermore, a suite of sensitivity analyses, including MR‒Egger and weighted median approaches, were concurrently employed to fortify the robustness of the conclusive findings. Finally, reverse MR analysis was conducted to evaluate the plausibility of reverse causation between hypertrophic scar and the cytokines identified in our study. In inflammatory cytokines, there was evidence of inverse associations of osteoprotegerin(OPG) levels(OR = 0.59, 95% CI = 0.41 ∼ 0.85, p = 0.01), and leukemia inhibitory factor(LIF) levels(OR = 0.51, 95% CI = 0.32 ∼ 0.82, p = 0.01) are a nominally negative association with hypertrophic scar risk, while CUB domain-domain-containing protein 1(CDCP1) level(OR = 0.59, 95% CI = 0.41 ∼ 0.85, p = 0.01) glial cell line-derived neurotrophic factor(GDNF) levels(OR = 1.42, 95% CI = 1.03 ∼ 1.96, p = 0.01) and programmed cell death 1 ligand 1(PD-L1) levels(OR = 1.47, 95% CI = 1.92 ∼ 2.11, p = 0.04) showed a positive association with hypertrophic scar risk. These associations were similar in the sensitivity analyses. According to our MR findings, OPG and LIF have a protective effect on hypertrophic scar, while CDCP1, GDNF, and PD-L1 have a risk-increasing effect on Hypertrophic scar. Our study adds to the current knowledge on the role of specific inflammatory biomarker pathways in hypertrophic scar. Further validation is needed to assess the potential of these cytokines as pharmacological or lifestyle targets for hypertrophic scar prevention and treatment.

HOXC10 promotes hypertrophic scar fibroblast fibrosis through the regulation of STMN2 and the TGF-ß/Smad signaling pathway.

The pathophysiology of hypertrophic scar (HS) shares similarities with cancer. HOXC10, a gene significantly involved in cancer development, exhibits higher expression levels in HS than in normal skin (NS), suggesting its potential role in HS regulation. And the precise functions and mechanisms by which HOXC10 influences HS require further clarification. Gene and protein expressions were analyzed using raeal-time quantitative polymerase chain reaction (RT-qPCR) and western blot techniques. Cell proliferation and migration were evaluated using EdU proliferation assays, CCK-8 assays, scratch assays, and Transwell assays. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were conducted to investigate the interactions between HOXC10 and STMN2. HOXC10 and STMN2 expression levels were significantly higher in HS tissues compared with NS tissues. Silencing HOXC10 led to decreased activation, proliferation, migration, and fibrosis in hypertrophic scar fibroblasts (HSFs). Our findings also indicate that HOXC10 directly targets STMN2. The promotional effects of HOXC10 knockdown on HSF activation, proliferation, migration, and fibrosis were reversed by STMN2 overexpression. We further demonstrated that HOXC10 regulates HSF activity through the TGF-ß/Smad signaling pathway. HOXC10 induces the activation and fibrosis of HSFs by promoting the transcriptional activation of STMN2 and engaging the TGF-ß/Smad signaling pathway. This study suggests that HOXC10 could be a promising target for developing treatments for HS.

How to Keep Myofibroblasts under Control: Culture of Mouse Skin Fibroblasts on Soft Substrates.

During the physiological healing of skin wounds, fibroblasts recruited from the uninjured adjacent dermis and deeper subcutaneous fascia layers are transiently activated into myofibroblasts to first secrete and then contract collagen-rich extracellular matrix into a mechanically resistant scar. Scar tissue restores skin integrity after damage but comes at the expense of poor esthetics and loss of tissue function. Stiff scar matrix also mechanically activates various precursor cells into myofibroblasts in a positive feedback loop. Persistent myofibroblast activation results in pathologic accumulation of fibrous collagen and hypertrophic scarring, called fibrosis. Consequently, the mechanisms of fibroblast-to-myofibroblast activation and persistence are studied to develop antifibrotic and prohealing treatments. Mechanistic understanding often starts in a plastic cell culture dish. This can be problematic because contact of fibroblasts with tissue culture plastic or glass surfaces invariably generates myofibroblast phenotypes in standard culture. We describe a straight-forward method to produce soft cell culture surfaces for fibroblast isolation and continued culture and highlight key advantages and limitations of the approach. Adding a layer of elastic silicone polymer tunable to the softness of normal skin and the stiffness of pathologic scars allows to control mechanical fibroblast activation while preserving the simplicity of conventional 2-dimensional cell culture.

Exosomes Derived from Hypertrophic Scar Fibroblasts Suppress Melanogenesis in Normal Human Epidermal Melanocytes.

Post-burn hypertrophic scars often exhibit abnormal pigmentation. Exosomes play important roles in maintaining normal physiological homeostasis and in the pathological development of diseases. This study investigated the effects of the exosomes derived from hypertrophic scar fibroblasts (HTSFs) on melanocytes, which are pigment-producing cells. Normal fibroblasts (NFs) and HTSFs were isolated and cultured from normal skin and hypertrophic scar (HTS) tissue. Both the NF- and HTSF-exosomes were isolated from a cell culture medium and purified using a column-based technique. The normal human epidermal melanocytes were treated with both exosomes at a concentration of 100 µg/mL at different times. The cell proliferation, melanin content in the medium, apoptotic factors, transcription factors, melanin synthesis enzymes, signaling, signal transduction pathways, and activators of transcription factors (STAT) 1, 3, 5, and 6 were investigated. Compared with the Dulbecco's phosphate-buffered saline (DPBS)-treated controls and NF-exosomes, the HTSF-exosomes decreased the melanocyte proliferation and melanin secretion. The molecular patterns of apoptosis, proliferation, melanin synthesis, Smad and non-Smad signaling, and STATs were altered by the treatment with the HTSF-exosomes. No significant differences were observed between the DPBS-treated control and NF-exosome-treated cells. HTSF-derived exosomes may play a role in the pathological epidermal hypopigmentation observed in patients with HTS.

