Enhanced extracellular matrix synthesis using collagen dressings loaded with Artemisia absinthium plant extract

Abstract

The aim of this study was to develop three-dimensional porous composites of collagen (Col) incorporating polyphenolic-rich wormwood extract and to investigate their interaction with human skin cells, in order to optimize wound healing treatments. The scaffolds’ ultrastructure was observed by scanning electron microscopy, and biodegradability and bioactive compounds release were investigated in physiologic environment. Interaction of composites in direct and indirect contact with human skin cells was evaluated using two in vitro experimental models. ColWE scaffolds presented high porosity, swelling degree, and increased stability against enzymatic degradation, compared to Col scaffold. Composite scaffolds incorporating higher quantities of wormwood extract allowed better control of polyphenolics release. ColWE 0.5 variant favored the attachment and proliferation of human dermal fibroblasts and keratinocyte cells. In addition, the composite scaffold stimulated the synthesis of skin extracellular matrix components. All these results demonstrated that ColWE composites with improved physico-chemical and biological properties could be used in advanced wound healing applications.

Keywords

Composite material cytotoxicity extracellular matrix polyphenols skin cells wormwood

Introduction

Skin injuries pass through a complex healing process, consisting of four overlapping stages: inflammation, formation of the granulation tissue, re-epithelialization, and remodeling of the extracellular matrix (ECM).¹ External factors, such as bacterial infections or internal ones, like advanced age or disease of the patient can interfere in the normal healing process, leading to chronic wounds.

Several passive (gauzes, films, hydrocolloids, hydrogels, foams) and active (biocomponents loaded in polymeric matrices) dressings were tested on different skin wound types, in order to improve the repair processes.² The main properties required of a wound dressing are to maintain a moist environment while absorbing excessive amounts of exudate at the injury site, to allow gaseous exchange and to provide protection against bacterial infection. Also, it is important for a wound dressing to be easy removable from the injury, biodegradable, and biocompatible.^3,4

Collagen (Col) is the major structural component of ECM and the most studied natural polymer for tissue engineering applications.^5–7 Collagenic dressings were used to accelerate skin wound healing, due to their easy adherence and protection of the wound bed, swelling capacity, biodegradability, as well as high biocompatibility.⁸ In addition, Col matrices provided an optimal three-dimensional (3D) microenvironment for cellular adhesion and proliferation at the lesion sites, thus supporting the wound healing processes of granulation tissue formation, re-epithelization, and new ECM synthesis.⁹ Plant extracts and their biologically active components exerted positive effect in skin wound healing processes.¹⁰ Artemisia absinthium L. (wormwood) is traditionally used as antiseptic, antifungal, antimicrobial, carminative, antihelmintic, and febrifuge agent.¹¹ In addition, wormwood polyphenolic extracts presented antioxidant and free radical scavenging activities, which could enhance wound healing.^12–14

Currently, plant extract–polymeric constructs have attracted a wide attention in wound healing applications due to their pharmacological activity elicited by controlled release of active molecules and prolonged contact extent with skin lesions.^15–17 Col-based composite sheets loaded with plant-derived regenerative compounds or total plant extracts were recently developed for skin tissue engineering applications.^16–18 Col dressings incorporating polyphenols from Hamamelis virginiana exhibited higher capacity to inhibit chronic wound enzymes, such as myeloperoxidase and collagenase, than Col scaffolds.¹⁹ The 3D sponges of Col loaded with triphala herbal extract induced fast wound closure and tissue regeneration of infected dermal wounds in rats.²⁰ Col matrices incorporating Astragalus polysaccharides were developed as promising wound dressings with angiogenic properties, while Col-chitosan scaffolds supplemented with Aloe Vera gel presented improved physico-chemical and biological properties and the capacity for recruiting, attachment, and proliferation of fibroblasts.^21,22

The aim of this study was to evaluate the physico-chemical, structural, pharmacological, and biological properties of 3D Col dressings incorporating polyphenolic-rich wormwood extract (WE), in order to select the optimal variant for applications in advanced wound healing.

Materials and methods

Materials

All reagents were of analytical grade and were purchased from Sigma-Aldrich unless otherwise specified.

