Flexible Electromagnetic Coil Patch-enhanced Healing of Full-Thickness Skin Wounds in Rats

摘要

This protocol demonstrates a method for creating full-thickness circular skin wounds in rats and enhancingtheirhealingusing a flexible copper coilpatch that delivers apulsedelectromagnetic field.

摘要

Chronic and large skin wounds present significant clinical challenges due to delayed healing and high infection risk. Pulsed electromagnetic field (PEMF) therapy is a non-invasive approach that can promote tissue repair. This study describes a reproducible rat wound model protocol using a flexible printed copper coil patch connected to a custom PCB controller to deliver localized PEMF stimulation to full-thickness dorsal wounds. The contralateral wound served as an untreated control. Healing progression was assessed through regular photography and wound area measurements. The protocol provides a practical, adaptable system for evaluating and optimizing PEMF-based therapies in vivo across various conditions, with a focus on localized application and parameter adjustment for regenerative medicine. Parameter selection was based on an orthogonal CCK-8 screen of fibroblast proliferation that tested 0.25-1.3 mT, 10-40 Hz, and 30-90 min/day; main-effect analysis identified 1.3 mT, 30 Hz, and 60 min/day as the optimal levels, which were adopted for in vivo testing.

引言

Chronic cutaneous wounds (such as diabetic ulcers) are a growing health concern, often leading to impaired healing, infection, or amputation¹. Traditional treatments can be prolonged and costly, prompting interest in adjunctive therapies that can speed up tissue repair². Electromagnetic field therapy is one such approach, which is non-invasive and has demonstrated pro-healing effects in both preclinical and clinical settings^2,3,4,5. Pulsed electromagnetic fields (PEMFs) in the extremely low-frequency range (~1-100 Hz) can stimulate cellular processes important for repair, including angiogenesis and growth factor release^2,6,7.

For example, PEMF exposure significantly accelerated wound closure in diabetic and healthy rodents by upregulating fibroblast growth factor-2 (FGF-2) and increasing angiogenesis⁸. Similarly, in the impaired healing model (e.g., diabetic rats), PEMF treatment has been shown to improve healing rates and increase the tensile strength of newly formed tissue⁹. Clinically, PEMF therapy has been explored in patients with chronic wounds. A pilot randomized trial in diabetic foot ulcer patients reported the enhanced microcirculatory blood flow and a trend toward faster wound closure under PEMF exposure¹⁰. In general, PEMF has also shown beneficial effects in other wound types, for instance, improved healing was observed in burn wounds treated with pulsed magnetic fields¹¹. These studies collectively underscore the potential of electromagnetic stimulation as a complementary therapy for wound care^8,9,10,11.

Compared with conventional wired stimulators, magnetic actuation eliminates the need for transcutaneous connectors and reduces the risk of hardware-related complications. Wireless magnetic systems have achieved tissue-specific stimulation in other contexts, for example, a conductive nerve guidance conduit can generate induced microcurrents under an alternating magnetic field, with the current amplitude indirectly determined by the field parameters¹². Magnetic strategies can also support vascularization -- a critical bottleneck in skin healing -- because static or dynamic magnetic fields, alone or in combination with magnetically responsive biomaterials, have been shown to modulate endothelial behavior and angiogenic signaling¹³. These precedents motivated our development of a wearable approach: a conformal, removable coil patch that concentrates PEMF at the wound bed while preserving non-invasiveness and external programmability^12,13. In the following protocol, we detail the steps for implementing this PEMF therapy in a rat full-thickness wound model, including device application, stimulation regimen, and outcome assessment.

方案

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Jiangnan University (JN. No2025-0630S0360-915[408]). We implemented the protocol using male SD rats (6-8 weeks old, approximately 200-250 g). Prior to experimentation, rats were individually housed with free access to food and water, maintained on a 12 h light/dark cycle, in accordance with the Guide for the Care and Use of Laboratory Animals.

1. Creation of full-thickness excisional wounds

1. Prepare the animal and surgical field.
   1. Induce anesthesia with 3-4% isoflurane in an induction chamber. After loss of the righting reflex, maintain anesthesia at ~2% via nose cone.
   2. Place the rat in the prone position on a sterile surgical surface. Shave the dorsal thoracic region (shoulder blades to mid-lumbar area, including flanks) using electric clippers.
   3. NOTE Remove fur from at least a 5 × 5 cm area on each side of the midline to allow clear surgical access and coil placement.
   4. Disinfect the shaved area with 70% ethanol followed by iodophor solution. Repeat the ethanol/iodine scrub three times, ending with iodine.
   5. Drape the surgical field with sterile gauze, leaving only the planned wound sites exposed.
2. Create bilateral full-thickness excisional wounds.
   1. Outline two 2 cm diameter circles on the dorsal flanks using a sterile surgical marker, positioning one on each side of the midline behind the forelimbs.
      NOTE: Ensure both circles are equal in size and symmetrically positioned to enable within-animal comparison^14,15.
   2. Excise the marked circular skin area using sterile scissors or a scalpel. Remove the epidermis, dermis, and panniculus carnosus to create a full-thickness wound (~3.14 cm²).
   3. Achieve hemostasis by applying gentle pressure with sterile gauze. Create an identical 2 cm wound on the contralateral flank using the same technique.
      ​NOTE: The wound bed should consist of exposed fascia or muscle with no residual dermis.
   4. Administer postoperative analgesia immediately after wound creation (e.g., lidocaine 4 mg/kg subcutaneously).
3. Assign experimental groups.
   1. Use a within-subject design in which one wound receives PEMF treatment and the contralateral wound serves as a control.
   2. Include a separate cohort receiving a sham device (identical non-powered coil) to control for device presence.
   3. Maintain untreated or sham-treated controls to distinguish electromagnetic effects from handling or dressing effects.

