Design of a new generation wound dressing with pine bark extract

Abstract

Medical textiles are one of the fastest growing sectors in the technical textile market. Wound dress is one of the significant applications with the largest share in medical textiles. Active molecules doped in the dressings may be therapeutic agents, vitamins, antibiotics, minerals, and growth factors, which contribute to wound healing. Medical plants have a great potential with positive effects in wound care and accelerate the rate of wound healing. Pine bark, which is known to exhibit wound healing properties, is also used in the medical field. The purpose of this study is to design a new wound dressing enriched with Pinus brutia extract. Microwave-assisted extraction which is an environmentally friendly method was carried out at 70℃, 900 W for 10 min to obtain the extracts. Subsequently, P. brutia bark extract was embedded to the alginate gel dressing and characterized and evaluated by in vivo studies on rats. According to the results, the extract was rapidly released from the alginate gel in the first 6 h, whereas the release was slowly increased to 24 h and then reached a steady state. Therefore, P. brutia extract-embedded alginate gel dressings applied for in vivo studies were changed every 24 h, reaching a healing rate of 75.7%, whereas the control group showed a healing rate of 48.6% indicating the superiority of the newly designed wound healing dress enriched with pine bark extract.

Introduction

The use of textile materials in the field of health care is dated back to old times. Since then, population growth in developing countries with a significant increase in the elderly population, improvements in living standards, and increased demands for health-care products have led to the development and growth of the medical textiles sector. Wound dressing, which has the largest share of medical textiles, is one of the significant applications. Wound healing is the process of repairing and reshaping the damaged tissue. Recently, treatments with natural and medicinal plants have become the focus of research due to the limitations and adverse effects of synthetic drugs [1]. Therefore, an increasing trend is observed for the usage of medicinal and aromatic plants as natural sources of antioxidants, antimicrobial, and anti-inflammatory compounds. Although it has been used for many years, pine bark has recently holds a wide area of application in nutrition health and medicine. The high content of phenolic compounds and procyanidin flavonoids in Pinus brutia extract [2] dictates its potential in medical industry. Phenolic compounds and flavonoids directly contribute to the antioxidant property [3] and play preventive role in wound healing process. The main purpose of using a wound dressing is to accelerate wound healing by preventing bacterial infection while enhancing tissue regeneration. Wound dressing materials should have essential properties such as the facility to prevent water loss, flexibility, gas permeability, and durability [4]. Alginate as an anionic hydrophilic polysaccharide is used in the fabrication of hydrocolloid film wound dressings due to its biocompatibility, biodegradation, and excellent film-forming properties. Alginate hydrogels can retain and create a moist environment around the wound to promote wound healing and epidermal regeneration [5]. In the presence of water, alginate-based hydrogel films with various degrees of crosslinking can be prepared by replacing the sodium ions of sodium alginate (SA) with di- and trivalent cations such as Ca²⁺, Cu²⁺, Ba²⁺, and A1³⁺ [6,7].

The objective of this study is to design a new wound dressing for acute wounds. For this, alginate solution was prepared by incorporating pine bark extract known to exhibit antioxidant and wound healing properties. By applying this solution to cotton fabrics, gelation was achieved on the surface of the fabrics and characterized in terms of release profile and functional groups. The ultimate aim was to design a wound dressing, and we hypothesized that if a moist environment for the wound can be sustained with pine bark extract-embedded alginate gel, then the released phenolic compounds and flavonoids can accelerate acute wound healing which was validated by the in vivo animal study.

Material and methods

Materials and reagents

In this study, scoured, rinsed, and plain weave cotton fabric (145.0 g/m² weight) was used for textile application. Fabric tightness was 21 thread/cm in weft direction and 29 thread/cm in warp direction. Yarn linear density was Ne 21 in weft direction and Ne 20 in warp direction. P. brutia barks were collected from Izmir-Deliomer (N: 38°10′17.0″, E: 27° 03′ 46.7″, altitude: 120 m) in Turkey. The barks were dried at room temperature, ground by using a conventional grinder, and stored at +4℃. Alginate were purchased from CHT Group. All other chemicals were used in analytical purity.

