Treatment of acute wounds in hand with Calendula officinalis L.: A randomized trial

ABSTRACT

Most injuries in the hand and fingers, especially on the digital pulps, are suited for healing by secondary intention. Nevertheless, delay in epithelization seems to unfavorably restrict this technique. The purpose of this controlled randomized clinical trial is to analyze by means of photo planimetry the progression of the healing process by secondary intention in acute wounds of the hand using the standardized extract of Calendula officinalis L. (SEC). The cohort of eligible participants included two groups of 20 patients with skin loss in the hand and fingers treated by secondary intention. Control group (CG) used mineral oil and intervention group (IG) received SEC. Wound pictures were captured at each outpatient assessment until epithelization was achieved and measured with ImageJ. Intervention group (IG) and control group (CG) with 19 wounds each, primarily formed by men in their 40’s with wounds in their index and ring fingers on the left side, showed homogeneous variables and similar initial wound areas. Epithelization time was shorter and healing speed was faster in IG (IG = 8.6 ± 4.7 days and 9.5 ± 5.8%day versus CG = 13.2 ± 7.4 days and 6.2 ± 2.9%day, ƿ < 0.05), leading to the conclusion that healing by secondary intention in acute wounds of the hand and fingers with SEC led to a faster epithelization.

Introduction

Injuries in the hand and wrist represent approximately 20% of all hospital emergencies, and 4 in each 10 cases correspond to finger injuries.^1–3 Many of these lesions involve loss of skin which demands healing by secondary intention. To find a procedure that speeds the re-epithelization process is of utmost importance, so patients can resume their professional and daily activities in the shortest possible time.⁴

Wound healing process is the result of complex interactions expressed in inflammation, repair and remodeling phases. Healing by secondary intention depends on the wound contraction by means of the centripetal movement of the normal skin.⁵ Proliferation, uniform migration and differentiation of keratinocytes depend on the adhesion between adjacent cells and the underlying surface regulated by tight junctional integral membrane proteins, such as claudins, occludin and junctional adhesion molecules, during the process of rebuilding the lost epidermis.⁶

The indication for secondary intention consists of the lack of exposure of the deep tissues (bone, vein, nerve, and tendon), which tend to pull the wound and lead to esthetic defects, contractures, and limitations of movements, especially close to the joint areas.⁵ Small and superficial skin loss or surfaces where healing by primary intention is incomplete may require healing by secondary intention as a plausible and successful alternative.^5,7,8 Skin losses on hands and fingers are appropriate for this type of treatment. However, the unprecise and many times prolonged time for the epithelization of the wounds in the hand and fingers is an unfavorable aspect of the use of secondary healing.⁴

Calendula officinalis L., a plant of the Asteraceae family, popularly known as calendula, is used in several medicinal products and represents an important source of compounds that may serve as new and efficient products for the wound care.⁹ Extracts obtained from the calendula flower are rich in saponins, flavonoids, sesquiterpenes, alcohol, triterpenoids, hydroxycoumarin, carotenoids, tannin, and essential oils, responsible for the antioxidative, anti-inflammatory, wound healing, antibacterial, antifungal, antiviral, antiedematous and immunomodulating factors.^10,11 Faradiol monoesters (triterpenoids) are considered the main active components of the calendula’s anti-inflammatory activity.¹² They can induce fibroplasia and angiogenesis and positively contribute to the wound healing processes in vitro and in vivo.^13,14 In animal models, the use of Calendula officinalis L. in skin loss presents encouraging results in histopathological analysis of wounds, such as angiogenesis stimulation and increase of hydroxyproline and hexosamine, which impact the formation of collagen and the decrease of epithelization time when compared with the control group.¹⁵ Similarly, good results in the use of calendula extracts have been reported in the management of complicated and chronic wounds, such as the ones resulting from venous, pressure and diabetic ulcers, due to its initial anti-inflammatory property and positive interference in the collagen synthesis.^9,15,16 Nevertheless, the authors could not identify in the researched studies the use of the calendula extract in clinical trials of traumatic skin loss of the hand and fingers treated with healing by secondary intention. Hence, the question that is raised is whether, according to its potential wound healing properties, Calendula officinalis L. can accelerate the epithelization of acute wounds and promote the prompt return of the functional activities of the hand.

