Multicomponent nanofibrous electrospun dressings, evaluated for the healing of full-thickness excision wounds in a porcine model

Abstract

The first layer of the advanced wound dressing comprises a 3 mm layer of copper (oxide) nanoparticles synthesized with the leaf extracts of M. oleifera and Musa, mixed with Cochlospermum gossypium and Acacia arabica gums, and coated on the cotton fabric. By electrospinning at 20 kV, a 9-layered nanofibrous framework of the biopolymers cellulose acetate, gelatin, and marine chitosan, impregnated with bioactives such as carbon dots derived from medicinal plants, green synthesized copper nanoparticles, and phytochemicals extracted with acetone and isopropanol was woven. The core, shell and active layer nanofibers of different polymers, with a fiber diameter ranging from 252 nm to 3.1 µm, were grafted onto the CuNP-coated cotton fabric. Four wound dressings viz., WD1 (L)/WD2 (H) with poly l-lysine in low and high concentrations, and WD3 (L)/WD4 (H) with polyethylene oxide in low and high concentrations, were fabricated to improve the piezoelectric behavior. Of them, WD2 and WD4 showed promising biological characteristics of anti-inflammatory, anti-oxidant, and anti-microbial potential. They were endowed with piezoelectric constants of 24.28 pm/V (WD2) and 0.28 nm/V (WD4), and zeta potentials of 57.7 mV (WD2) and 94.6 mV (WD4). Within 8 days of causing full-thickness wounds, the two test items were able to effectively cause quick wound contraction, epithelial tissue regrowth, and elimination of inflammation; by 34 days, normalcy had returned.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-025-04460-w.

Introduction

The largest barrier of defense against the outside world is the skin. The wounds, which are disruptions in a part or the entire thickness of skin, are classified as open or closed, acute or chronic, clean or contaminated, internal or exterior, and piercing (stab wounds, cuts, surgical wounds etc.)/non-penetrating (abrasions, lacerations, bruises, concussions) (Fout and Plotzke 2022; Schlereth et al. 2022; Yu et al. 2022). Medical treatment and health-care-costs for all wounds, including surgical and diabetic ulcers, range from $28.1 to $96.8 billion (Ajit and Ambika Gopalankutty 2021; De Francesco et al. 2022). In addition, the regenerated skin tissue frequently has misaligned scar tissue and is devoid of important components like nerves, glandulae sebaceae, and hair follicles (Lv et al. 2022).

Wound healing is a complex process and is influenced by several factors that fall predominantly under environmental, anatomical, biochemical, immunochemical and others in the wound environment. It is therefore impossible to make a “one plug—all challenges” kind of wound care material. Instead, multicomponent wound dressings enriched with different natural and/or synthetic polymers viz., chitosan, polyvinyl alcohol, polylactic acid, polyacrylic acid, polyvinyl pyrrolidone, polyethyleneimine, polycaprolactone, polyethylene glycol, polyethylene oxide, poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P34HB), collagen, silk fibroin, and pullulan (Agarwal et al. 2021; Alizadeh et al. 2023; Altinkok et al. 2023; Campa-Siqueiros et al. 2023; Dogan et al. 2023; Goswami et al. 2021; Jia et al. 2024; Khataei et al. 2023; Luo et al. 2024; Shadman-Manesh et al. 2023; Virijevic et al. 2022), and bioactives that alleviate infections and inflammation, and improve the healing capacity are the need of the hour. The use of antibiotics in dressings often induces resistance in bacteria, and to overcome this critical problem, phytoextracts like Portulaca oleracea, Capparis spinosa and Plantago major extracts have been effectively encapsulated in electrospun nanofibers. The integration of plant derived bioactive compounds into fibrous nanoscaffolds enhanced the healing efficacy of compressive materials by augmenting their bactericidal and ROS-neutralization characteristics (Anaya-Mancipe et al. 2023; Stoyanova et al. 2023; Zhu et al. 2023). The incorporation of nanoparticles such as cerium oxide, boron nitride, silver and zinc oxide (Dogan et al. 2023; Fereydouni et al. 2023; Gao et al. 2023; Lai et al. 2021) to improve the efficiency of wound dressings was tried. As the nanoparticles in wound dressings come in close contact with the wound, there is an imperative need to evaluate their biocompatibility. The most often reported transdermal effects of nanoparticles are skin irritation and allergies. Moreover, research has shown that the use of nanoparticles on the skin's surface can enhance many skin disorders, such as psoriasis, oxidative stress, autophagy, and programmed cell death in fibroblasts and keratinocytes (Lai et al. 2021; Wang et al. 2018; Xiao and Zhang 2020). Considering these factors, several studies world-wide have been initiated and a few of the observed results, relevant to the current study, are reported here.

Nanofibers, produced using a core of PVA-chitosan and a shell of PVP-maltodextrin, were incorporated with ethereal oil derived from Satureja mutica/Oliveria decumbens for the purpose of managing bacterial infections (Barzegar et al. 2021). The process of coaxial electrospinning, which involves putting the shell and core solutions in separate syringes, has many benefits, including a high encapsulation efficacy, a wide range of material options, improved mechanical strength, usability, and low cost (Gao et al. 2023). By using poly (l-lactide-co-ε-caprolactone) (PLCL) as the shell layer and polyethylene oxide (PEO) or sodium tanshinone IIA sulfonate (STS)/PEO as the core layer, Li et al. created nanofibers using coaxial electrospinning. The fibers possessed good mechanical qualities, higher strengths of suture retention, and appropriate degradability; they were proposed to be employed as tissue engineering materials with promising results for vascular grafts and other applications (Li et al. 2023).

