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Introduction

Skin grafting is a well-established surgical technique used to restore areas of skin loss resulting from burns, trauma, ulcers, or surgical excisions. Among the various grafting methods Split-Thickness Skin Grafts (STSG) are most employed, involving the removal of the epidermis and part of the dermis from a donor site and transplanting it to the wound bed. Although this procedure effectively promotes wound closure and functional recovery, it also creates a secondary wound at the donor site, which can cause significant postoperative pain, often more intense than that experienced at the recipient site. Effective management of this donor site pain is. therefore. crucial not only for improving patient comfort but also for facilitating early mobilisation, reducing analgesic dependency, and promoting optimal wound healing. Despite advancements in dressing materials and analgesic protocols, donor site pain remains a persistent clinical challenge.¹

Methylene Blue (MB), traditionally known as a biological dye, has recently gained attention for its potential analgesic, anti-inflammatory, and neurolytic properties. It is a non-toxic phenothiazine compound that can be safely administered via oral, intravenous, or subcutaneous routes, most commonly as a 1% solution. Doses below 2mg/kg are considered safe for human use, with established applications in treating methemoglobinemia, malaria, vasoplegic syndrome and as an intraoperative marker during parathyroid surgery to aid in gland identification.^2–4 Despite its long-standing use in medicine, there remains a lack of standardised dosing protocols or administration guidelines for intradermal injection of MB for pain management, particularly in surgical settings, such as skin grafting.

The mechanism of action underlying MB’s analgesic effect is multifactorial. MB is known to inhibit the nitric oxide (NO) pathway, a critical mediator of vasodilatation and inflammation. Under physiological conditions, NO plays an essential role in vascular smooth muscle relaxation. However, under pathological or inflammatory conditions, the overproduction of NO contributes to pain and tissue inflammation through the up regulation of inducible nitric oxide synthase (iNOS). MB acts as a selective inhibitor of iNOS, thereby suppressing excessive NO production and reducing inflammation and pain at the tissue level.^5,6 Additionally, its vasoconstrictive effect limits local blood flow and edema, which may further alleviate discomfort and promote hemostasis at the donor site. This dual anti-inflammatory and vasoconstrictive action provides a mechanistic rationale for exploring MB as a novel local analgesic agent.

Several clinical studies have demonstrated the efficacy of MB in pain reduction across various medical and surgical conditions. Randomised controlled trials (RCTs) have reported significant decreases in postoperative pain scores following MB administration in procedures such as hemorrhoidectomy, osteoarthritis, and interventions for chronic discogenic low back pain.^7-9 The findings suggest that MB’s analgesic properties may extend to other postoperative settings, including donor site pain management after skin grafting. Reported adverse effects of intradermal MB are generally mild and transient, such as localised redness, swelling and temporary blue discoloration of the skin or urine.^10,11 While some studies have raised concerns about potential effects on re-epithelialisation or scar quality, these effects have not shown any long-term clinical significance.¹²

Given this growing body of evidence, the present randomised controlled trial was designed to evaluate the efficacy and safety of a 1% intradermal injection of MB administered at the donor site following STSG. The study aimed to compare postoperative pain outcomes, analgesic requirements and wound healing parameters between patients receiving MB injections and those receiving the standard tulle gras dressing. Through this comparative assessment, the trial sought to establish whether MB could serve as a safe, effective, and low-cost adjunct for donor site pain relief, potentially transforming a common yet under-addressed aspect of postoperative care in reconstructive surgery.

Methods

Study design and setting

This study was designed as a prospective, randomised, parallel-group, active-controlled clinical trial conducted in the Department of General Surgery at Lokmanya Tilak Municipal Medical College and General Hospital, Mumbai, India. The hospital provides tertiary-level care and is equipped with the necessary infrastructure to manage elective and emergency surgical procedures, including skin grafting. The study was conducted over a period of 18 months, ensuring adequate recruitment, intervention and follow-up. The detailed protocol and methodological framework of this trial have been published previously by the authors, and a summary of the methodology is presented below.¹³

Sample size

The sample size for this trial was determined a priori and has been described in detail in the published study protocol. Briefly, the calculation was based on detecting a clinically meaningful difference in postoperative pain intensity, measured using the Visual Analogue Scale (VAS), between the two groups. Assuming a mean difference of −0.82 with corresponding standard deviations of 0.61 and 0.69, a two-sided α of 0.05, and 80% power, a minimum of 18 participants (9 per group) was required.¹⁴ To account for potential attrition, the sample size was conservatively increased to 60 participants, with 30 allocated to each study arm.