Based on these considerations, the objective of this controlled randomized clinical study is to analyze the progression and speed of the healing process by secondary intention in acute wounds of the hand and fingers by using the standardized extract of Calendula officinalis L. (SEC), assessed by photo planimetry.

Material and methods

Recruitment

Control and intervention groups were formed by patients seen at the emergency room of the School Hospital who presented acute trauma in the hand and fingers, mainly with skin loss or wounds in which primary healing was not possible. Each group was composed of 20 patients, under the age of 18, presenting acute wounds smaller than 10 cm² in the hand and fingers, whose treatment by the emergency physician was defined as secondary intention healing. Randomization was performed by using sealed envelopes in the first outpatient evaluation under the supervision of one of the authors.

Wounds in more than one finger of the same patient were included. Exclusion criteria consisted of wounds with exposed bones, nerves or tendons, lack of adherence to treatment evidenced by inappropriate wound cleansing, clinical signals of wound infection, adverse reaction to prescribed medication, absence of signed informed consent or treatment withdrawal. Patients’ data about diabetes control and smoking were also considered. The research was approved by the Committee of Ethics and Research (number: 28118614.3.0000.0020) and registered at ReBEC (Brazilian Registry of Clinical Trials) RBR-3sg8mbc.

Products

In the intervention group, 2% standardized extract of Calendula officinalis L. (SEC) was used twice a day, prepared by state-licensed compounding pharmacies, according to the legal criteria of the National Agency of Sanitary Surveillance (ANVISA). This product is extracted from the flowers of Calendula officinalis L. (Plenusdermax®/Phytoplenus Bioativos S.A.), using specific extractors (polyethylene glycol hydroxytoluene, paraben, ethanol) under controlled pressure, frequency, and temperature. Mineral oil (Farmacêutica Cearense Ltda Chemical Industry) routinely provided by our institution, was used as control product¹⁷ and, like SEC, provides hydration to the wound and prevents trans-epidermal fluid loss during the epithelization process.¹⁸

In both groups, patients and their families received oral and written instructions about the daily sequence of wound management, the drying of the wound edges with paper towel, and product application. Similar dispensers were used for both products delivered to patients. After the sprinkling of the product on the wound, they were recommended to wait for natural drying and apply conventional dressing (dry gauze and bandage). These recommendations were verified and photographed at each outpatient consultation until complete epithelization was achieved. We have considered a fully epithelialized wound as the one that appears firm and granular and presented no granulation tissue.¹⁹ As exemplified in Figure 1, the epithelialized tissue presents a pearl pink color like an opaque and frosted finish.

Figure 1.

Final stage of wound healing by second intention in the index finger. (a): Exemplifies an area in process of epithelization that keeps a central small granulation area (b): Close detail of the small granulation zone measured with ImageJ software. In black, the outline of the margins between the granulation process and the area already epithelialized. The measurement of this granulation area was the one used for statistical calculations (c): Epithelialized wound.

Planimetry

Disposable stickers with a uniform area (176.7145 mm²) and a different color for each group were used as a gauge for further measurement of the wounds, which were photographed with an 8-megapixel digital camera (Canon, Powershot SX20is). About ten pictures were taken perpendicular to the wounds, in a 90° angle, approximately 50 cm far, and close attention was paid to the formation of shades, variation of lighting and focus. Both researchers that assessed the wounds advised the patients about daily care in the use of products, and those who took the pictures maintained confidentiality about the identification of groups.

The wound planimetry phase was performed by the main researcher, who was blinded about the correspondence between the gauge color used in the photodocumentation, the intervention and control. Image J (National Institute of Health/ USA)²⁰ software was used to measure the wound areas. Recommendations by Aragón-Sánchez et al were used to standardize the outline of the wounds and their measurements.²¹ To measure the delimited area between the epithelized or dry necrosed margins and the granulation or wet necrosed area in the wounds, images obtained from each outpatient evaluation were used. These images were amplified using the same software program to obtain better delimitation of the areas. Three readings of the gauge and the wound areas were performed from the images captured in each one of the days during the healing process by secondary intention. The mean value of the three readings in Mega Pixels was transformed into square millimeter. The planimetry of the wound performed each day during the outpatient assessment enabled the calculation of the daily contraction of the wound and its healing speed.