To fulfill the increasing need for efficacious biomedical dressings, an optimal preclinical animal model is a requisite for evaluating wound heal and care (Horrobin 2003; Singer and McClain 2003). The effectiveness of various dressing materials, using animal models that replicate various human skin wound diseases, are investigated (Ansell et al. 2012; Chu et al. 2010; Davidson 1998; Lindblad 2000; Perez and Davis 2008; Ramos et al. 2008). Small animals like the “Glires” (rodents and rabbits) are often selected for their low housing space and cost (Ansell et al. 2012; Chu et al. 2010; Davidson 1998; Lindblad 2000; Perez and Davis 2008). However, a few critical drawbacks of the glires include loosened and limited skin surface area accompanied by quicker wound closure via contraction, and more importantly large anatomical variation from human skin. Further, small animal studies with provision for limited number of wounds, warrant multiple animals to collect enough data, which in turn may cause errors and discrepancies in the result analyses (Han et al. 2017; Lin et al. 2011). The significant advantages of porcine models include similar tissue-restorative mechanisms, comparable to humans (Kwak et al. 2014; Sullivan et al. 2001; Swindle et al. 2012). In addition, the pig possesses a larger surface area on its back, which allows for the creation of multiple wounds. This increases the accuracy of the findings by eliminating unrelated variables associated with different species. With a thick outer layer (epidermis), distinct ridges (rete pegs), and a complex network of collagen and elastin fibers in the deeper layer (dermis), the pig's skin structure is very similar to that of humans. Like humans, pigs have strong individual hairs in place of fur and skin that is firmly attached to the underlying structures (De Vries et al. 1994; Hollander et al. 2003; Zhu et al. 2007).

The current study set out to investigate the safety and effectiveness of new bioactive-enriched electrospun nanofibrous multilayered core/shell/core–shell (ENM) dressing materials, also known as WDs, for use in full-thickness wounds made on the dorsal skin of Yorkshire pigs at the thoraco-lumbar region, which is the area between the paravertebral region above the rib tips and the boundaries of both armpits. For mechanical strength, the ENM is made of synthetic polymers including cellulose acetate, polyvinyl alcohol, and polyethylene oxide in addition to natural polymers like chitosan, gelatin, and keratin; poly l-lysine to provide positive charge to the nanofibers; Taurine (secondary amino-acid) to enhance piezoelectricity and faster tissue regeneration, bioactives viz. phytoextracts, green synthesized copper/copper oxide nanoparticles (Cu(O)NPs) and co-doped carbon dots (FN-CDs) for biological functions. Maintaining the right wound size and the ideal distance between adjacent wounds may provide a high-throughput platform for precisely assessing ENM's therapeutic potential.

The creation of a one-pot synthesis process that directly carbonizes plant materials in the presence of ethanol—a solvent that acts as a medium and a reactant—represents a fresh innovative step. The solvent preserves the integrity of the components produced from plants while promoting the synthesis of carbon dots. This technique (one pot synthesis method) streamlines the procedure and increases the production of premium carbon dots. Using an effective dopant, such as 4, 5 difluoro 1, 2 benzenediamine (DFBD), to carefully dope nitrogen and fluorine into the carbon dots during synthesis is another creative step. The ratio of plant material to carbonization temperature can be changed to manage the doping and produce carbon dots with certain qualities. With 480 mg of dopant (DFBD) and 8 h at 180 °C (controlled co-doping), we were able to optimize the weight of the plant material to 1.5 g. Similar to this, the green production of copper nanoparticles using medicinal plant extracts to cap them and use them in biopolymers improved the antibacterial capability and decreased any systemic toxicity of the Cu(O)NPs.

The efficacy and performance of two out of four fabricated wound dressings hereafter denoted as WD2 and WD4, was tested on the Yorkshire breed of Sus scrofa, while concurrently looking at the underlying biochemical and histological changes. To override the toxicity effects of the metal nanoparticles, green synthesized metal (copper) nanoparticles were encapsulated in the electrospun nanofibrous dressings and evaluated for their in vitro cytotoxicity for deployment in the current study.

Materials and methods

Synthesis of copper/copper oxide nanoparticles and FN-doped carbon dots

The steps outlined in our earlier work were followed to green synthesize copper/copper oxide nanoparticles (Cu(O)NPs) (Fig. S1) and FN-doped carbon quantum dots (FN-CDs) (Fig. S2) (Kota et al. 2023, 2024) with medicinal plants known for their vital activity in wound healing. The details of the protocols are outlined in the supplementary file.

Preparation of gel for coating of wound dressing cotton fabric

Using a magnetic stirrer, dissolved 0.22 g of Cochlospermum gossypium (kondagogu) in 20 ml of warm water, then added 0.8 g of gum acacia and 100 mg of manufactured Cu(O)NPs. Stirred for one hour. The natural gums aid in the dispersion of Cu(O)NPs and also have proven medicinal value in traditional medicine. The white cotton fabric from local textiles was soaked in water overnight, air dried and the process repeated one more time, to remove all the starchy material that is normally found on a new cloth. The dry cloth was pressed to remove wrinkles/folds, if any, and cut in pieces of 10 cm × 10 cm width and length. The fabric was then placed on top of the plate in the film coater and coated with CuNP-loaded natural gum-containing gel using the film coater fitted with the calipers, to a thickness of 3 mm (Fig. 1). After coating, the fabric was dried by placing the machine in an auto-mode/manual mode with the heater on, or in a hot-air-oven.