Participants

A total of 60 adult patients (≥18 years) undergoing STSG were enrolled. Inclusion criteria comprised patients undergoing elective grafting with donor sites located on the anterolateral thigh and willing to provide informed consent. Patients with known hypersensitivity to MB, impaired wound healing (such as uncontrolled diabetes), systemic infections and those receiving immunosuppressive therapy were excluded from participation.

Randomisation and blinding

Participants were randomised in a 1:1 ratio to either the intervention group (intradermal MB injection) or the control group (standard tulle gras dressing) using computer-generated random numbers. Allocation concealment was ensured using the Sequentially Numbered Opaque Sealed Envelopes (SNOSE) method. Due to visible skin staining following MB injection, blinding patients and treating clinicians was not feasible. However, outcome assessors responsible for pain scoring and wound evaluation were blinded to group allocation to minimise detection and performance bias.

Surgical procedure and intervention

All participants underwent STSG performed by the same surgical team to ensure procedural uniformity. Grafts were harvested from the anterolateral thigh using a Humby’s knife, maintaining uniform thickness and consistent donor site size. Following the graft harvest, the donor site was managed according to group allocation. In the intervention group, a 1% solution of MB was injected intradermally at the donor site immediately after graft harvest, followed by conventional tulle gras dressing. The injection was administered using a 26-gauge needle in a grid-like pattern, with a maximum total volume of 5mL per patient. The dose of MB was adjusted to wound size: wounds up to 10cm received approximately 2.5mL, while those between 10–20cm received up to 5mL, though no patient required more than 3mL in practice. A preoperative intradermal sensitivity test was performed on the forearm in all patients to exclude hypersensitivity. No allergic or systemic adverse reactions were observed. In the control group, standard postoperative management involved application of tulle gras dressing to the donor site without any MB injection. The application has been represented in Figures 1 and 2.

 

Figure 1: Donor Site of STSG on the thigh

 

Figure 2: Schematic representation of intradermal MB injection at the STSG donor site: Depicting the technique of intradermal administration of MB at the skin graft donor site, demonstrating the injection plane, distribution within the dermis, and coverage of the donor area for postoperative pain management

 

Postoperative care and assessments

All patients received routine postoperative analgesia comprising intravenous paracetamol (1g every 8hours). Opioids were prescribed as rescue analgesics when required, primarily in patients with larger donor sites (>20 cm). Dressing changes were performed on postoperative days (POD) 5, 7 and 15. During each change, pain, wound healing and potential complications were assessed. Pain intensity was evaluated using the VAS, a 10-point scale ranging from 0 (no pain) to 10 (worst imaginable pain). Patients were specifically instructed to rate the pain experienced during dressing changes, as this reflects donor site sensitivity.^15,16 Healing was assessed by clinical examination and defined as complete epithelialisation of the donor site. Scar quality was evaluated on day 15 using clinical assessment. In the intervention group, the duration of visible blue staining at the donor site was recorded.

Outcome measures

The primary outcome was the intensity of donor site pain during POD 5, 7, and 15. Secondary outcomes included duration of analgesic use, requirement of rescue analgesia, time to complete epithelialisation, scar quality at one month, persistence of staining, and occurrence of adverse effects such as hypersensitivity, infection or delayed healing.

Data management and statistical analysis

All data were collected prospectively using standardised case record forms (CRFs), anonymised, and securely stored. Statistical analyses were performed using SPSS version 31. Continuous variables were expressed as mean ± standard deviation (SD), and categorical variables as frequencies and percentages. Intergroup comparisons were performed using the independent t-test for normally distributed variables and the Mann–Whitney U-test for non-normally distributed data. The magnitude of treatment effect on pain intensity was quantified using Cohen’s d for the overall mean VAS score. A p-value of <0.05 was considered statistically significant.

Results

A total of 60 patients undergoing STSG were enrolled and randomised equally into the intervention group (n=30), which received intradermal 1% MB at the donor site, and the control group (n=30), which received standard tulle gras dressing without MB.