2. Application of the PEMF device

1. Dress the wound and position the coil.
   1. Place sterile thin gauze or semipermeable film directly over the wound designated for PEMF treatment. Ensure complete coverage of the wound bed.
   2. Position the flexible copper coil patch over the dressed wound (Figure 1). Center the coil so that the wound lies beneath the middle of the coil.
      NOTE: The coil diameter (~5 cm) may extend beyond the wound margins; ensure the wound remains within the effective magnetic field zone.
2. Secure the coil.
   1. Secure the coil using medical-grade adhesive tape wrapped gently around the torso. Maintain close contact between the coil and dressing without compressing the thorax.
   2. Place a flexible aluminum strip over the coil to stabilize positioning if needed. Avoid restricting respiration or local blood flow.
   3. Route lead wires away from the body (e.g., toward the head) to prevent entanglement.
3. Administer PEMF stimulation.
   1. Connect the coil to the PCB controller (Figure 2) and activate the device. Set stimulation parameters to 30 Hz frequency and ~1.3 mT magnetic field strength for 60 min/day.
   2. Perform stimulation sessions at the same time each day (±1 h) to maintain consistent intervals.
      NOTE: These parameters were selected based on orthogonal CCK-8 screening (0.25-1.3 mT, 10-40 Hz, 30-90 min/day, Table 1) identifying 1.3 mT, 30 Hz, and 60 min/day as optimal (Table 2).
   3. Place an identical inactive coil over the control wound for 1 h to provide sham treatment. Clearly mark the sham coil to avoid confusion.
4. Remove the device and inspect the wound.
   1. Turn off the controller after 60 min. Remove the coil and adhesive materials carefully.
   2. Inspect the wound for bleeding, edema, or adverse effects. Replace sterile gauze if necessary.
      ​NOTE: Coil surface temperature remains near ambient during 1 h stimulation; no significant heating was observed.
5. Provide posttreatment care.
   1. Fit an Elizabethan collar to prevent self-trauma. Ensure proper fit without impairing respiration or feeding.
   2. Wrap gauze lightly around the hind limbs to reduce scratching of dorsal wounds.
   3. Monitor the rat until fully recovered from anesthesia. Continue analgesia (e.g., lidocaine 4 mg/kg every 12 h for 2-3 days or per institutional protocol).
   4. Repeat daily PEMF treatments for the study duration (e.g., 14 days).

3. Wound monitoring and data collection

1. Capture wound images.
   1. Photograph wounds at 0, 3, 7, 10, and 14 days post wounding. Lightly anesthetize animals if necessary to prevent movement.
   2. Include a measurement scale in each image and position the camera perpendicular to the wound at a fixed distance^14,15.
2. Measure wound area.
   1. Calibrate image scale in ImageJ using the reference ruler. Trace wound margins to calculate area in mm².
   2. Record wound area at each time point and calculate percent wound closure relative to baseline.
   3. Compare PEMF-treated and control wounds quantitatively.
3. Harvest tissue for histology (optional).
   1. Euthanize animals at the experimental endpoint according to IACUC-approved procedures.
   2. Excise wound tissue samples and fix in formalin for histological processing.
   3. Perform paraffin embedding and staining (e.g., H&E, Masson's trichrome) to assess collagen deposition, re-epithelialization, and angiogenesis^13,16.

4. Data analysis and interpretation

1. Generate wound closure curves.
   1. Plot percent wound closure over time for each group.
   2. Calculate healing rate and time to predefined closure thresholds.
2. Perform statistical analysis.
   1. Compile wound area data for all animals and calculate mean ± SD at each time point.
   2. Confirm normality (Shapiro-Wilk test) and homogeneity of variance (Levene's test).
   3. Analyze differences using two-way ANOVA with factors "Treatment" and "Time." Perform Tukey's post hoc testing if significant interaction is detected.
   4. Define statistical significance as *p < 0.05 and **p < 0.01.

结果

In this full-thickness rat wound model, daily PEMF therapy via a flexible coil patch resulted in significantly faster wound closure compared to controls. To rigorously evaluate the effect, rats were allocated into three groups based on the experimental design outlined in the Protocol section: a within-subject control group (where each rat had one PEMF-treated wound and one contralateral untreated wound, data pooled for analysis), a sham control group (PATCH: wound covered with an unpowered coil), and an untreated control...