Fig. 1.

Copper nanoparticles synthesized with the leaf extracts, coated on the cotton fabric with the automatic film coater (aka doctor’s blade)

Preparation of phytochemical concoction

Using a soxhlet apparatus, 150 ml of each of the solvents of increasing polarity—acetone (0.355) and isopropanol (0.546)—were used to sequentially extract the equiproportionate mixture of leaf powders of Azadirachta indica L. (Neem), Moringa oleifera L. (Drumstick), Tinospora cordifolia L. (Heart-leaved moon seed/Guduchi), Tridax procumbens L. (coat button/Tridax daisy), and Trigonella foenum-graecum (fenugreek) L. Soxhlet extraction was carried out 5 °C to 10 °C less than their boiling points (acetone: BP-56 °C and Isopropanol: BP-82.3 °C) and the extract concentrated in a rotary vacuum evaporator to one-fifth of the initial volume (30 ml). These two solvents were chosen as they predominantly extracted the wound healing phytochemicals, and for their compatibility with other constituents used in electrospinning of the wound dressings.

Electrospinning of the multilayered nanofibrous scaffold

On top of the cotton fabric coated with Cu(O)NPs, the core and shell solutions were coaxially electrospun using an electrospinning machine (built at Royal Enterprise, Chennai, India) (Fig. 2). The compositions of core and shell solutions (Table 1) and process parameters viz. rate of flow, voltage and distance between the needle and the collector, were optimized through several iterations and combinations of the aforementioned factors (Alven et al. 2023; Sharaf et al. 2022).

Fig. 2.

Fabrication of core/shell/core–shell/active nanofibrous layers of the dressing pads with the double syringe pump fit electrospinning unit

Table 1.

Preparation of core and shell solutions used in fabrication of wound dressings

S. No.	Components	Quantity
		Core	Shell
1.	Pure acetone	20 ml	20 ml
2.	Acetone-extracted phytochemicals (concoction)	10 ml	10 ml
3.	Pure isopropanol	5 ml	5 ml
4.	Isopropanol extracted phytochemicals (concoction)	10 ml	10 ml
5.	DMSO	2 ml	2 ml
6.	Taurine	0.04	–
7.	Glacial acetic acid	–	2 ml
8.	Keratin (1 g of keratin in 5 ml of 40% TBAH)	–	0.5 ml
9.	Moringa and Chromolaena FN-doped carbon dots	0.5 ml	0.5 ml
10.	Cellulose acetate	4 g	4 g

Active ingredients in fabrication of wound dressings (WD1 to WD4)

Out of the four wound dressings fabricated (Table 2), WD2 and WD4 were chosen owing to their physico-chemical characteristics and functional efficiency observed from preliminary evaluation results obtained for full-thickness-wound-healing-studies. Multilayered electrospun nanofibrous Scaffold/Mat was fabricated “layer by layer” on the CuNP-coated cotton fabric. The multilayered nanofibrous scaffolds were observed by Micro-CT-Scan, with contrast agent(s) viz. Iodine, bismuth oxide, and lanthanum oxide (Amariei et al. 2018; Neisiany et al. 2020; Wang et al. 2017).

Table 2.

Active ingredients in the fabrication of wound dressings WD1 to WD4 with the electrospinning parameters

S. No.	Solution ingredient	Type of wound dressing
		WD 1	WD 2	WD 3	WD 4
1	D.I. water	25 ml
2	Glacial acetic acid	5 ml
3	Chitosan	0.5 g
4	Gelatin	0.5 g
5	Poly vinyl alcohol	2 g		1 g
6	Poly-l-lysine	300 µg	750 µg	–	–
7	Polyethylene oxide	–	–	0.3 g	0.6 g
8	Distance between needle tip and collector plate	20 cm
9	Flow rate	1 ml/hr		3 ml/hr
10	Voltage	20 kV

Characterization of electrospun wound dressings

The diameters of the nanofibers and their elemental mapping, as analyzed by FESEM-EDS (AMETEK, advanced microanalysis solutions), provided insights into the architecture of the fabricated wound dressings. With the aid of a nanoparticle analyzer (Horiba, SZ-100), the zeta-potential distribution was assessed and a micro-CT-scan (Bruker), provided the layered organization of the scaffold, slice by slice.

In vivo study in Sus scrofa

The FDA Good Laboratory Practice (GLP) Regulations, 21 CFR Part 58, 2022, and the Organization for Economic Co-operation and Development's (OECD) Principles of Good Laboratory Practice (GLP) and Compliance Monitoring (as amended in 1997) [C(97)186/Final and ENV/MC/CHEM(98)17] were adhered to in this study. The Institutional Animals Ethics Committee (IAEC) of Palamur Biosciences Private Limited supported the current study on April 3, 2023, with Proposal No. PAL/IAEC/2023/04/01/17, and CCSEA authorized it on May 8, 2023. The Compendium of CPCSEA 2018: Guidelines for laboratory animal facility, published in 2015(7) (pp. 561–96), contains ethical guidelines and regulations that were adhered to in order to conduct all related experiments in accordance with the Biological Evaluation of Medical Devices-Part 2: Animal welfare requirements, ISO 10993-2:2022 (E). All the experimental data from the animal study were subjected to statistical analysis (ANOVA/Student’s ‘T’ test) and the level of significance at 95% confidential limits is represented in respective tables and figures. ANOVA results for data included in Tables 5 and 6 are provided in the supplementary file as appendix 3.

