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.

Effectiveness of microneedle fractional radiofrequency combined with transcutaneous delivery of botulinum toxin in the management of post-acne scars.

BACKGROUND: Post-acne scars are a common sequela of acne, especially prevalent among young people. Delayed treatment not only affects self-perception of beauty but also affects the mental health of patients. OBJECTIVE: This study aims to investigate the clinical efficacy of microneedle fractional radiofrequency (MFR) combined with botulinum toxin A (BoNT/A) in managing post-acne scars. METHODS: This retrospective study involved 63 adult patients with post-acne scars, divided into two groups: group 1 (n = 30) and group 2 (n = 33). Group 1 received treatment with MFR combined with transcutaneous delivery of BoNT/A, whereas group 2 received treatment with MFR alone. The study observed the clinical outcomes in both groups. RESULTS: Based on experimental analysis, the combination of MFR with transcutaneous delivery of BoNT/A demonstrated superior clinical efficacy compared with group 2. There were no significant differences in baseline data or treatment-related pain and adverse reactions between the two groups. However, group 1 exhibited a higher effectiveness rate, lower ECCA score after treatment, higher satisfaction levels, and statistically significant differences compared to group 2. CONCLUSION: MFR combined with transcutaneous delivery of BoNT/A represents an effective and safe alternative for treating acne scars with minimal side effects and complications. SUMMARY STATEMENT: Post-acne scars are a common sequela of acne and combination therapy proves beneficial. Microneedle fractional radiofrequency (MFR) combined with transcutaneous delivery of BoNT/A can be considered an effective and safe alternative for the treatment of acne scars with minimal side effects and complications. It works together through microneedles, radiofrequency, and botulinum toxin. MFR combined with transcutaneous delivery of BoNT/A is based on the direct action of MFR on acne scars and the use of microneedle to create a transient skin microchannel, facilitating BoNT/A penetration into the skin.

Composite microneedles loaded with Astragalus membranaceus polysaccharide nanoparticles promote wound healing by curbing the ROS/NF-κB pathway to regulate macrophage polarization.

The healing of chronic diabetic wounds remains a formidable challenge in modern times. In this study, a novel traditional Chinese medicine microneedle patch was designed based on the physiological characteristics of wounds, with properties including hemostasis, anti-inflammatory, antioxidant, antimicrobial, and induction of angiogenesis. Initially, white peony polysaccharide (BSP) with hemostatic properties and carboxymethyl chitosan (CMCS) with antimicrobial capabilities were used as materials for microneedle fabrication. To endow it with antimicrobial, procoagulant, and adhesive properties. Among them, loaded with ROS-sensitive nanoparticles of Astragalus polysaccharides (APS) based on effective components baicalein (Bai) and berberine (Ber) from Scutellaria baicalensis (SB) and Coptis chinensis (CC) drugs (APB@Ber). Together, they are constructed into multifunctional traditional Chinese medicine composite microneedles (C/B@APB@Ber). Bai and Ber synergistically exert anti-inflammatory and antimicrobial effects. Microneedle patches loaded with BSP and APS exhibited significant effects on cell proliferation and angiogenesis induction. The combination of composite polysaccharides enabled the microneedles to adhere stably to wounds and provide sufficient strength to penetrate the biofilm and induce dispersion. The combination of composite polysaccharides enabled the microneedles to adhere stably to wounds and provide sufficient strength to penetrate the biofilm and induce dispersion. Therefore, traditional Chinese medicine multifunctional microneedle patches offer potential medical value in promoting the healing of diabetic wounds.

INDIVIDUAL ARTICLE: Real-World Patient Cases Using Triple Lipid-Containing Cream for Cutaneous Healing Post Laser or Microneedling Radiofrequency Treatment.

Lipids play an essential role in skin barrier health. With age, there is a natural reduction of physiological lipids such as fatty acids, ceramides, and cholesterol. The triple lipid restore cream is a moisturizer that contains an optimized lipid ratio for aging skin. The cream contains a 2:4:2 ratio of ceramides, cholesterol, and fatty acids that have been shown to best support aging skin. The triple lipid restore cream has been used in combination with energy-based procedures, to provide patients with comprehensive integrated skincare regimens. With limited clinical data and guidelines available in regenerative medicine, real-world cases serve as an invaluable guide for patients and dermatologists in navigating rejuvenation treatment plans. J Drugs Dermatol. 2024;23:9(Suppl 1):s3-14.