WE was prepared from aerial parts of Artemisia absinthium (voucher specimen No. 637250 at Botanical Garden Cluj-Napoca, Romania) using the maceration procedure, as previously described.²³ Briefly, dried powdered material was extracted with ethanol 70% (v/v), in a ratio material: solvent of 1:10 (w/v), on a shaker at 200 r/min, room temperature, for 8 h. The supernatant was separated from the vegetal residue by filtration on Whatman No. 1 filter paper, evaporated under reduced pressure at a rotary evaporator and lyophilized using Gamma 1-16 LSC freeze-drying equipment (Christ, Osterode am Harz, Germany). The chemical composition of ethanolic WE was determined by high-performance liquid chromatography (HPLC) analysis using DAD detection and standards of phenolic acids (gallic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid) and flavonoids (rutin, luteolin, quercetin, myricetin, apigenin).²³

Col type I was enzymatically extracted from bovine tendons by digestion with 0.5 mg/mL pepsin in 0.5 M acetic acid, purified by precipitation with 2.4 M NaCl, and dialyzed against distilled water.²⁴

Preparation of ColWE matrices

A solution of 0.5% (w/w) Col type I, pH 5.5 was stirred with WE, in different ratios of 20:1, 10:1, 5:1, and 2:1 (w/w) on a magnetic plate, for 2 h. Then, the mixtures were poured into round polypropylene molds and lyophilized using Gamma 1-16 LSC freeze-dryer (Christ, Germany) to obtain 3D porous scaffolds. Samples of 0.5 × 0.5 × 0.5 cm³ were sealed in plastic bags and sterilized by ultraviolet (UV) irradiation, for 4 h. A control scaffold was prepared using the same quantity of Col processed in similar conditions.

Scanning electron microscopy

The ultrastructure was examined on surface and transversal cross sections of ColWE scaffolds by scanning electron microscopy (SEM). Samples were mounted on carbon pads and visualized at an environmental SEM XL-30 apparatus (Philips, Netherlands), operated at 25 kV, in low vacuum mode.

Porosity and swelling degree measurements

The porosity of ColWE scaffolds was determined by liquid displacement method.²⁵ Samples of known weight were immersed in a graded test tube having a known volume of water (v₁). The scaffolds were kept for 3 h and pressed to force air from the matrix, in order to allow the water to fill the pores. The total volume of water plus water–impregnated sample was recorded as v₂. The scaffold was removed from the test tube and the remaining water volume was recorded as v₃. The porosity was calculated using the following equation

P o r o s i t y (ε) = \frac{(v_{1} - v_{3})}{(v_{2} - v_{3})} \times 100

(1)

The swelling degree of ColWE matrices was measured by the gravimetric method.¹⁹ The samples were weighted (Wi) and placed in phosphate-buffered saline (PBS), at room temperature. After 24 h, the hydrated scaffolds were removed, gently dried with filter paper and weighted again (Ws). The following equation was used

S w e l l i n g d e g r e e (%) = \frac{(W s - W i)}{W i} \times 100

(2)

Biodegradability test

ColWE scaffolds (10 mg) were incubated in 5 mL TES buffer, containing 50 mM CaCl₂, at 37°C, for 30 min. Then, 100-µL solution of collagenase type IA (100 µg/mL) was added and the mixture was incubated at 37°C, for 6 h. The reaction was stopped by cooling on ice. The supernatants were centrifuged at 6000g, for 10 min, and analyzed for amino acids content by ninhydrin method, as previously described.²⁶ Biodegradability was calculated as percent reported to Col matrix (control), considered 100% degraded.

In vitro release assay

ColWE samples (10 mg) were incubated in 2 mL PBS, pH 7.4, with gentle mixing (60g), at 37°C, similar to in vivo conditions. At predetermined periods of time, the scaffolds were placed in fresh PBS in the same conditions, while the incubation buffer was removed and analyzed for total phenolics content by Folin–Ciocalteu method.²⁷ In vitro release of phenolic compounds was reported as percent of caffeic acid equivalents (CAEs) from total phenolics content considered 100%.