Table 5.

Clinical biochemistry data of the study animals on day 4 (baseline) and day 34 (terminal day) with the student’s ‘T’ test performed between the means of the three animals’ parameters (p ≤ 0.05 considered significant and is indicated in bold with an asterisk)

Parameters	Baseline (day 4)				Terminal (day 34)				Student’s ‘T’ test
	P1	P2	P3	Mean ± SD	P1	P2	P3	Mean ± SD
ALB (g/l)	30.2	30.7	31.1	30.7 ± 0.45	33.4	33.2	30.9	32.5 ± 1.39	t = 2.839 p = 0.02*
ALP (U/l)	140	96	145	127.0 ± 26.96	142	130	368	213.3 ± 134.08	t = 1.701 p = 0.08
ALT (U/l)	28	27	33	29.3 ± 3.21	26	30	32	29.3 ± 3.06	t = 0 p = 0.5
AST (U/l)	50	57	55	54.0 ± 3.61	33	37	73	47.7 ± 22.03	t = − 0.713 p = 0.25
Calcium (mmol/l)	2.89	3.28	2.96	3.0 ± 0.21	3.14	3.09	3.07	3.1 ± 0.04	t = 0.824 p = 0.23
T. Cholesterol (mmol/l)	1.88	2.11	1.89	2.0 ± 0.13	1.89	1.67	1.82	1.8 ± 0.11	t = − 2.349 p = 0.03*
Creatinine (µmol/l)	60	63	56	59.7 ± 3.51	66	84	59	69.7 ± 12.9	t = 2.349 p = 0.04*
GGT (U/l)	61	41	75	59.0 ± 17.09	68	51	58	59.0 ± 8.54	t = 0 p = 0.5
Glucose (mmol/l)	4.11	5.39	2.54	4.0 ± 1.43	4.48	4.49	4.07	4.3 ± 0.24	t = 0.594 p = 0.29
Pi (mmol/l)	1.74	1.72	1.42	1.6 ± 0.19	1.83	1.93	1.48	1.7 ± 0.24	t = 3.725 p = 0.01*
T. Bilirubin (µmol/l)	3.3	3.24	2.59	3.0 ± 0.39	2.6	3.09	3.27	3.0 ± 0.35	t = − 0.154 p = 0.442
Total protein (g/l)	76.4	89.1	74.8	80.1 ± 7.84	79.1	87.1	76.2	80.8 ± 5.65	t = 0.677 p = 0.267
Triglycerides (mmol/l)	0.33	0.72	0.37	0.5 ± 0.21	0.63	0.82	0.49	0.6 ± 0.17	t = 1.139 p = 0.159
BUN (mmol/l)	1.66	2.01	2.13	1.9 ± 0.24	2.51	3.86	3.22	3.2 ± 0.68	t = 5.914 p = 0.002*
Sodium (mmol/l)	142.3	140.7	141.2	141.4 ± 0.82	140.8	139.3	139.1	139.7 ± 0.93	t = − 13.446 p = 0.00*
Potassium (mmol/l)	3.44	3.25	3.31	3.3 ± 0.1	3.93	3.97	3.62	3.8 ± 0.19	t = 7.102 p = 0.00*
Chloride (mmol/l)	112.5	108.6	107.9	109.7 ± 2.48	104.2	104.5	103.6	104.1 ± 0.46	t = − 4.673 p = 0.004*

ALB albumin, ALP alkaline phosphatase, ALT alanine transaminase, AST aspartate transaminase, GGT gamma-glutamyl transferase, Pi inorganic phosphorus, BUN blood urea nitrogen, g gram, U units, L liter, mmol millimole, µmol micromole

Table 6.

Hematological parameters of the three experimental animals measured on day 4 (baseline) and day 34 (terminal) tested by Student’s ‘T’ test to understand the significant differences, if any (p ≤ 0.05 considered significant and is indicated in bold with an asterisk)

Parameters	Baseline (day 4)				Terminal (day 34)				Student’s ‘T’ test
	P1	P2	P3	Mean ± SD	P1	P2	P3	Mean ± SD
WBC (109/l)	20.93	11.71	10.44	14.36 ± 5.73	16.47	12.56	11.24	13.42 ± 2.72	t = − 0.330 p = 0.378
RBC (1012/l)	5.99	5.72	5.24	5.65 ± 0.38	5.23	5.64	4.87	5.25 ± 0.39	t = − 3.723 p = 0.01*
HGB (g/l)	103.00	105.00	106.00	104.67 ± 1.53	97.00	105.00	102.00	101.33 ± 4.04	–
HCT (l/l)	0.33	0.32	0.34	0.33 ± 0.01	0.30	0.32	0.32	0.31 ± 0.01	t = − 3.674 p = 0.01*
PLT (109/l)	410.00	373.00	334.00	372.33 ± 38.00	422.00	368.00	299.00	363.00 ± 61.65	–
NEUT (109/l)	48.30	19.20	33.30	33.60 ± 14.55	32.00	15.70	28.60	25.43 ± 8.6	t = − 2.788 p = 0.02*
LYMPHO (109/l)	44.70	69.40	57.60	57.23 ± 12.35	61.10	76.20	64.70	67.33 ± 7.89	t = 4.531 p = 0.005*
MONO (109/l)	4.60	8.70	5.50	6.27 ± 2.15	4.30	6.80	4.80	5.30 ± 1.32	t = − 2.603 p = 0.03*
EOS (109/l)	0.40	0.80	1.20	0.80 ± 0.4	0.30	0.60	0.50	0.47 ± 0.15	t = − 2.578 p = 0.03*
LUC (%)	1.50	1.80	2.20	1.83 ± 0.35	2.10	0.70	1.10	1.30 ± 0.72	t = − 1.603 p = 0.092
BASO (109/l)	0.40	0.10	0.20	0.23 ± 0.15	0.20	0.10	0.20	0.17 ± 0.06	t = − 1.381 p = 0.119
RETIC (%)	1.15	1.03	3.22	1.80 ± 1.23	1.45	0.64	0.65	0.91 ± 0.46	t = − 1.667 p = 0.085