The wound contract (WCd), expressed in mm²/day, was measured as the difference between the initial area (iA) and the final area (fA) in mm², divided by the time in days, considering the photo documentation of the initial area and of the last one before epithelization, as shown in the expression below:

$$W{C}_d=\frac{\left(iA-fA\right)}{Time}$$

Healing speed (HS%d), expressed in daily percentage, represents epithelization rate, and was calculated considering the interval between the initial and final area and the time used to capture the images, as expressed below:

$$H{S}_{\mathrm{\%}d}=\frac{\left(100\ast \frac{\left(iA-fA\right)}{iA}\right)}{Time}$$

Statistical analysis

IBM SPSS Statistics v.20.0 (Armonk, NY: IBM Corp.) was used for statistical calculations and p < .05 values were considered statistically significant. The results of the quantitative variables were expressed by the mean, standard deviation and interquartile interval, and the qualitative variables were described by frequencies and percentages. The last photo documented area before complete epithelization was considered the final one as the final time was the day of the last picture taken before the healing (Figure 1(c)). The comparison between the groups (control and intervention) in relation to the quantitative variables age and healing speed was performed using Student’s t test for independent samples with equal variances not assumed (Levene’s test). Non-parametric Mann-Whitney U test was used to analyze the other quantitative variables – initial and final areas of the wounds, number, and interval between the collection of photo documentation, treatment time by secondary intention and contraction of wounds, due to the lack of normality of these variables. Normality was evaluated with the Shapiro-Wilk test. Comparison between the groups in relation to categorical variables was performed using either Fisher’s exact test or Chi-Square Test.

The relationship between the treatment effect within the groups and the healing speed was achieved using a simple linear regression model, considering the group treatment as the explanatory variable. In further analysis, the multivariate model was adjusted, including the covariables gender, age, diabetes, and smoking.

The time elapsed until the observation of the last area before epithelization (outcome) was described by Kaplan-Meier curves. Logrank test was used to compare the curves defined by the control and intervention groups.

Results

The 40 randomized patients of both groups totalized 40 wounds, 22 in the intervention group (IG), which used standardized extract of Calendula Officinalis L. (SEC), and 20 in the control group (CG), treated with mineral oil. After the measurement of the initial wound with the ImageJ software, patients of the IG with wounds larger than 1000 mm² were excluded. Three wounds were excluded for not presenting previous measures of epithelization (2 wounds from the IG and 1 from the CG), that is, in the second outpatient visit epithelization was already present, preventing the accurate measurement of the area before epithelization and determination of the wound healing speed. Hence, the study cohort consisted of 17 patients with 19 IG wounds and 19 CG wounds. Table 1 shows the distribution of the variables in both groups. No significant differences were observed in the distribution of the control and intervention groups for the variables age, gender, diabetes, and smoking (Table 1).

Table 1.

Comparison of groups in relation to age and categorical variables (gender, presence of diabetes and smoking)

Variable	Classification	Group			p*
		Control (19 patients)	Intervention (17 patients)
Age (years)	Mean ± SD	43,4 ± 20.3		41.2 ± 18.2	0.736
	Median (IQR)	40 (42)		35 (33.5)
Gender (%)	Female	26.3		17.6
	Male	73.7		82.4	0.695
Diabetes (%)	No	89.5		94.1
	Yes	10.5		5.9	1
Smoking (%)	No	84.2		76.5
	Yes	15.8		23.5	0.684

*Student’s t test for independent variables (age); Fisher’s exact test or Chi-Square test (categorical variables); p < .05; SD: standard deviation; IQR: interquartile range.