Comparing the efficacy and safety of microneedling and its combination with other treatments in patients with acne scars: a network meta-analysis of randomized controlled trials.

This study aimed to analyze the efficacy and safety of microneedling (MN), both alone and in combination with other treatments, to refine the approach for treating acne scars using MN. We systematically searched Pubmed, Cochrane Library, Embase, and Web of Science for randomized controlled trials examining MN or its combinations in patients with acne scars. All statistical analyses were performed using Stata 18 software. A total of 24 studies involving 1546 participants were included. The analysis revealed that MN combined with chemical peels (CP) exhibited the best results in terms of degree of improvement, patient satisfaction, and treatment efficacy compared to other treatments examined, including MN alone, MN with hyaluronic acid (HA), MN with botulinum toxin­A (TA), MN with platelet-rich plasma (PRP), PRP alone, CP, and laser therapy. The results for MN combined with additional treatments were obviously better than for MN alone. Side effects such as erythema, pain, and post-inflammatory hyperpigmentation showed no significant differences across all treatments assessed.

A retrospective study of non-insulated microneedle radiofrequency on wrinkles of facial photoaging subjects.

Non-insulated microneedle radiofrequency (NIMNRF) is a method of promoting dermal collagen shrinking and remodeling with minor injury reducing wrinkles. We conducted a 3-years retrospective observation on wrinkles of facial photoaging subjects treated with NIMNRF in Chinese subjects to demonstrate the efficacy and side effects. Chinese subjects clinically diagnosed as facial photoaging treated with MNRF in the Laser Center of The First Hospital of China Medical University and Guangzhou Mylike Medical Cosmetic Hospital from Jan 1, 2018 to Dec 31, 2021 were enrolled in this study. Inclusion criteria included. Each subject was treated with NIMNRF for 1-3 sessions, with a 3-month interval. At baseline and 3 months after each treatment, a Wrinkle Assessment Scale (WAS) was used to score the wrinkles in 10 areas. The total WAS score and WAS improvement rate was assessed at each time point. A total of 96 subjects, aged 25-65 years old, received at least one session of NIMNRF were enrolled. 63, 24, 9 of them received 1, 2 or 3 sessions, respectively. The total WAS score decreased from 14.65 ± 9.20 to 11.51 ± 8.70 after Session 1, from 15.92 ± 9.48 to 12.17 ± 8.83 after Session 2 and from 17.56 ± 6.99 to 11.11 ± 7.13 after Session 3 (P < 0.01). The WAS improvement rate was 25.61%, 30.69% and 39.82% after 1, 2, 3 sessions, respectively. As for subjects in different age groups, the improvement rate decreased with age, from 39.13% in 25-30 years old group to 16.39% in over 60 years old group after Session 1 (P < 0.05). Better efficacy and less sessions of treatments were conducted in younger subjects. NIMNRF can be used in the treatment of facial wrinkles in photoaging subjects, especially in youngster as better efficacy.

M2 macrophage-polarized anti-inflammatory microneedle patch for accelerating biofilm-infected diabetic wound healing via modulating the insulin pathway.

Macrophages play a pivotal role in the healing of diabetic ulcers. The sustained elevation of glucose levels damages the insulin signaling pathway in macrophages, leading to dysfunctional macrophages that struggle to transition from pro-inflammatory (M1) to reparative (M2) states. Therefore, modulating macrophage inflammatory responses via the insulin pathway holds promise for diabetic ulcer treatment. Additionally, the presence of biofilm impedes drug penetration, and the resulting immunosuppressive microenvironment exacerbates the persistent infiltration of pro-inflammatory M1 macrophages. Therefore, we designed an array of dissolvable microneedle (denoted as NPF@MN) loaded with self-assembled nanoparticles that could deliver NPF nanoparticles, acid-sensitive NPF-releasing Protocatechualdehyde (PA) with hypoglycemic and insulin-like effects, regulating macrophage polarization to an anti-inflammatory M2 phenotype. Additionally, this study extensively examined the mechanism by which NPF@MN accelerates the healing of diabetic ulcers through the activation of the insulin signaling pathway. Through RNA-seq and GSEA analysis, we identified a reduction in the expression of pathway-related factors such as IR, IRS-1, IRS-2, and SHC. Our work presents an innovative therapeutic approach targeting the insulin pathway in diabetic ulcers and underscores its translational potential for clinical management.