WBC white blood cells, RBC red blood cells, HGB hemoglobin, HCT hematocrit, PLT platelets, NEUT neutrophils, LYMPHO lymphocytes, MONO monocytes, EOS eosinophils, BASO basophils, LUC leukocytes, RETIC reticulocytes, g gram, L liter, % percentage

Wound creation, application of wound dressing, wound inspection and euthanasia

The three experimental animals were anaesthetized with 15 mg/kg ketamine IM, 2.5 mg/kg Xylazine IM, and 0.05 mg/kg of Atropine IM followed by 1–2.5% Isoflurane inhalation through face mask. Wounds were created (Fig. 3) with the help of a scalpel blade, after marking the wound areas with the help of a marker (Fig. 4A, B) and were subject to baseline planimetry on day 4 (L1 × B1 = A1, L2 × B2 = A2, mean area = A1 + A2/2). Photography was also done for the same (Fig. 4C, D). A total of 8 wounds (4 on each side), each of 2.5 cm × 2.5 cm (square shaped), and 6–10 mm deep full thickness, were created on the back of the animal with sufficient space between adjacent wounds (5–10 cm).

Fig. 3.

Schematic of wound creation on the back of the experimental animal, Yorkshire Pig

Fig. 4.

a Wound markings on left side. b Wound markings on right side. c Wound creation on left side. d Wound creation on right side. e Primary and secondary wound dressing on left side. f Primary and secondary wound dressing on right side. g Crape bandage application on animal. h Jacket application on animal

The wound dressing (WD) was applied twice weekly with direct contact to the wound site. Standard care refers to the application of sterile dressing material with betadine solution (Fig. 4E, F). Negative control means sterile dressing without any material or any healing substance. WDs were applied as per the wound matrix, designed to nullify the variations between the study animals that might be implicated because of position, movements of the body, sliding of the protective jacket etc. For animal P1, wounds 1 and 2 were applied with standard care, wounds 3 and 4 were applied with wound dressing WD2, and wounds 5 and 6 denote WD4, while wounds 7 and 8 represent negative control. The wound matrix for animal P2 is different and stands as wounds 1 and 2 for negative control; wounds 3 and 4 for standard care; wounds 5 and 6 for WD2, and wounds 7 and 8 for WD4. For animal P3, wounds 1 and 2 were applied with wound dressing 4 (WD4), wounds 3 and 4 denote negative control, wounds 5 and 6 represent standard care, and wounds 7 and 8 refer to the application of wound dressing 2 (WD2). The wounds were always covered with a sterile, non-adherent primary dressing once the dressing was applied, followed by secondary dressing with transparent film, vet-wrap, crape bandage (Fig. 4G), and a waterproof jacket was put on after completion of dressing (Fig. 4H). Wound inspection, planimetry and photography were performed on day 11, 18, 25 and 34. Animals were mildly anaesthetized for dressing change and removal of exudates from wounds, if any. On day 34, the above mentioned procedure was sequentially performed and finally euthanized with 100 mg/kg of Thiopental Sodium (Neon Lab) intravenous injection in the marginal ear vein for necropsy and biopsy collection of wound tissues for histopathology. A detailed gross pathological examination was conducted on all the organs for abnormal lesions.

Wound healing assessment

Wound area (%) = (wound area remaining/original wound area) × 100.

Wound healing area (%) = [(original wound area − wound area at each time point)/(original wound area)] × 100.

Histopathological studies

All the wound tissues were processed to a section thickness of 3–5 μm and stained by hematoxylin and eosin (H&E) stains to observe the skin structure. The stained slides were examined under the light microscope and micrographs taken.

Epidermal characteristics were evaluated based on the extent of epithelialization and restoration of rete-ridges. Orientation of collagen fibers, their density and maturity were together quantified and indicated with a score for dermis restoration. Further, neovascularization and scar scores were determined from microscopic observations and quantified, as indicated in Appendices 1 to 3 (Altavilla et al. 2001; Beausang et al. 1998; Jung et al. 2013; Theunissen et al. 2016).

Results

The spherical copper nanoparticles prepared with the leaf extracts (Kota et al. 2023) in the size range of 35–100 nm (Fig. 5A) and the FN-doped carbon dots (Kota et al. 2024) in the size range of 15–20 nm (Fig. 5B) were added to the biopolymer solution prepared for the fabrication of wound dressing by electrospinning.

Fig. 5.