The distribution of the wounds in the groups regarding wound location and laterality is shown in Table 2. In the CG, 31.6% of the patients suffered injuries on their way to or during their paid work. Similar proportion was observed in the IG (35.3% of the patients).

Table 2.

Distribution of wounds of the control and intervention groups, in relation to the wound location and laterality.

	Control Group 19 lesions		Intervention Group 19 lesions
Location	right side	left side	right side	left side
1st finger	2	2	1	2
2nd finger	1	3	3	2
3rd finger	1	1	2	1
4th finger	2	3	2	2
5th finger	0	2	1	2
Palm (N = 3)	1	1	0	1

Table 3 shows the analysis of the quantitative variables related to the 19 trauma wounds of each group. Considering the initial and final area of the wounds and the interval between the days for outpatient wound assessments and capturing of photographs (sampling interval) there was no difference between the groups. Epithelialization time and number of samplings were significantly lower in IG (ƿ = 0.046 and ƿ = 0.018, respectively) and healing speed was higher (p = .036).

Table 3.

Quantitative variables of the 19 wounds in each group (control and intervention).

	Control Group n = 19		Intervention Group n = 19		ƿ* Value
VARIABLE	Mean ± SD	Median (IQR)	Mean ± SD	Median (IQR)
Initial Wound Area mm2	182.3 ± 183.5	146.2 (120.9)	171.3 ± 121.2	153.7 (117.1)	0.686
Number of Samplings	4.4 ± 1.8	4 (2)	3.1 ± 1.3	3 (2)	0.018
Sampling Interval mean of days	4.1 ± 1.9	3.5 (0.8)	4.2 ± 1.6	3.5 (0.5)	0.931
Final Area** mm2	41 ± 40.6	28.7 (51,1)	64.2 ± 72.3	37.2 (64.7)	0.311
Time days	13.2 ± 7.4	11 (14)	8.6 ± 4.7	7 (7)	0.046
Wound Contraction mm2/day	12.5 ± 18.5	7 (9.3)	13.8 ± 8.9	8.8 (14.3)	0.103
Healing Speed % day	6.2 ± 2.9	6.2 (3.9)	9.5 ± 5.8	6.9 (9.3)	0.036

SD: standard deviation; IQR: interquartile range. * p value in Student’s t test for independent variables or in non-parametric Mann-Whitney test. **Final area was the last assessment before the wound epithelization.

The result of the univariate linear regression model for the healing speed (Table 4 – model 1) indicated that there was a significant association between the treatment groups and the healing speed (ƿ = 0.033). The attained angular coefficient of 3.33 indicates that the healing speed in the intervention group is 3.33 percentage points per day faster when compared to the control group.

Table 4.

Models of Linear Regression.

	Explanatory Variable	ƿ	Estimated Coefficient	CI 95%
Model 1	Treatment in group	0.033	3.33	0.27–6.38
Model 2	Treatment in group	0.048	3.20	0.02–6.37
	Gender	0.325	1.90	−1.98–5.78
	Age	0.700	−0.02	−0.10–0.07
	Diabetes	0.907	0.35	−5.67–6.36
	Smoking	0.257	−2.35	−6.50–1.79

Model 1: Univariate analysis, considering the dependent variable as the healing speed (% day) and the treatment in groups as the explanatory variable. The healing speed in the intervention was 3.33 percentage points higher than the one in the control group

Model 2: Multivariate analysis, including as co-variables: gender, age, presence of diabetes and smoking. Healing speed remained faster in the intervention group, p = 0,048, regardless of the inclusion of the covariables.

No changes in the treatment effects were observed when covariables such as gender, age, diabetes, and smoking were added to this regression model, that is, the healing speed remained significantly faster in the intervention group (ƿ = 0.048; angular coefficient = 3.20). (Table 4 – model 2)

Figure 2 demonstrates the Kaplan Meier curves using as parameters the proportion of epithelialized wounds in the groups related to time in days. The outcome was the observation of the final area (last area photographed before epithelialization of the wounds). A significant difference was observed between the curves of the two groups in the longrank test (ƿ = 0.02). Epithelialization time was shorter in the intervention group when compared to the control group.