Microneedle patch casting using a micromachined carbon master for enhanced drug delivery.

For successful treatment of diseases, sufficient therapeutics must be provided to the body. Microneedle applications in therapeutic delivery and analytics sampling are restricted because of various issues, including smaller area for drug loading and analytics sampling. To achieve sufficient drug loading and analytics sampling and improve drug penetration while maintaining painless administration, patch-type microneedle arrays were designed and fabricated using polymer casting from a conical cavity mold. Microcavities were formed on a carbon plate via micromechanical machining. A porous polymer layer was coated on a microneedle patch (MNP). The pores of the porous polymer layer provided space and channels for drug delivery. A pH-sensitive polymer layer was employed to cap the porous polymer layer, which prevented drug leakage during storage and provided a stimulus drug release in response to body pH conditions. The drug can be delivered through holes connected to both sides of the patch. The drug release of the MNP was investigated in vitro and in vivo and showed conceptual proof that these MNs have the potential to enhance treatment protocols for various diseases with the flexibility of coating and therapeutic materials and offer significant scope for further variations and advancement.

Comparison of the Cobalt Content in Needles Stainless Steel.

Cobalt (Co) alloys are used extensively in a wide range of medical devices due to their biocompatibility, durability, and properties. In 2017 the European Chemical Agency (ECHA) proposed to classify Co metal as a category 1B carcinogen (i.e. presumed to have carcinogenic potential for humans), as a reproductive hazard category 1B (i.e. presumed human reproductive toxicant) and as a category 2 mutagen. Even more, the European Medical Device Regulation (MDR) requires that medical devices contain > 0,1 w/w of substances that are category 1 A and 1B (CMR). As far as the European Medical Device Regulation, it is not specified if the Co content and concentration had to be determined/measured on the entire product or only on the product components. The object of this work is the comparison of Co profile in three different suppliers of stainless-steel needles as is and after being processed (i.e. sterilized) and then provide the proper interpretation and application of MDR requirements. The study and the extractable profile demonstrate the low cobalt content on needles and on a total syringe, thus suggesting to consider the Co content only on the syringe component.

Finite Element Analysis of Skin Deformation and Puncture for Microneedle Array Design.

The mechanics of microneedle insertion have thus far been studied in a limited manner. Previous work has focused on buckling and failure of microneedle devices, while providing little insight into skin deformation, puncture, and the final positioning of needle tips under full microneedle arrays. The current study aims to develop a numerical approach capable of evaluating deformation and puncture conditions for full microneedle array designs. The analysis included a series of finite element submodels used to calibrate the microneedle-epidermal interface for failure properties using traction-separation laws. The single needle model is validated using experimental data and imaging, including results from a customized nanoindentation procedure to measure loads and displacements during microneedle insertion. Upon validation, full microneedle arrays are implemented in a 3 D finite element model and a design framework is developed, allowing evaluation of different design variables (i.e. needle shape, material, spacing) with respect to outputs relevant to successful microneedle performance. Results from the model include skin deformation, force to puncture, penetration depth, and the punctured state at each microneedle tip. In addition to microneedle parameters, patient parameters such as subcutaneous tissue thickness are included to evaluate the sensitivity of different microneedle designs to expected patient and anatomical region variability.

A Novel Combined Approach to Better Predict Sealing Performance of Luer Lock Connectivity.