In vitro cytotoxicity test

A primary culture of human dermal fibroblasts was obtained from skin biopsies by enzymatic method.²⁸ The protocol and volunteer consent have complied with ethics norms in force. Dermal fibroblasts were used in experiments at passages 5–9. Human epidermal keratinocyte HaCaT cell line was purchased from AddexBio (San Diego, CA, USA).

ColWE scaffolds placed in 24-well culture plates were injected with a suspension of human dermal fibroblasts and HaCaT keratinocytes (AddexBio), at a density of 5 × 10⁴ cells/mL and cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) and RPMI-1640 culture medium, respectively, supplemented with 10% fetal serum, at 37°C, for 5 days. Their cytotoxicity was evaluated using CellTiter 96 Aqueous MTS Reagent kit, in the presence of phenazine methosulfate (PMS), according to the manufacturer protocol (Promega, Madison, WI, USA). Briefly, viable cells reacted with MTS salt yielding a colored product and its optical density was read at 490 nm using a microplate reader (Tecan, Männedorf, Switzerland). A calibration curve was built with known cell densities.

Fluorescence microscopy

ColWE-cell constructs were cultivated in standard conditions, for 7 days and cell viability was evaluated by Live/Dead fluorescent assay (Molecular Probes, Eugene, OR, USA). Briefly, non-adhered cells were removed by washing in PBS and, then, constructs were incubated with calcein-AM and ethidium homodimer-1, at room temperature, in the dark, for 30 min. The samples were washed in PBS and observed at an Axiostar Plus fluorescence microscope (Zeiss, Gottingen, Germany).

ECM synthesis

Human dermal fibroblasts and HaCaT keratinocytes were seeded in 24-well culture plates, at a density of 5 × 10⁴ cells/mL. After 24 h, sterile ColWE scaffolds were added in the culture medium and the plates were incubated at 37°C and 5% CO₂. After 5 days of cultivation, the culture medium was harvested, centrifuged, and the supernatant was used to analyze the synthesis of total collagens and fibronectin. Col content secreted in the culture medium was measured using Sircol soluble collagen assay (Biocolor Assays, Carrickfergus, UK). The absorbance of samples was measured at 555 nm using a Sunrise Microplate Reader (Tecan). Untreated cell culture served as control. In order to eliminate interferences from samples containing Col, each variant was separately incubated in PBS and processed in similar conditions, in order to serve as blank. Fibronectin secretion in the culture supernatant was measured using sandwich enzyme-linked immunosorbent assay (ELISA) assay. Incubation steps were performed according to the manufacturer’s protocol (Abcam, Cambridge, MA, USA) using specific anti-fibronectin antibody precoated onto 96-well culture plate and biotinylated secondary antibody. Results of total collagens and fibronectin production were reported to total protein content determined by Bradford assay.²⁹

Statistical analysis

Data were reported as mean ± standard deviation (SD). Statistical analysis was performed on control–sample pairs of interest using Student’s t test. Significant differences were considered at p < 0.05.

Results and discussion

Polyphenols-rich WE was characterized by a total phenolic content of 178.76 mg caffeic acid/g dry extract and a high content in quercetin (2.707 mg/g dry extract) and luteolin (0.677 mg/g dry extract) determined by HPLC.^23,30 In addition, WE exhibited antioxidant activity in stressed fibroblasts and antimicrobial activity against Klebsiella pneumoniae, Acinetobacter baumannii, and Staphylococcus aureus, requiring a conditioning form for treatment of skin disorders.^23,31 Lyophilized matrices obtained after mixing with Col were noted ColWE 0.25, ColWE 0.5, ColWE 1, and ColWE 2.5, according to WE final concentrations (mg/mL).

Ultrastructure of ColWE scaffolds

ColWE scaffolds presented a 3D sponge appearance, characteristic for freeze-dried polymeric matrices. SEM observations of their surface (Figure 1(a)–(d)) showed a random microporous structure, with small-sized pores, similar to Col scaffold (Figure 1(e)). Transversal cross section of ColWE scaffolds presented a regular porous structure (Figure 1(f)–(i)), similar to that of Col scaffold (Figure 1(j)). Interconnected pores were present within the entire structure of ColWE scaffolds and their sizes varied between 50 and 600 µm. The ultrastructure and pore size and morphology in composite scaffolds were similar to those in Col scaffold, indicating that Col gel and WE formed a homogeneous mixture before lyophilization.