Figure 2.

Kaplan Meier curve: Evolution of epithelialization in groups. The curves demonstrate the cumulative proportion of wounds that epithelized over time. Solid line represents intervention group and dotted line represents control group. Logrank test ƿ = 0.02.

Figure 3 exemplifies the clinical evolution of patients in the intervention group.

Figure 3.

Intervention group clinical evolution. (a): Shows initial area of 418.1 mm². (b): Clinical aspect at 7 days. (c): Epithelization at 14 days. Male, 23 years old.

Discussion

This randomized clinical trial analyzed the evolution of the healing process by secondary intention in hand and finger wounds. By means of image planimetry, it was possible to verify the progressive decrease in the wound area by comparing the healing speed reported in the study groups. The aspect raised by the authors was the necessary acceleration of the process, mainly in the hand and finger wounds, relevant for the performance of most daily activities. The similarity of the samples in both groups enabled the authors to identify a shorter epithelization time and faster healing speed in the intervention group (Table 3).

It should be pointed out that few articles report the secondary intention healing process in acute wounds, although it is a common practice among surgeons who recommend it for the treatment of superficial wounds. Much of what we found in the literature referred to the treatment of acute wounds in experimental studies^17,22,23 and in resection of skin tumors.^24–26 A study by Bosley et al about skin loss on the dorsal part of the hand and fingers in 48 patients who underwent resection of squamous cell carcinoma, between 0.8 and 6 cm, reports excellent results based on functional ability, sensitivity and esthetics after 10 months of healing by secondary intention.⁸ They stress out that healing by secondary intention is an effective option in the repair of skin losses in the hand and fingers, simplifies wound care procedures, minimizes complex surgical techniques, and reduces treatment costs. Similarly, Ehrlich and Hunt in their thorough review on wound contraction mechanism conclude that a faster secondary intention process would avoid discomfort and secondary defects caused by skin grafting.⁵ Additionally, as reported by Dias and Garcia Elias, outpatient procedures with shorter hospitalization time help decrease direct and indirect costs for the treatment of wounds in the hand.²⁷ There are reports of good results from the use of secondary intention in acute wounds without exposure of deep tissues, superior to skin grafting, especially in tip amputations. Kakar cited that wound with skin loss progress in the process of granulation and epithelialization by secondary intention with complete return of sensitivity and discrimination between two points, providing better results when compared to the use of surgical skin grafts.⁴

Importance should be given to recent studies on the role of tight junction (TJ) proteins in the wound healing process. Knockdown experiments in primary human keratinocytes revealed delayed migration and reduced proliferation due to the decreased expression of claudins.⁶ The authors concluded that the effects caused by changes in the location and expression of claudin-1 and occludin KD in human keratinocytes may be an important feature in the pathogenesis of non-healing wounds. Although TJ proteins are expressed by epithelial cells at wound edges and regulate signal transduction, modulate cellular processes and also interfere on the immune response and homeostasis, little is known about the complete action of TJ proteins in the healing process. TJs may be more essential to wound remodeling than early healing stages. Shi et al raised several questions about the role of TJs in the different phases of wound healing and the way in which the expression of TJ proteins impacts the inflammatory response in healing and in non-healing wounds.²⁸

Calendula officinalis L. was selected to be used in this study as it is known for accelerating epithelization and offering advantages during the healing process.^13,14,29 Variation in the composition and concentration of the plant extract make it difficult to compare the effect of Calendula officinalis L. in wound healing in vivo.^9,15 Givol et al, in their five studies with animals and a randomized clinical trial with episiotomy wounds stated that healing of acute wounds was faster at the inflammation stage with the increase in the production of granulation tissue in the groups treated with calendula extract, most of them ethanolic with no specifications about its composition. In chronic wounds, they reported variable results with the use of calendula extract, in general. However, the lack of specifications regarding its composition and concentration may have influenced the results.¹⁵ The authors also mention the use of the standardized extract of Calendula Officinalis L. of this study in a clinical trial which reports a significant decrease in the wound area when compared to the control group for the treatment of venous ulcers.¹⁶ The option for the use of the SEC in this research study, in glycolic and non-ethanol vehicle, may be accounted for by the higher security in obtaining the plant bioactive compounds and in the topical use of the product, due to its humectant properties for the skin. Conversely, the ethanolic extract tends to dehydrate the wounded tissue and cause discomfort when applied.