Role of Emerin in regulating fibroblast differentiation and migration at the substrate of stiffness coupled topology.

In hypertrophic scars, the differentiation and migration of fibroblasts are influenced by the extracellular matrix microenvironment, which includes factors such as stiffness, restraint, and tensile force. These mechanical stresses incite alterations in cell behavior, accompanied by cytoskeletal protein reorganization. However, the role of nucleo-skeletal proteins in this context remains underexplored. In this study, we use a polyacrylamide hydrogel (PAA) to simulate the mechanical stress experienced by cells in scar tissue and investigate the impact of Emerin on cell behavior. We utilize atomic force microscopy (AFM) and RNA interference technology to analyze cell differentiation, migration, and stiffness. Our findings reveal that rigid substrates and cellular restriction elevate Emerin expression and diminish differentiation. Conversely, reducing Emerin expression leads to attenuated cell differentiation, where stiffness and constraining factors exert no notable influence. Furthermore, a softening of cells and an enhanced migration rate are also markedly observed. These observations indicate that variations in nuclear skeletal proteins, prompted by diverse matrix microenvironments, play a pivotal role in the pathogenesis of hypertrophic scars (HSs). This research offers novel insights and a reference point for understanding scar fibrosis formation mechanisms and preventing fibrosis.

Graphene Quantum Dots Reduce Hypertrophic Scar by Inducing Myofibroblasts To Be a Quiescent State.

Contrary to the initial belief that myofibroblasts are terminally differentiated cells, myofibroblasts have now been widely recognized as an activation state that is reversible. Therefore, strategies targeting myofibroblast to be a quiescent state may be an effective way for antihypertrophic scar therapy. Graphene quantum dots (GQDs), a novel zero-dimensional and carbon-based nanomaterial, have recently garnered significant interest in nanobiomedicine, owing to their excellent biocompatibility, tunable photoluminescence, and superior physiological stability. Although multiple nanoparticles have been used to alleviate hypertrophic scars, a GQD-based therapy has not been reported. Our in vivo studies showed that GQDs exhibited significant antiscar efficacy, with scar appearance improvement, collagen reduction and rearrangement, and inhibition of myofibroblast overproliferation. Further in vitro experiments revealed that GQDs inhibited α-SMA expression, collagen synthesis, and cell proliferation and migration, inducing myofibroblasts to become quiescent fibroblasts. Mechanistic studies have demonstrated that the effect of GQDs on myofibroblast proliferation blocked cell cycle progression by disrupting the cyclin-CDK-E2F axis. This study suggests that GQDs, which promote myofibroblast-to-fibroblast transition, could be a novel antiscar nanomedicine for the treatment of hypertrophic scars and other types of pathological fibrosis.

Decorin attenuates hypertrophic scar fibrosis via TGFß/Smad signalling.

The management of hypertrophic scars (HSs), characterized by excessive collagen production, involves various nonsurgical and surgical interventions. However, the absence of a well-defined molecular mechanism governing hypertrophic scarring has led to less-than-ideal results in clinical antifibrotic treatments. Therefore, our study focused on the role of decorin (DCN) and its regulatory role in the TGF-ß/Smad signalling pathway in the development of HSs. In our research, we observed a decrease in DCN expression within hypertrophic scar tissue and its derived cells (HSFc) compared to that in normal tissue. Then, the inhibitory effect of DCN on collagen synthesis was confirmed in Fc and HSFc via the detection of fibrosis markers such as COL-1 and COL-3 after the overexpression and knockdown of DCN. Moreover, functional assessments revealed that DCN suppresses the proliferation, migration and invasion of HSFc. We discovered that DCN significantly inhibits the TGF-ß1/Smad3 pathway by suppressing TGF-ß1 expression, as well as the formation and phosphorylation of Smad3. This finding suggested that DCN regulates the synthesis of collagen-based extracellular matrix and fibrosis through the TGF-ß1/Smad3 pathway.

Integrative Analysis of Lactylome and Proteome of Hypertrophic Scar To Identify Pathways or Proteins Associated with Disease Development.

Lactylation (Kla), a recently discovered post-translational modification derived from lactate, plays crucial roles in various cellular processes. However, the specific influence of lactylation on the biological processes underlying hypertrophic scar formation remains unclear. In this study, we present a comprehensive profiling of the lactylome and proteome in both hypertrophic scars and adjacent normal skin tissues. A total of 1023 Kla sites originating from 338 nonhistone proteins were identified based on lactylome analysis. Proteome analysis in hypertrophic scar and adjacent skin samples revealed the identification of 2008 proteins. It is worth noting that Kla exhibits a preference for genes associated with ribosome function as well as glycolysis/gluconeogenesis in both normal skin and hypertrophic scar tissues. Furthermore, the functional enrichment analysis demonstrated that differentially lactyled proteins are primarily involved in proteoglycans, HIF-1, and AMPK signaling pathways. The combined analysis of the lactylome and proteome data highlighted a significant upregulation of 14 lactylation sites in hypertrophic scar tissues. Overall, our investigation unveiled the significant involvement of protein lactylation in the regulation of ribosome function as well as glycolysis/gluconeogenesis, potentially contributing to the formation of hypertrophic scars.