Formation and morphology of pores was the result of water crystals sublimation during the freeze-drying process. Lyophilization was used in this study in order to obtain good interaction with different cell types and controlled release of bioactive molecules. It was previously showed that freeze-dried biomaterials cultivated with cells ensured nutrients diffusion and stimulated cell proliferation and ECM production, in a higher proportion than two-dimensional (2D) materials or loose hydrogels, due to its 3D porous structure.³² Moreover, their pore size and microstructure could be further tailored by variation of freezing temperature between −80°C and −20°C.³³ It was showed that cell infiltration, adhesion, and proliferation within a scaffold are favored by certain pore sizes.³⁴

The non-homogeneity in pore size obtained in our composite materials is also present in skin.³⁵ This complex tissue has macropores that usually serve as space for tissue growth and vascularization, while micropores enhance scaffold’s mechanics and allow specific cell migration during tissue regeneration and treatment of defects.³⁶ On the other hand, the differences between surface and inside pore morphology were due to the lyophilization process and vacuum exposure of the upper part, while the lower part was in direct contact with the plastic mold.³⁷

Physico-chemical characterization of ColWE scaffolds

Total porosity of ColWE scaffold variants was determined by liquid displacement method using water as solvent. The small water molecules diffused into different pore sizes of the scaffolds and were forced by pressure to rapidly occupy the void volume. The predominantly hydrophobic Col molecule and the polyphenols-rich WE with low water solubility delayed samples swelling during the incubation time. The results are shown in Table 1. The values ranged between 80% porosity for ColWE 0.25 and ColWE 0.5% and 70% porosity for ColWE 1 and ColWE 2.5. Col scaffold prepared by freeze-drying presented a porosity value of 97% (Table 1). Incorporation of increasing WE induced a proportional and significant (p < 0.05) decrease in porosity, compared to that of Col scaffold. Still, ColWE scaffolds’ porosity above 70% could favor cell colonization and application in skin tissue engineering.³² The decrease in porosity could be due to cross-linking that took place between Col and polyphenols chains and formation of multiple hydrogen bonds that alter the ultrastructure of the porous scaffold.³⁸

Table 1.

The porosity and biodegradability of composite variants (ColWE) and collagen (Col) scaffold.

Sample	Porosity (%)	Swelling degree (%)	Biodegradability (%)
ColWE 0.25	78.90 ± 4.83*	2342 ± 14.42*	86.83 ± 3.22*
ColWE 0.5	80.08 ± 3.19*	2240 ± 15.71*	63.54 ± 4.24*
ColWE 1	71.57 ± 3.24*	2146 ± 15.09*	49.53 ± 4.08*
ColWE 2.5	69.80 ± 3.95*	1936 ± 10.14*	35.61 ± 3.20*
Col	97.06 ± 2.54	1977 ± 14.93	100 ± 4.57

p < 0.05, compared to Col scaffold.

The swelling of Col scaffolds represents their ability to increase their volume, absorbing water for at least 1500% from their initial volume.³⁹ Composite material consisting of Col loaded with increasing quantities of polyphenols-rich WE, which contained a significant number of free hydroxyl groups on the hydrophobic backbone, should present increased hydrophilicity and improved swelling degree. In this respect, the swelling degree of ColWE composite variants was analyzed and the results are presented in Table 1. The values for ColWE dressings were significantly (p < 0.05) higher than that of Col scaffold, except ColWE 2.5 (Table 1). The decrease in the swelling degree from 2342% for ColWE 0.25% to 1936% in ColWE 2.5 corresponded to WE concentration increase. This could be due to formation of cross-links between Col and polyphenols chains, leading to a decrease in free amino and hydroxyl side groups with hydrophilic properties and smaller swelling degree. This variation was in accordance to porosity decrease. However, ColWE 0.25, ColWE 0.5, and ColWE 1 variants with improved swelling properties should be able to facilitate exudate absorption at the wounded sites. Similar studies have reported an increased water uptake of Col-polyphenolics biomaterials compared to Col²⁰ and a decrease in swelling degree with increasing concentrations of vegetal polysaccharides added to form a collagenic hybrid material.⁴⁰