Methods

Preparation of extracts

About 2 g of P. brutia (dried parts) bark submerged in ethanol was transferred into the vessel of microwave equipment (Sineo Microwave Chemistry Technology Co., Shanghai). The extraction was carried out at 70℃, 900 W for 10 min. Then, the vessel was opened by with using a rotary vacuum evaporator (Hahnvapor RS2005V-N) and then stored at +4℃.

Preparation of wound dress

In this study, 2% SA solution was prepared with 2% P. brutia bark extract. The prepared solution was impregnated into cotton fabrics. After the removal of excess solution, the fabric was immersed in 10% CaCl₂ solution to allow crosslinking and formation of ionic gelation on the surface of the fabric. Samples were left to dry for 4 h and then packed and sterilized.

Fourier transform infrared spectroscopy analysis and confocal Raman microscopy

Infrared spectra in the range of 4000–650 cm⁻¹ were taken using attenuated total reflectance-Fourier transform infrared spectroscopy (FTIR) (Spectrum 100, Perkin Elmer) device for characterization of wound dressings with and without P. brutia bark extracts. Also, wound dressings with P. brutia bark extracts were analyzed using confocal Raman microscopy (Renishaw).

Release studies

For the release studies, alginate gel dressing with P. brutia bark extract was placed in a dialysis membrane and merged in the beaker containing 100 ml of phosphate-buffered saline (PBS) solution, covered with parafilm and shaken on a magnetic stirrer at 37℃ in the incubator. Samples were taken at certain time intervals, and 1 ml of PBS buffer solution was added in order not to affect the concentration due to the withdrawn samples. At 1, 2, 3, 4, 5, 6, 24, 48 and 72 h, absorbances of the solutions at 285-nm wavelength were measured by using ultraviolet spectrophotometer for the determination of the amount of the extract in the samples.

In vivo studies

Wound healing analysis

After anesthesia provided by intraperitoneal (IP) injections of xylazine (8 mg/kg) and ketamine (75 mg/kg), the rats were fixed in the sternal position and the dorsal region was shaved. A single dose of clindamycin phosphate was injected 7.5 mg/kg intramuscular (IM) as prophylaxis against infection. The skin was prepared for surgery with povidone–iodine solution and then sterile covers were placed. In the right mid dorsal region, the skin and the lower Musculus Cutaneus Trunci muscle were excised with a scalpel tip of 11 which contain all the layers 1 × 1 cm in size. Hemostasis was achieved with compresses made with sterile surgical gauze. Wounds were observed and dressed on a daily basis. For the dressing, the wound was cleaned with saline solution, and then the antisepsis was made with iodine solution.

Group 1: alginate gel dressing with P. brutia extract (n = 8),

Group 2: Control group (n = 8)

Alginate gel dressings with P. brutia extract were applied to Group 1, and no band dressing was applied to Group 2 and then covered with sterile gauze and plaster. On the 0, 7, 14, and 21st days, wound area was calculated as a result of markings made with special software developed for the purpose of planimetry on wound photographs taken in animals under anesthesia. This area is defined as full wound area. It was then referred to as the non-healing wound area by marking the boundaries of the progressive epithelium. The wound healing rate was calculated with a two-step process:

Step 1:

$$\text{Accordingtothefirstdayopenwoundarea}=\frac{\text{Openwoundarea}(\text{Measurementday})\times 100}{\text{Completewoundareaonday}1(\text{day}0)}$$

Step 2:

$$\text{Measurementdayfullrecoveryarea}\%=100-\text{firstdaytoopenarea}\%[8]$$

All data and mean value comparisons between the groups were made by using analysis of variance (variance) repetitive measurement model. In case of differences, Duncan's multiple-range test was used. SPSS 13.0 was used in all analyses. In determining the number of animals, 3R principles and related studies [9] were taken into consideration and the lowest number was determined. This study was carried out with the approval of the Local Ethics Committee of Animal Experiments of Ege University with the approval numbered 2018-005. The animals were observed daily during the study period, and all kinds of findings were recorded. The animals were examined at Ege University Laboratory Animal Research and Application Center during the research. Without feed restriction, water is housed in a free environment in a 12-h/12-h dark setting at 21℃ (Figure 1).