A a: FESEM pattern of green synthesized copper nanoparticles. b: EDS peaks denoting the presence of copper and elements in organic plant material coating the CuNPs. B a: FESEM pattern of FN-CDs which predominantly appear oblong/oval b: EDS pattern of FN-CDs indicating the presence of F and N. C Zeta potential distribution of bioactive enriched biopolymer nanofibrous dressing scaffolds dissolved in 1:1 pure acetone and isopropanol a: WD1, b: WD2, c: WD3, d: WD4. D a, d: Field emission scanning electron micrographs depicting morphology of WD2 and WD4, b, e: Elemental composition of WD2 and WD4, c, f: Elemental mapping of WD2 and WD4. E Piezoresponse force microscopy (PFM) of a: WD2 (24.28 pm/V), b: WD4 (0.28 nm/V)

Electrospun nanofibrous nonalayered scaffold

The zeta potential distributions of WD1 to WD4 are clear indications that the samples were polydisperse with multiple charged species of constituent polymers with bioactives, and the cumulative zeta potentials were—44.0 mV, 57.7 mV, − 118.3 mV and 94.6 mV respectively. Further, zeta potential of a sample is significantly influenced by factors like pH/ionic strength, concentration of the material, double layer thickness, and particular interactions. WD2 and WD4 with 57.7 mV and 94.6 mV, respectively, were selected for efficacy study in animals (Fig. 5C). The FESEM characterization of electrospun nanofibers revealed the encapsulation of Cu(O)NPs and FN-CDs, with the fiber diameter ranging between 252 nm and 3.1 µm and the EDX examination of the elemental composition revealed the presence of fluorine, copper, and nitrogen. (Fig. 5D). The piezoelectric coefficient of WD2 is 24.28 pm/V and WD4 is 0.28 nm/V (Fig. 5E). Micro-CT-Scan of the fabricated nanofibers demonstrated nine layers of shell, core and core–shell layers, interspersed with bioactive enriched biopolymer layers (Fig. 6).

Fig. 6.

Multilayered dressing pad with bioactives-impregnated electrospun biopolymer nanofibers (as drawn from micro-CT scan)

Body weights of the three pigs on test day 4 (prior to surgery) were found to be 25.5, 25.8 and 25.2 kg for P1, P2 and P3 animals, respectively making it easier to handle and take up less space in the feeding area. Following the creation of wounds, wound dressing WD2 or WD4 is applied as per the previously designed matrix model and then the wounds were covered with sterile non-adherent primary dressing, followed by secondary dressing with transparent film dressing, vet-wrap, and crape bandage (Fig. 4G, H) to cover the entire wound array region. The body weights were taken thereafter weekly once till day 34. On day 34 i.e. before necropsy, the body weight was found to be 27.3, 28.2 and 27.2 kg for P1, P2 and P3 animals, respectively. There was uniform increment in weight according to feed given as per standard requirements; the mean values of the body weights of the three animals with the standard deviations are presented in Table 3. There is no loss of weight in the animals at the end of the 34-day study period. On the contrary, there was a significant increase in the body weight of the experimental animals, as revealed by the student’s ‘T’ test, corroborating with the physical observations of the animals, viz., remaining healthy, active, and feeding normally in the entire study period. There was no morbidity/mortality observed during the procedure, and throughout the study duration from day 4 till day 34.

Table 3.

Recorded body weights of animals P1, P2 and P3 on different days of the study with the results of statistical test 

Animal number	Body weights (kg)						Student’s T test
	Day 4	Day 11	Day 18	Day 25	Day 34	Mean ± SD
P1	25.5	25.8	26.8	26.5	27.3	26.4 ± 0.7	t = 6.917 p = 0.001*
P2	25.8	26.0	27.1	27.1	28.2	26.8 ± 1.0
P3	25.2	25.6	26.2	26.2	27.2	26.1 ± 0.8
Mean ± SD	25.5 ± 0.3	25.8 ± 0.2	26.7 ± 0.5	26.6 ± 0.5	27.6 ± 0.6	26.4 ± 0.8

(p< 0.05 denotes statistically significant difference between the samples tested by Student's 'T' test)

Monitoring of animal pre, intra and postoperatively

Animals were observed during the procedure and during recovery from anesthesia and until day 34, for ECG/SpO₂, respiration rate, for defecation, urination and behavior. Animals consumed food and water normally with normal urine and fecal output. On day 4 (the day of the procedure), the pain score was 2 (moderate), 1 (mild) on day 5, 6 till day 8, and 0 (no pain) from day 9 to day 34 (Table 4).

Table 4.

Pain scores observed in the animals during experimentation

Animal number	Acclimatization phase			Experiment Phase
	04012	04015	04014	P1	P2	P3
Sex	Male
Days of observation	Pain Score
Acclimatization Phase (day 1–3)	0/3			–
Experiment phase (day 4–8)	–			2/3 (day 4); 1/3 (day 5 to day 8)
Experiment phase (day 9–34)	–			0/3

Wound healing evaluation

Clinical biochemistry and hematology

On day 4 (before the procedure) and the last day, blood samples were taken in order to use a hematology analyzer and a clinical biochemistry analyzer to examine hematological and clinical biochemistry parameters. The clinical biochemistry and the baseline hematology indicators were within acceptable limits on day 4 and day 34 (final day), and no abnormalities were seen. The clinical analysis of biochemical tests done on day 4 and day 34 showed a notable increase in albumin by 1.8 g/l, creatinine by 10 µmol/l, blood urea nitrogen (BUN) by 1.3 mmol/l, inorganic phosphate by 0.1 mmol/l, and potassium by 0.5 mmol/l. At the same time, there was a significant decrease in total cholesterol by 0.2 mmol/l, sodium by 1.4 mmol/l, and chloride by 5.6 mmol/l. These changes are usually due to the body's adjustments during recovery. On the other hand, a significant decline was noticed in total cholesterol by 0.2 mmol/l, sodium by 1.4 mmol/l, and chloride by 5.6 mmol/l. These statistically significant variations are typically attributable to the systemic changes during convalescence (Table 5).