Luer systems, for example Luer-needle hub with syringe's Luer cone tip and its Luer lock Adapter, are common interface on medical devices. One of the key questions in this application is about the safety guaranty and dose accuracy. It is then crucial to study the sealing between these elements. In this study we combine the use of Finite Element Analysis (FEA) and Multiscale Contact Mechanics (MCM) to analyze the connectivity and sealing performance of a glass syringe and a plastic needle Luer hub.This methodology has been applied before to the contact between glass and rubber and this is the first time that it is used for the contact between glass and plastic materials. The use of FEA allows to calculate the contact pressures and the nominal area of contact. The surface topographies of the two surfaces were measured, over a wide wavelength range (mm to nm). Subsequently, the air and liquid interfacial flow (leakage) is calculated using Persson's MCM theory which considers the roughness and elasto-plasticity of the interfacial surfaces. The theoretical predictions are compared to experimental leak measurements by pressure decay method. Further analysis is conducted, evidencing the key features that are responsible for a good sealing.

Glucose detection: In-situ colorimetric analysis with double-layer hydrogel microneedle patch based on polyvinyl alcohol and carboxymethyl chitosan.

Skin interstitial fluid (ISF) has emerged as a significant reservoir of biomarkers for disease diagnosis and prevention. Microneedle (MN) patches are regarded as an optimal platform for ISF extraction from the skin due to their non-invasive nature. However, challenges such as prolonged sampling durations and complex detection procedures impede timely metabolic analysis. In this investigation, we amalgamated MN technology with immobilized enzyme technology to fabricate a dual-layer MN patch integrating sampling and detection functionalities, thereby enabling in-situ colorimetric detection of hyperglycemia. The tip layer of the patch, comprising polyvinyl alcohol/carboxymethyl chitosan (PVA/CMCS) MN, was synthesized utilizing a chemical crosslinking approach for the first time, with glucose oxidase (GOx) being incorporated. The hydrophilicity of CMCS expedited the extraction process, facilitating the retrieval of approximately 10 mg of ISF within 10 min. The backing layer consisted of an immobilized polyvinyl alcohol-chitosan-horseradish peroxidase (PVA-CS-HRP) hydrogel film loaded with 3,3', 5,5'-tetramethylbenzidine (TMB). Incorporating macromolecular polymer PVA and CS for HRP immobilization addressed the issue of poor stability associated with traditional natural enzymes, thereby enhancing the sensitivity of the reaction system. The in-situ colorimetric sensor facilitated minimally invasive ISF extraction and swift conversion of glucose levels into detectable color changes.

Development of Transdermal Formulation Integrating Polymer-Based Solid Microneedles and Thermoresponsive Gel Fucoidan for Antiaging: Proof of Concept Study.

Skin can be damaged by intense and prolonged exposure to ultraviolet (UV) radiation. Photoaging and acute damage from sun exposure result in collagen degradation and enzymatic activity decline in the skin. Fucoidan (FUC) exhibits potential antiaging properties, including collagen synthesis promotion and enzyme activity inhibition. However, FUC's limited ability to penetrate the skin layers due to its large molecular weight makes it a challenge for topical application. In this study, we successfully developed a new approach by integrating thermoresponsive gel (TRG) containing FUC with solid microneedles (SMNs) as a delivery system. TRG is formulated using a combination of Pluronic F127 (PF127) and Pluronic F68 (PF68) polymers, while SMNs are made from a mixture of poly(vinyl alcohol) (PVA) and poly(vinylpyrrolidone) (PVP) polymers with a variety of cross-linkers. Based on the results of ex vivo testing, it was shown that more than 80% of FUC can be delivered using the optimized formula. Furthermore, the results of the in vitro blood hemolytic test showed that TRG-FUC-SMNs were relatively biocompatible. In vivo antiaging activity tests using a rat model exposed to UV for 14 days showed that histological assessment, skin elasticity measurement, wrinkle evaluation, and skin moisture content had no significant differences (p < 0.05) compared to the positive control group. In contrast, a significant difference (p < 0.05) was observed when comparing the TRG-FUC-SMNs group with the group that received only TRG-FUC without pretreatment and negative controls. These findings suggest that FUC has potential to be delivered using the TRG system in combination with SMNs to harness its antiaging properties.

Microneedles-Based Theranostic Platform: From the Past to the Future.