Collagenic wound dressings are exposed in vivo to enzymatic degradation. In this study, the biodegradability test was carried out using collagenase type IA, specific for the cleavage of -X-Gly-Pro sequence from connective tissue components. The results showed that ColWE scaffolds were degraded to a lesser extent (35%–86%), compared to Col matrix (100%) (Table 1). Moreover, increasing concentrations of WE induced higher stability of scaffolds against collagenase attack. Thus, ColWE 0.25 was 86% degraded, ColWE 1 scaffold was 50% degraded, while ColWE 2.5 was only 35% degraded (Table 1). This could be explained by the high content in hydroxyl and carboxyl groups of the polyphenolic extract, favoring multiple hydrogen bonds with proteins, including Col.⁴¹ Similar results were previously obtained for Col sponges loaded with polyphenols from Hamamelis virginiana, which showed improved stability toward chronic wound enzymes.¹⁹

In vitro release of biologically active compounds

The polyphenolics release from ColWE porous scaffolds was studied in conditions mimicking in vivo environment, using PBS pH 7.4, at 37°C. The results are presented in Figure 2.

The data indicated that large amounts of polyphenolics were gradually released, in the first 8 h of incubation (Figure 2(a)). The slope of release profile decreased between 8 and 24 h of incubation and further incubation resulted in a plateau of polyphenolics release (Figure 2(b)). At the end of incubation period, the percentage of released polyphenolic compounds reached high values of 45.42% and 76.43% for ColWE 0.5 and ColWE 0.25, respectively. ColWE 1 and ColWE 2.5 scaffolds released lower percent of phenolics (16.13% and 8.85%, respectively) in the physiologic environment. The obtained results could be due to scaffolds’ biodegradability; thus, smaller percent of bioactive compounds release was registered in slower degraded variants. However, the actual amounts of phenolics released in the physiologic environment were within a narrow range. This similarity in released amounts probably corresponded to the parts not involved in physico-chemical interactions with Col molecules.³⁸

ColWE scaffold interaction with human skin cells

ColWE effect on cell proliferation and viability

Preliminary cytotoxicity test of ColWE variants was performed in a culture of fibroblasts (L929 clone cell line), according to SR EN ISO 10993-5/2009. The results indicated high biocompatibility (>80% cell viability) of ColWE 0.25 and ColWE 0.5 and slight cytotoxicity (>70% cell viability) of ColWE 1 and cytotoxicity (~30% cell viability) of ColWE 2.5 variant, after 24 h of cultivation. Therefore, only ColWE 0.25, ColWE 0.5, and ColWE 1 were selected for further studies in cell culture. The cytotoxicity of ColWE 2.5 could be due to the high phenolics amount released into the cell culture medium and insufficient Col amount to protect skin cells. In addition, a steep increase in release profile could correspond to the enlargement of the surface–volume ratio of the sample used in cell culture studies.⁴²

Two types of human skin cells, fibroblasts and keratinocytes, were cultured in direct contact with ColWE scaffolds in order to evaluate cell proliferation. The results showed that the number of fibroblast cells gradually increased during the entire period of cultivation (Figure 3(a)). The cells proliferated to a higher extent in 3D ColWE scaffolds than on plastic (control) and the cell number was significantly (p < 0.05) higher in ColWE 0.25 and ColWE 0.5 variants, at each period of cultivation. ColWE 1 scaffold allowed cell proliferation in a similar extent to Col scaffold, but higher than 2D control group at 7 days of cultivation.