The demographic and clinical profiles of the two groups were comparable at baseline and are presented in Table 1. The mean age of participants was 43.50±12.73 years in the intervention group and 43.03±14.37 years in the control group (p=0.89). The proportion of male participants was slightly higher in the control group (83%) compared to the intervention group (67%), although this difference was not statistically significant (p=0.233). The indications for grafting included cellulitis (57% in the intervention group versus 67% in control), necrotising fasciitis (37% versus 13%), and other causes (6% versus. 20%), with no significant group differences (p=0.108). A total of three patients (10%) in the intervention required additional analgesia compared to eight patients (26%) in the control group (p=0.09). Despite the use of additional analgesics, patients in the case group consistently reported lower VAS scores compared to the control group across all assessed time points. Although wound size distribution did not differ significantly between groups, a trend toward larger donor sites in the control group was observed. This imbalance may have contributed to higher pain scores in the control arm. However, despite this potential confounder, the intervention group demonstrated consistently lower pain scores across all postoperative assessments, suggesting a robust analgesic effect.

 

Table 1. Comparison of baseline characteristics, pain scores and clinical outcomes of intervention and control groups after STSG

 

Pain intensity assessed using the VAS on POD 5, 7 and 15 is presented in Table 2. The intervention group demonstrated significantly lower pain scores at all times. On POD 5, the mean VAS score in the intervention group was 3.9±1.97, compared to 8.6±1.42 in the control group (p<0.00001). By POD 7, the intervention group further improved to 1.96±0.92, whereas the control group reported a VAS score of 6±1.38 (p<0.00001). By POD 15, pain in the intervention group had reduced to near-baseline levels (VAS 0.86±1.27), while the control group continued to report moderate pain levels (VAS 2.76±1.0), with the difference remaining statistically significant (p<0.00001).

 

Table 2. Distribution of postoperative pain intensity (VAS Scores)
between intervention and control froups following STSG

 

The average pain score over the three time points was 2.2±1.06 (95% CI: 1.82–2.58) in the intervention group versus 6±0.9 (95% CI: 5.68–6.32) in the control group (p<0.00001). This corresponded to a very large effect size (Cohen’s d=3.86; 95% CI: 4.73–3) indicating a marked reduction in donor-site pain in the intervention arm, favoring intradermal MB as clinically significant over control treatment. These findings were seen despite both groups receiving added analgesia and confirmed that patients receiving intradermal MB experienced faster and more substantial pain relief.

Discussion

Effective pain management following STSG remains a persistent clinical challenge, with donor site pain often exceeding discomfort at the recipient site. Conventional pain control strategies, including topical agents, systemic analgesics, and regional anesthesia, frequently provide incomplete or short-lived relief. In this context, MB, a compound traditionally used as a biological dye and antidote, has gained increasing attention for its analgesic and anti-inflammatory properties in diverse clinical settings, such as idiopathic pruritus ani, discogenic low back pain, osteoarthritis and postoperative surgical pain.^7,8,17 Building on this growing evidence base, the present randomised controlled trial evaluated the role of intradermal MB as a local analgesic for donor site pain following STSG.

The findings of this trial demonstrate a consistent and clinically meaningful reduction in pain intensity among patients receiving intradermal MB, with significantly lower VAS scores observed at all postoperative time points compared with standard dressing alone. This reduction was evident despite both groups receiving standardised baseline analgesia, suggesting an additive analgesic effect attributable to MB. While pain assessment relied primarily on patient-reported VAS scores, the parallel reduction in rescue analgesic requirement in the intervention group supports the clinical relevance of the observed pain relief and provides an indirect objective correlate. Given the visible blue staining associated with MB, patient and clinician blinding was not feasible; however, outcome assessors were blinded, and pain reduction was sustained across multiple postoperative assessments, reducing the likelihood that expectation bias alone explains the magnitude of effect.

Mechanistically, the analgesic benefit observed is biologically plausible. MB inhibits NO–mediated inflammatory signaling through suppression of inducible NOS and may exert localised neurolytic effects on peripheral nociceptive fibers.⁵ These mechanisms are particularly relevant in donor site wounds, where inflammation and superficial nerve exposure contribute substantially to postoperative pain. Importantly, the analgesic effect appeared independent of donor site size, although a trend toward larger wounds in the control group may have modestly amplified pain differences.