Figure 1.

Images depicting would dressing applications during in vivo studies.

Result and discussion

Microwave-assisted extraction was carried out at 70℃, 900 W for 10 min to obtain the extracts. Subsequently, P. brutia bark extract was embedded to the alginate gel dressing and characterized and evaluated by in vivo studies on rats.

Release studies

Release studies have been carried out to investigate the diffusion of active compounds in pine bark extracts embedded in alginate gel dressing. The release profile of pine bark extract from the wound dressing showed a rapid release in the first 6 h. After 6 h, the release was slowly increased until 24 h and then reached a steady state (Figure 2). Based on this result, the number of daily dressings and the total number of dressings to be applied to the rats were determined. According to the results, the wound dressings were decided to be replaced and renewed every 24 h.

Figure 2.

The release profile of Pinus brutia bark extract from wound dressing.

Fourier transform infrared spectroscopy

P. brutia has a high tannin content, with active groups, namely, polyhydroxy and polyphenol tannin groups. These structures have different polar functional groups such as alcohol, aldehyde, ketone, carboxyl, phenol, and ether [10]. FTIR spectroscopy (PerkinElmer, Spectrum 100) was used to analyze the functional groups, and the spectrum of P. brutia extract is given in Figure 3(a). The band at 3250 cm⁻¹ is assigned as OH stretch vibration in phenolic and aliphatic structures, and the width of this band indicates the presence of strong hydrogen bonds. Small peaks at 2950 and 2850 cm⁻¹ originate from CH stretch vibration in aromatic methoxyl groups and in methyl and methylene groups of side chains. The bands detected at 1518 cm⁻¹ are assigned to C=C bonds in aromatic rings vibration and 1440 cm⁻¹ corresponding to C–O bonds, and peaks at about 1200 cm⁻¹ indicate ketone C–C bonds [11]. The peak at 1100 cm⁻¹ may be caused by alcohol C–O bonds and the peak at 1054 cm⁻¹ from C–O–C bonds [12]. Finally, aromatic C–Hn bonds are about 766 cm⁻¹ [13]. Alginate gel dressings with and without pine bark extracts were analyzed and compared in terms of functional groups as well (Figure 3(b)). In both spectra, the wide band seen at 3310 cm⁻¹ represented the OH bonds in the structure. The spectrum of alginate gel dressing without pine bark extract indicated that the bands detected at 1634 cm⁻¹ were assigned to –COO (asymmetric) and at 1412 cm⁻¹—COO (symmetric) bonds. Moreover, C–O–C peaks were detected at 1085 and 1031 cm⁻¹. In regard to the spectrum of the alginate gel dressing with pine bark extract, an increase in the width of the peak band was observed at 1410 cm⁻¹, which might be due to the increase in C–O bonds from the extract.

Figure 3.

FTIR spectrum of Pinus brutia extract (a) and FTIR spectra of alginate gel dressing with (a) and without Pinus brutia bark extract (b) (b).

In comparison to the spectrum of the alginate gel dressing without pine bark extract, a very intense peak at about 1600 cm⁻¹ was observed, which corresponds to the increase in carboxylic C=O bonds. Furthermore, the presence of pine bark extract in the wound dressing resulted in the ketone C–C stretch from the active compound taxifolin around 1200 cm⁻¹.

Confocal Raman microscopy

Confocal Raman microscopy was used to determine the gelation on the surface. A proper gelation was achieved and the layer on the surface of the wound dress provides the necessary moisture for wounds (Figure 4).

Figure 4.

Confocal Raman spectral image of alginate gel wound dressing.