Statistical analysis of the hematological data measured at day 4 and day 34 indicated a significant decline in RBC count, hematocrit value, and the count of neutrophils, monocytes, and eosinophils, while the lymphocytes showed a significant increase. These changes suggest a potential alteration in the immune response and overall blood health of the subjects over the observed period. Further investigation is warranted to understand the underlying mechanisms driving these hematological shifts. These variations in the hematological parameters clearly suggest that there are no symptoms of infection and that the animals are dealing with the slight inflammation that would obviously persist in the long wound-healing process (Table 6).

Wound creation, application of wound dressing, wound inspection and euthanasia

Wound healing was assessed by planimetry and quantitative assessment of wound scarring with photography on day 4, 11, 18, 25 and 34 (Fig. 7A). The wound healing progress from day 4 to day 34 showed significant improvement across all groups. The negative control group also displayed positive changes, a reduction of mean wound area from 6.25 to 0.08 cm², 0.84 cm² in P1 and P2 animals, while in P3 animal, it was completely healed with 0.00 cm² mean wound area. Standard Care witnessed a notable reduction in mean and showed complete healing with 0.00 cm² mean wound area. WD2 exhibited complete healing, transitioning from 6.25 to 0.00 cm² mean wound area in P1 and P3, while in P2, it almost healed with the mean wound area of 0.69 cm² on day 34. Similarly, WD4 demonstrated a remarkable decrease in the mean wound area from 6.25 to 0.00 cm² in P1 and P3, while in P2, it almost healed with the wound area of 0.52 cm² on day 34 The corresponding percent reduction in wound areas are depicted in Fig. 7B, C. Figure 7D displays the proportion of wound contraction/healing achieved with the wound dressings WD2, WD4, in contrast to the negative and standard while Fig. 7E displays the proportion of wound healing area in percent for WD2 and WD4. In day 11 and day 18 samples, WD2 was significantly better than WD4. However, no significant difference is visible between WD2 and WD4 over the study period of 34 days.

Fig. 7.

A Wound healing progress in the three animals, P1, P2 and P3, upon treatment with WD2 and WD4 from day 4 till day 34, in comparison with the standard and negative control. B Wound area percentage of WD2, WD4 in comparison with standard and negative control (* denotes p ≤ 0.05 from the results of ANOVA, suggestive of significant difference between the samples under consideration). C Wound area percentage achieved with the dressing pads WD2 and WD4 during the study period of 34 days (* denotes p ≤ 0.05 from the results of ANOVA, suggestive of significant difference between the two test samples only on day 11). D Percentage of wound healing area as observed with the negative, standard and the two test samples (* denotes p ≤ 0.05 from the results of ANOVA, suggestive of significant difference between the samples under consideration on day 11). E A comparison between the test items WD2 and WD4 in terms of wound healing area percentage on different days of observation during the study period (* denotes p ≤ 0.05 from the results of ANOVA, suggestive of significant difference between the samples on day 11)

Pathology, euthanasia, necropsy and microscopic findings

Animals were humanely euthanized by Thiopental sodium (100 mg/kg, IV) injection in marginal ear vein on day 34. Gross necropsy was done for wound areas and all the organs after euthanasia. In addition, a detailed gross pathological examination was conducted on all the organs for abnormal lesions. No abnormal lesions were found in the systemic organs. The hematoxylin and Eosin (H&E) stained slides were examined under the light microscope by the pathologist for evaluation of wound healing (Fig. 8A–H).

Fig. 8.

A Negative control, B standard care, C P1—WD2, D P1—WD4, E P2—WD2, F P2—WD4, G P1—WD2, H P1—WD4. E = epidermis (epithelialization); D = dermis; ▲ = neovascularization; Ic = immature collagen; *inflammation

Wounds in WD2, WD4, Standard Care, and Negative Control had mean epithelialization (neo-epithelialization) scores of 2.33, 3.00, 3.00, and 2.83, respectively. In negative control, standard care, WD2, and WD4, the average restoration of rete-ridges score (epidermis) of the wound was 1.33 for each therapy. In negative control, standard care, WD2, and WD4, the mean overall score for collagen fiber orientation, density, and maturity (Dermis) of the wound was 6.17, 5.17, 5.33, and 5.00, respectively. The mean neovascularization score (Dermis) of wounds in negative control, standard care, WD2 and WD4 was 2.17, 1.67, 2.00 and 1.83, respectively. The mean inflammation score (Dermis) of wound in negative control, standard care, WD2 and WD4 was 1.50, 0.50, 0.67 and 0.33, respectively. The mean scab score (Dermis) of wounds in negative control, standard care, WD2 and WD4 was 0.50, 0.17, 0.00 and 0.00, respectively (Table 7).

Table 7.