In regard to HaCaT keratinocyte cells, no differences were registered between tested groups, at 2 days of cultivation (Figure 3(b)). After 5 days of cultivation, the results showed that keratinocytes proliferated intense within ColWE 0.5 (46.9 × 10⁴ cells) (Figure 3(b)). ColWE 0.25 and ColWE 1 stimulated the cell growth (37.2 × 10⁴ cells and 34.6 × 10⁴ cells, respectively) to a higher extent than Col scaffold (28.5 × 10⁴ cells) and cells cultivated on plastic (18.2 × 10⁴ cells) (control). After 7 days of cultivation, the number of keratinocyte cells was also significantly (p < 0.05) higher than in Col scaffold. However, it decreased in all tested groups, including control, probably due to cell overpopulation, which rapidly consumed the available nutrients.

All these data demonstrated that certain variants of ColWE composites, like ColWE 0.25 and ColWE 0.5, were more efficient as 3D scaffolds for skin cells, especially for keratinocytes, compared to Col scaffold, based on synergistic effect of its components. ColWE 1 dressing was better than Col scaffold only in HaCaT keratinocyte cell culture. The stimulative effect of ColWE composites could be due to their porous architecture, which ensured an efficient absorption of nutrients from the culture medium. Moreover, certain amounts of WE incorporated in composites and phenolics released in the biological medium supported the proliferation of both types of human cells. Previous studies showed that polyphenolics present in Artemisia genus could stimulate skin cell proliferation.⁴³ On the other side, Col with its unique triple helical structure is known to favor the attachment and proliferation of dermal fibroblast cells when conditioned as 3D scaffolds.⁴⁴ No reports on interaction of WE-loaded Col dressings with skin cells were found.

Fluorescence microscopy observations confirmed high number of viable cells that colonized ColWE scaffolds without signs of cytotoxicity (Figure 4). Dermal fibroblasts migrated and infiltrated within the whole structure (Figure 4(a)–(d)), while keratinocytes formed isolated colonies on the surface of scaffolds (Figure 4(e)–(h)).

ColWE effect on ECM synthesis by skin cells

First, total level of collagens secreted in the culture medium was determined after dermal fibroblasts cultivation in the presence of composite scaffolds. The results indicated that ColWE 0.25 and ColWE 0.5 enhanced threefold and fourfold, respectively, the production of collagens, compared to control cell culture. Moreover, the values were significantly (p < 0.05) higher than those registered for Col scaffold (Figure 5(a)). ColWE 1 and Col scaffolds exerted a positive effect on collagens synthesis by fibroblast cells, compared to control cells, but registered low values.

Similar results were encountered in case of collagens synthesized by HaCaT keratinocytes cultured in the presence of ColWE scaffolds (Figure 5(a)).

All these data demonstrated that ColWE scaffolds had stimulative effect on collagens synthesis in human skin cells due to certain concentrations of WE. Previous studies showed that flavonoid glycosides (isoquercitrin, quercetin-3-O-β-d-glucoside, quercetin-3-O-rhamnoglucoside, isorhamnetin-3-O-rhamnoglucoside, isorhamnetin-3-glucoside) present in Artemisia genus had the capacity to enhance Col production in human skin fibroblasts culture.^45–47

Another main component of ECM, fibronectin, was synthesized by dermal fibroblasts cultivated in the presence of ColWE scaffolds. Its production was significantly (p < 0.05) increased by ColWE 0.5 and ColWE 0.25 variants, compared to Col scaffold and control group (Figure 5(b)). Fibroblasts cultivated with ColWE 1 scaffold synthesized a similar level of fibronectin to that of Col scaffold group. In HaCaT keratinocyte cells, ColWE 0.5 scaffold induced a significantly (p < 0.05) higher value of synthesized fibronectin, compared to Col scaffold group and control cells (Figure 5(b)).

Cell culture results demonstrated that ColWE 0.5 scaffold was optimal for skin cells proliferation and metabolism, stimulating ECM production.

Conclusion

ColWE composite scaffolds exhibited optimal architecture and porosity for cell infiltration, adhesion, and proliferation. They were stable against collagenase attack and released bioactive compounds in a controlled manner. Skin cells adhered and proliferated within ColWE composite scaffolds and synthesized more ECM components than Col scaffold. All these results demonstrated that ColWE composite scaffolds had improved physico-chemical and biological properties and could be used in wound healing treatments.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was financially supported by a grant of the Romanian National Authority for Scientific Research and Innovation within Programme Nucleu, project no. 18-190.

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