讨论

Critical steps include creating symmetric full-thickness wounds, ensuring the coil patch lies flat over a sterile barrier without gaps, and maintaining consistent daily exposure; deviations can reduce field delivery and increase variability in healing outcomes.

This work has several limitations that should be considered when interpreting the findings and assessing translational potential. First, the protocol was demonstrated in healthy male SD rats (6-8 weeks, 200-250 g) with acute excisional ...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
96孔细胞培养板	白帝	H908002	用于CCK-8检测
ADHDESION 显微镜载玻片	CITOTESTO	邮编/编号80312-3161	设备及nbsp;H&E/马森染色
镇痛药（盐酸利多卡因注射）	胡阿穆	70011657	阿片类止痛药，4 mg/kg（用于止痛）
麻醉机	后轮驱动生命科学（深圳，中国）	R520-等离子型;	带氧气供应的异氟醚蒸发器
自动免疫组化染色机	徕卡	21.2201	自动免疫组化染色
活检冲头（20毫米）	英特格拉·米尔特克斯	#33-37	圆形皮肤冲孔器，直径2厘米
数码相机	华为Mate 70 Pro	无	≥12 MP 用于创伤成像
CD31抗体	阿布卡姆	AB182981	IHC的内皮标志物（图4B）
CD34抗体	阿布卡姆	AB81289	IHC的内皮标志物（图4B）
细胞计数套件-8（CCK-8）	生物锐利	BS350B	用于体外PEMF参数筛查（OD450）
细胞培养皿（10厘米）	白帝	H802004	10厘米（100毫米）组织培养皿用于成纤维细胞培养
细胞培养基（例如DMEM+FBS）	白帝	L100-500	用于CCK-8筛查中的成纤维细胞培养
二氧化碳培养箱	热力学	BB150-2TCS-L	对于成纤维细胞培养
线圈，柔性铜	定制制造（内部制造）	无	螺旋打印线圈（直径5厘米，聚酰亚胺）
控制板（PEMF发生器）	定制（内部设计）	无	定制PCB，驱动线圈频率30 Hz，~1.3 mT
电动理发器	Wahl 或 Oster（宠物美容）	9160型及nbsp;	用于剃毛的动物美容剪
伊丽莎白时代的项圈（啮齿动物）	MukeBio	MK-EC-01	防止伤口干扰
嵌入站	Epredia HistoStar	A81000002	组织学处理（如已进行）
口罩	维登	V531596	保持麻醉
成纤维细胞（细胞系L929）	Procell	CL-0137	用于CCK-8增殖筛查的细胞
有限元仿真软件（例如，COMSOL Multiphysics）	COMSOL	CM010004	磁场仿真（图5B-C）
柔性铝条式夹板	亲爱的	1592	防止线圈位移的支撑
镊子	雄伟	01625	手术时处理皮肤和敷料
血液素与伊欧辛（H&E）自动染色和封层 uni	DRS-棱镜-P+薄膜	61770249	用于常规组织学染色的消耗品
图像分析软件	美国国立卫生研究院	版本 1.54	用于伤口面积测量（例如，ImageJ）
图像分析软件	美国国立卫生研究院	版本 1.54	用于伤口面积测量（例如，ImageJ）
感应室	后轮驱动生命科学（深圳，中国）	802-00132-00	异氟醚麻醉诱导
红外热成像相机	HIKMICRO	H11	监测线圈表面温度（图5A）
碘聚维酮消毒剂 10%	海诺特	无	皮肤消毒剂（碘素）
异氟醚	后轮驱动生命科学（深圳，中国）	20037015	吸入麻醉剂（USP级）
利多卡因注射 10%	桑马	无	术后止痛
马松的三色染色套件	巴索	C250202	用于胶原蛋白/ECM可视化的消耗品
医疗皮肤标记与尺	通道斯	无	伤口照片中包含比例
微板读器	热力学	A51119500C	用于CCK-8测定中的OD450测量
切片机	Epredia HM 340E	23900670	组织学/IHC切片（如果进行）
聚甲醛组织固定剂	分享简介	SB-C009	动物组织标本的固定
老鼠饲养笼	凤桥	CP-JM	标准的老鼠笼子用于单独饲养。
Rats，Sprague Dawley（雄性，6岁及ndash;8周）	魏景宇苏州	SCXK2024-0004	200–250克;单独住宿;IACUC批准的使用
手术刀	金环	35W0602	用于切除伤口的创立
半透膜涂料	ZD	Q20189	替代伤口屏障
统计分析软件（例如 GraphPad Prism 或 R）	软件马凯夫	10.6.9(890)	双因子方差分析及事后多重比较
无菌纱布	ZD	3141626	伤口敷料/保护屏障
无菌手术布	ZD	V271617	皮肤准备后为手术区域布置
外科剪刀	雄伟	01627	用于切除伤口的创立
注射器和针头	米南克	20193141643	用于皮下止痛
整片扫描仪	3DHISTECH	无	数字幻灯片扫描