In vivo application of alginate gel wound dressing

Pine bark extract is rich in polyphenols and has important flavonoid compounds such as catechin, epicatechin, and proanthicin [14]. Particularly, P. brutia extract was reported to possess high amounts of taxifolin and catechin [15] with a high total phenol value and a radical scavenging activity [16]. These phenolic compounds exhibit antioxidant properties that not only protect the connective tissue but also maintain the integrity and flexibility. Besides, such compounds are utilized in health applications due to the ability of protecting the elasticity of the skin, preventing wrinkle formation and accelerating wound healing [17]. Antioxidant compounds applied for wound healing have been reported to protect tissues from oxidative damage [18]. As wound healing is defined as the completion of the closure of the clinically injured skin area [19], the anti-inflammatory activity of the compounds is of prime importance as well. In a previous study, the anti-inflammatory activity of P. brutia bark extract was investigated in a rat model of carrageenan induced paw edema. Saline (control group), pine bark extract, and indomethacine (positive control) were administered intraperitoneally prior to subplantar application of carrageenan to rats. Paw volume was measured for 6 h before and after the injection of carrageenan. Finally, the anti-inflammatory activity of P. brutia extract was assessed. No acute toxicity was identified in intraplantar injection of the extract at a dose of 2000 mg/kg. Therefore, P. brutia bark extract was reported to be utilized as an anti-inflammatory agent [20].

In this study, alginate gel dressing with P. brutia extract with a high phenolic content was tested on rats. Subsequent to the application, the effects of wound dressing on the rats have been evaluated in terms of wound healing and the ability to accelerate the wound healing process. There were two groups: one of which was experimental and the other one was control group. Each groups was consisted of eight animals. The areas of open wounds in rats were measured by a digital camera system on days 0, 7, 14, and 21. Average wound healing percentages were calculated for each group before and after treatment. Between 0 and 7 days, the rats had healed significantly, but the wounds were not completely closed.

At the end of the first week, 75.7% healing rate was observed in the experimental group treated with pine bark extract-embedded alginate gel dressing, while the control group showed a 48.6% healing rate (Figure 5). At the end of the second week, the wounds were completely closed in the experimental group, whereas the control group exhibited a healing rate of 88.9%. These findings are also supported by the images of the wounds for the experimental (Figure 6(a)) and the control group (Figure 6(b)). It is worth to mention that the wounds of the rats in the control group were closed on day 18. As a result of the statistical evaluation, a significant difference was found in the percentage of wound area between the experimental and the control group at the end of day 7 (p < 0.05) which indicated a more rapid recovery in the experimental group treated with pine bark extract-embedded alginate gel wound dressing.

Figure 5.

Average healing area (%) during 0–7 and 7–14 days.

Figure 6.

Experimental (a) and control groups (b) for healing phase.

At the end of the second week (p > 0.05), there was no statistically significant difference between the rats in the experimental and the control groups. The results revealed that wound healing rates were higher and reepithelialized times were shorter with obtained wound dress as compared to the control group.

Conclusion

Novel wound dressings are required for successful wound treatments. Active substances embedded in wound dressings may include therapeutic agents, vitamins, antibiotics, minerals, and growth factors to contribute to wound healing [21]. The use of medicinal plants to improve wound healing is an extensive practice, and the high content of phenolic compounds in P. brutia extract makes it a potential candidate for such applications. As known, antioxidants can accelerate the wound healing process. The results of the in vivo studies revealed the superiority of the wound healing properties of P. brutia bark extracts embedded in alginate gel wound dressing. The observed effect is attributed not only to the phenolic compounds and taxifolin in the pine bark extract but also to the ability of alginate to create a moist wound environment [22]. Indeed, the calcium in the alginate and sodium in the wound exudate and blood form a hydrophilic gel on the wound surface by an ion exchange reaction. This hydrophilic gel ensures a moist wound environment, which supports healing and epidermal regeneration [23,24]. The in vivo results on the rats are very promising from preclinical point of view and further studies can be conducted to observe the effects of pine bark extract-embedded alginate gel dressings in humans. Additionally, proposed wound dressing can be applied for the treatment of burn wounds.