Importantly, MB was well tolerated by this cohort. No hypersensitivity reactions, infections, delayed epithelialisation  or adverse scar outcomes were observed, and donor site healing progressed similarly in both groups. The only notable side effect was transient for local staining, which resolved spontaneously within a few weeks and did not interfere with wound assessment or healing. Although systemic absorption and biochemical markers of inflammation were not specifically evaluated, the low intradermal dose used, absence of systemic symptoms, and established safety profile of MB at doses below 2mg/kg support its clinical tolerability.^2–4 These findings are consistent with prior reports of MB use in anorectal and spinal procedures, where local staining was temporary and not associated with long-term adverse effects.^7,18

The present results align well with existing literature supporting MB’s analgesic efficacy. A recent meta-analysis including 598 patients demonstrated that MB significantly reduced postoperative pain within the first 12 hours following hemorrhoid surgery without increasing complication rates.¹⁹ Similarly, studies in total knee arthroplasty have reported sustained reductions in pain scores from 24 hours up to 28 days postoperatively in MB-treated patients compared with controls.²⁰ Surgical studies, such as those by Mandovra et al, further report decreased postoperative pain and reduced analgesic requirements with MB use, reinforcing its role as an effective adjunct in surgical pain management.²¹ Our study extends these findings to the domain of skin graft donor sites, an area where high-quality interventional evidence remains limited.

Overall, intradermal MB appears to offer a safe, clinically significant and low-cost adjunct for donor site pain control following split-thickness skin grafting. While future studies incorporating double-dummy designs, objective inflammatory or neurophysiological markers, and longer-term safety assessments would further strengthen the evidence base, the current findings provide clinically relevant support for its use in routine surgical practice, particularly in resource-limited settings.

Strengths and limitations of the study

This study has several notable strengths. The prospective design standardised surgical technique performed by a single surgical team, and blinded outcome assessment strengthens internal validity and reduces measurement bias. In addition, careful safety monitoring, including preoperative sensitivity testing and structured follow-up, demonstrated a favorable tolerability profile for intradermal MB without adverse effects on wound healing or scar quality. However, certain limitations should be acknowledged. Although baseline characteristics were comparable, a trend toward larger donor site wounds in the control group may have contributed to higher pain scores. The relatively small sample size and single center setting limit generalisability. Furthermore, reliance on subjective pain assessment using the VAS, while clinically relevant, may be influenced by patient perception and expectation bias. The absence of objective biomarkers of inflammation, nerve recovery, or long-term scar assessment represents another limitation. Future multicenter studies with larger sample sizes, stratified randomisation based on wound size, extended follow-up, and incorporation of objective outcome measures are warranted to validate and expand upon these findings.

Conclusions

This study demonstrates that MB is an effective and safe adjunct in reducing pain following STSG, particularly in patients with smaller grafts. Its incorporation into routine postoperative protocols could enhance patient comfort, reduce reliance on opioids, and improve overall recovery following reconstructive procedures.

This research contributes to the growing body of knowledge on alternative analgesic therapies and could potentially redefine pain management protocols in dermatological and reconstructive surgery. By bridging the gap between preclinical findings and clinical application, the study validated MB as a viable option for improving pain management outcomes in patients undergoing STSG.

Conflict of interest

The authors declare no conflicts of interest.

Ethics statement

The study protocol was reviewed and approved by the Institutional Ethics Committee (IEC) of Lokmanya Tilak Municipal General Hospital and Lokmanya Tilak Municipal Medical College. The trial was conducted in accordance with the principles of the Declaration of Helsinki (2013) and Good Clinical Practice guidelines. All participants provided written informed consent prior to enrolment.

Funding

The authors received no funding for this study.

Author contribution

AS: Conceptualisation; Investigation; Data curation; Formal analysis; Writing (original draft). SI: Conceptualisation; Supervision; Validation; Writing ( review and editing). KF: Investigation; Data curation; Resources; Writing (review and editing). RS: Methodology; Formal analysis; Writing (original draft). MS: Investigation; Data curation; Validation; Writing (review and editing). All authors approved the final manuscript and agree to be accountable for all aspects of the work.

Author(s)

Ashvi U Solanki¹, Sandhya Iyer¹, Kausar Fakih¹, Riddhi U Solanki²*, Mansha Singh^1
1Department of General Surgery, Lokmanya Tilak Municipal Medical College and General Hospital, Sion, Mumbai, India
²Indian Institute of Public Health Gandhinagar, India

*Corresponding author email riddhis195@gmail.com

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