Fully integrated theranostic devices are highly esteemed in clinical applications, offering immense potential in real-time disease monitoring and personalized care. Microneedles (MNs), as innovative and wearable devices, boast important advantages in biosensing and therapy, thus holding significant promise in the advancement of diagnostic and therapeutic platforms. Encouragingly, advancements in electrochemical sensing technology, micronano fabrication, and biocompatible materials are propelling momentum for MNs-based closed-loop systems, enhancing detection capabilities, biocompatibility, and cost-effectiveness. Moreover, the notable progress in integrating MN chips with other biochips signifies a frontier for growth. Successful clinical trials in target molecule monitoring and drug delivery domains herald excellent clinical translational prospects for the aforementioned theranostic platform. Finally, we delineate both challenges and opportunities in the development of integrated diagnostic and therapeutic MN systems, including continuous monitoring, intelligent control algorithms, safety, and regulatory considerations.

Effect of thumbtack needle on functional constipation: A pragmatic randomized controlled trial.

OBJECTIVE: Thumbtack Needling (TN) has been employed in the treatment of functional constipation (FC), although the existing evidence supporting its effectiveness is limited. This study is to evaluate the efficacy of TN in ameliorating FC. METHOD: A total of 482 eligible patients were recruited and randomly assigned to the TN group or the Mosapride Citrate (MC) group. The TN was buried once for three days, rest for one day after two consecutive burials, followed by a 4-week follow-up. The primary outcome measure was the score for Complete and spontaneous bowel movement score (CSBMs). Secondary outcome measures included the Bristol Stool Form Scale (BSFS), Cleveland Clinic Score (CCS), and the Patient Assessment of Constipation Quality of Life Questionnaire (PAC-QOL). RESULTS: Out of the 482 patients randomized, 241 were allocated to each group. Of these, 216 patients (89.6 %) in both groups completed the intervention and follow-up. Compared with the baseline, the differences of CSBMs in TN group [1.76(95 % CI, 1.61 to 1.91)] and MC group [1.35(95 % CI, 1.20 to 1.50)] at week 4 meet the threshold for minimal clinically important difference (MCID). However, there were no clinical difference from baseline at week 2 and week 8 in both groups. Mean CSBMs at week 4 was 3.35 ± 0.99 in the TN group and 3 ± 1.03 in the MC group (adjusted difference between groups, 0.37 points [95 % CI, 0.18 to 0.55]; P < 0.001), although differences between the two groups did not meet the MCID threshold. CONCLUSION: Compared with mosapride citrate, thumbtack needling produced a greater improvement in CSBMs, although the difference from control was not clinically significant. GOV IDENTIFIER: ChiCTR2100043684.

Evaluation of different irrigation activation techniques for the removal of various medicaments from a simulated internal resorption cavity: an in vitro study.

OBJECTIVES: This study aimed to assess the efficacy of different activation techniques in removing calcium hydroxide (Ultracal XS), Ledermix, and Bio-C Temp from simulated internal root resorption (IRR) cavities. MATERIALS AND METHODS: 108 single-rooted maxillary incisors were prepared using Reciproc R50 files. Simulated IRR cavities, 2 mm in diameter and located 8 mm from the apex, were created. Ultracal XS, Ledermix, and Bio-C Temp were applied to the samples, grouped by irrigation activation techniques: Standard Needle Irrigation (SNI), EDDY, Passive Ultrasonic Irrigation (PUI), and XP-endo Finisher (XPF). Medicament removal efficacy was evaluated using a standardized scoring system. Statistical analysis was performed using the Kruskal-Wallis test. RESULTS: XPF and PUI were more effective than SNI in medicament removal across the groups, with no significant difference. EDDY showed no significant difference than other groups. Ledermix was more effectively removed in all activation groups compared to Bio-C Temp. The XPF was superior in removing Ultracal XS compared to Bio-C Temp. However, none of the groups achieved complete medicament removal. CONCLUSIONS: XPF and PUI techniques enhance medicament removal efficacy. Bio-C Temp was more difficult to remove from the IRR cavities than other medicaments. CLINICAL RELEVANCE: Bio-C Temp could be removed from the canals less effectively compared to calcium hydroxide and Ledermix. Among the tested irrigation activation methods, XPF and PUI were found to be more effective at removing the tested medicaments.

Subcision with platelet-rich plasma and microneedling versus subcision with saline and micro-needling in posttraumatic scars.