Kharat et al, studying infection as a barrier to wound healing added Calendula officinalis L. to chitosan/polyethylene oxide (CS/PEO) scaffolds by electrospinning. They observed an improvement in the mechanical properties of CS/PEO nanofibers and a significant reduction in Gram positive and negative bacteria (96% and 94%, respectively). The authors reported in vitro studies of fibroblast cells using CS/PEO loaded with Calendula officinalis L. that resulted in increased proliferation, growth and cell adhesion. Moreover, histological analysis of acute wounds in rats showed that CS/PEO/Calendula officinalis L. dressings acted positively in collagen synthesis, epithelialization and tissue remodeling resulting in excellent wound healing.³⁰

Few studies report the healing speed of acute wounds until the epithelization phase. Some articles address the importance to measure the wound areas mainly when referring to wound chronicity. Flanagan, in a narrative review, suggested that the most efficient way to monitor healing is by calculating the reduction in the percentage of the wound area over time, mainly in the first weeks, which will provide the early identification of the factors that could delay it.³¹ Khoo & Jansen also emphasize the importance of the decrease in the wound area between 20% and 40% in the first weeks as predictors of healing.³² They also suggest that larger than 10 cm² areas tend to be distorted in the captured images for computer program analysis, the reason why we have limited the inclusion of larger wounds in our study.^31,32 One factor that can lead to some difficulties in the measurements was the date of the patient’s last assessment that would not always match with the precise time of the epithelization evidence. Therefore, in our study we used the measurement of the area and time of the last outpatient appointment before verifying the epithelialized wound.

It is also noteworthy that the total number of samplings was significantly lower in the intervention group, due to the shorter time of epithelialization in these lesions. We also emphasize that acute and uncomplicated wounds tend to heal by second intention, evolving to granulation, contraction and epidermal migration in a progressive and expected way,^5,31 resulting in equivalent wound contraction in both groups. (Table 3)

In this randomized clinical trial, both groups were mostly made up of men in their forties. The majority answered “no” to the use of drugs for the control of diabetes (CG: 89.5%, IG: 94.1%) and almost one fourth reported daily use of cigarettes (CG: 15.8%, IG: 23.5%) (Table 1). DiPietro, as a coauthor in narrative reviews, suggested that local and systemic factors interfere in the wound healing process, both acute and chronic.^33,34 Oxygenation, infection, presence of foreign bodies and venous insufficiency are some of the referred local factors. We also point out diabetes and smoking among the systemic factors that may affect the healing of skin wounds, leading to complications such as infection and chronicity. There was no difference in the healing speed of the patients with diabetes or smoking habit in our study, verified in the multivariate regression model (Table 4). The healing speed in the intervention group (SEC) remained faster regardless of the inclusion of variables such as diabetes, smoking and even age or gender (Table 4). Indexes that grade the severity of diabetes and daily smoking of our patients were not measured, representing one of the limitations of our study. We also refer to the limitations of the wound measurement techniques and the examiner-dependent effect variations, although minimized by the previous training performed by the main author of this research.

Despite the promising results reported in this study and the limited knowledge available about the interference of TJ proteins in wound healing, further studies on the standard extract of Calendula officinalis L. are recommended, mainly to explore its role in barrier function and interactions between epithelial cells during the wound healing process.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are openly available in ReBEC (Brazilian Registration Platform of Clinical Trials) at https://ensaiosclinicos.gov.br/, reference number RBR-3sg8mbc.

Authors’ contributions

GSG: Conceptualization, Methodology, Formal analysis, Software, Validation, Writing- Original draft preparation; EMN: Data curation, Visualization, Investigation Writing- Reviewing and Editing; LCGS: Writing- Reviewing and Editing, Supervision

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website