BACKGROUND: In treating post-traumatic scars, this study compared the safety and effectiveness of combined subcision with saline and microneedling versus combined subcision with platelet-rich plasma and microneedling. Combined subcision with saline and microneedling or combined subcision with platelet-rich plasma and microneedling were used to treat 36 consecutive individuals with post-traumatic scarring. The Modified Manchester score was used to assess texture change, pigmentation, and surface distortion changes. Each change was given a score between 1 and 4. A lower score (range: 3-12) indicates a better result. The mean of the three individual scores was determined. For best outcomes, each patient needed four treatment sessions for each scar, with a one-month follow-up period following the final treatment. The three variables in group B had mean scores of 1.4 ± 0.5, 2 ± 0.8, and 2.2 ± 0.9, respectively, for texture change, pigmentation, and surface distortion. With a mean score of 1.4 ± 0.5, texture change had the best response out of the three variables we evaluated. The investigator determined that the mean improvement score for patients in group B's overall appearance was 5.61 ± 1.19. The study has shown that the combination of subcision with platelet-rich plasma, and microneedling appears to be a promising treatment for posttraumatic scars due to its low risk and high efficacy. Our findings suggest that this is a safe method for treating posttraumatic scars, with few side effects and a low chance of recurrence. IRB LOCAL APPROVAL NUMBER: 04-2023-300279. CLINICAL TRIAL REGISTRY: NCT06135480.

Removable Photocatalysis Microneedle Reactor for Carbon Monoxide Delivery to Enhance Chemosensitization.

Carbon monoxide (CO) has emerged as a promising therapeutic agent, yet ensuring safe and precise CO delivery remains challenging. Here, we report a removable hydrogel-forming microneedle (MN) reactor for CO delivery via photocatalysis, with an emphasis on chemosensitization. Upon application, body fluids absorbed by the MNs dissolve the effervescent agents, leading to the generation of carbon dioxide (CO2) and triggering the release of the chemotherapeutics cisplatin. Meanwhile, the photocatalysts (PCs) trapped within MNs convert CO2 to CO under 660 nm light irradiation. These PCs can be removed by hydrogel-forming MNs, thereby mitigating potential biological risks associated with residual PCs. Both in vitro and in vivo experiments showed that MN-mediated CO delivery significantly improved tumor sensitivity to cisplatin by suppressing DNA repair, using an A375/CDDP melanoma model. This removable photocatalysis MN reactor offers safe and precise local delivery of CO, potentially creating new opportunities for CO or its combination therapies.

Effect of sequential delivery of 1- and 2-MHz bipolar microneedling radiofrequency energy on thermal tissue reactions in a minipig model.

BACKGROUND: Bipolar microneedling radiofrequency (RF) treatment generates different patterns of thermal reactions, depending on the skin impedance and RF treatment parameters, including the frequency, power, conduction time, settings of sub-pulse packs, and penetrating depth and type of microneedles used. We compared the effect of sequential delivery of 1- and 2-MHz bipolar RF energy to in vivo minipig skin on thermal tissue reaction. METHODS: RF treatments at frequencies of 1 and 2 MHz were sequentially delivered to minipigs' skin in vivo. A histological study was performed to analyze RF-induced skin reactions at 1-h and at 3-, 7-, and 14-days post-treatment. RESULTS: The skin specimens demonstrated that the two different frequencies of RF treatment generated mixed patterns of the peri-electrode coagulative necrosis (PECN) according to the experimental settings and tissue impedance. In the PECN zone, the tissue coagulation induced by the first RF treatment was surrounded by the effect of the later RF treatment at the other RF frequency. In the inter-electrode non-necrotic thermal reaction zone, the effect of the latter RF treatment was widespread and deep through the dermis, which had received RF treatment at the other frequency first. The delivery of pulsed-type RF energy at sub-pulse packs of 6 or 10 provided effective RF delivery over long conduction time without excessive thermal damage of the epidermis. Nonetheless, by sequential delivery of two different RF frequencies, RF-induced tissue reactions were found to be markedly enhanced. CONCLUSION: The sequential delivery of 1- and 2-MHz RF energy induces novel histological patterns of tissue reactions, which can synergistically enhance the thermostimulatory effects of each RF setting. Moreover, variations in patterns of tissue reactions can be generated by regulating the order of frequencies and the number of sub-pulse packs of RF used.