Mean histopathological score computed for the two dressings WD2 and WD4, and in comparison with the negative control and the standard care

Treatment	Mean histopathological scores with the ‘t’ and ‘p’ values calculated (Student’s ‘T’ test)
	Epidermis		Dermis
	E	RRR	CFO	CFD	CFM	N	I	S
Negative control	2.33 ± 1.0	1.33 ± 0.5	2.5 ± 0.6	1.83 ± 0.8	1.83 ± 0.8	2.17 ± 1.0	1.50 ± 1.1	0.50 ± 0.8
			6.2 ± 1.8
Standard care	3.00 ± 0.0 (t = 1.581; p = 0.09)	1.33 ± 0.5 (t = 0; p = 0.5)	2.00 ± 0.0 (t = − 2.236; p = 0.04*)	1.83 ± 0.4 (t = 0; p = 0.5)	1.33 ± 0.5 (t = − 1.167; p = 0.15)	1.67 ± 0.8 (t = − 1.463; p = 0.1)	0.50 ± 0.6 (t = − 3.872; p =.005*)	0.17 ± 0.4 (t = − 1.581; p = 0.09)
			5.17 ± 0.8 (t = − 1.290; p = 0.13)
WD2	3.00 ± 0.0 (t = 1.581; p = 0.09) (t = 0; p = 0.05**)	1.33 ± 0.52 (t = 0; p = 0.5) (t = 0; p = 0.05**)	2.33 ± 0.5 (t = − 1; p = 0.18) (t = 1.581; p = 0.09)	1.67 ± 0.8 (t = − 0.542; p = 0.31) (t = − 0.542; p = 0.31)	1.33 ± 0.5 (t = − 2.236; p = 0.037*) (t = 0; p = 0.5)	2.00 ± 0.9 (t = − 0.415; p = 0.4) (t = 1.581; p = 0.1)	0.67 ± 0.5 (t = − 2.711; p = 0.02*) (t = 1; p = 0.2)	0.00 ± 0.0 (t = − 1.463; p = 0.1) (t = − 1; p = 0.2)
			5.33 ± 1.8 (t = − 1.386; p = 0.112) (t = 0.25; p = 0.40)
WD4	2.83 ± 0.4 (t = 1.463; p = 0.10) (t = − 1; p = 0.181) (t = − 1; p = 0.18)	1.33 ± 0.52 (t = 0; p = 0.5) (t = 0; p = 0.05) (t = 0; p = 0.5)	1.83 ± 0.8 (t = − 3.162; p = 0.012*) (t = − 0.542; p = 0.305) (t = − 2.236; p = 0.03#)	1.67 ± 0.5 (t = − 1; p = 0.18) (t = − 1; p = 0.181) (t = 0; p = 0.5)	1.5 ± 0.8 (t = − 1; p = 0.18) (t = 0.542; p = 0.31) (t = 1; p = 0.18)	1.83 ± 0.8 (t = − 1.581; p = 0.09) (t = 1; p = 0.181) (t = − 0.542; p = 0.31)	0.33 ± 0.5 (t = − 3.796; p = 0.006*) (t = − 1; p = 0.181) (t = − 1.581; p = 0.09)	0.00 ± 0.0 (t = − 1.463; p = 0.10) (t = − 1; p = 0.181) (t = 0; p = 0.5)
			5.00 ± 1.9 (t = − 2.150; p = 0.042) (t = − 0.237; p = 0.411) (t = − 1; p = 0.18)

RRR restoration of rete ridges, CFO collagen fiber orientation, CFD collagen fiber density, CFM collagen fiber maturity, N Neovascularization, I inflammation, S scab; * & # indicate significant difference between the samples under consideration as demonstrated by the value of 'p≤ 0.05' obtained from Student's 'T' test. The first set of 't' and 'p' values indicate the difference between the negative control and others; second set denote between standard care and dressing pads WD2 and WD4; third set denote between WD2 and WD4.

Discussion

Plants such as Moringa oleifera, Tridax procumbens, Tinospora cordifolia, Chromolaena odorata, Lantana camara have been shown to have high concentrations of several phytochemicals, including flavonoids, tannins, phenols, saponins, sterols and tocopherols. The secondary metabolites of these plants act as natural therapeutic agents for wound healing because of their phytochemical bioactives (scutellarein, Isosakuranetin, rutin, gingerol, vicenin-2, Niazirin, Quercetin) that serve as strong antioxidant, antimicrobial, anti-inflammatory and antidiabetic modulators (Kota et al. 2023). Medicinal plants produce an array of secondary metabolites like phenols, flavonoids, sterols and others. These phenolic compounds contain hydroxyl and ketone groups, contributing to the iron chelation, and subsequently demonstrate a strong antioxidant property. Green synthesis of nanoparticles increases stability, prevents agglomeration, and phytochemicals get deposited, encapsulating the nanoparticles. Copper is a lower-cost metal than Silver and Gold, which are widely employed as antimicrobial and conducting materials in their nanoparticulate form. Effective catalysts, copper nanoparticles are simple to make, readily oxidized to produce copper oxides, and capable of interacting with biological systems. Metallic nanoparticles in the sizes of 20, 40, and 80 nm in diameter, with often a spherical shape, stimulate endothelial cell migration in a dose- and size-dependent way, while fibroblast- and keratinocyte-cell proliferation occur at particular ratios and sites. Larger copper nanoparticles (Cu(O)NPs) (80 nm) activate collagens in cultured fibroblast-cells more effectively than smaller Cu(O)NPs (40 nm). In addition, it has been discovered that, in rat models, copper nanoparticles significantly improve the process of epidermal healing and neovascularization without having any adverse effects (Negut et al. 2018). The capacity of Cu(O)NPs to adsorb to the cell membrane and induce depolarization is the suggested mechanism for their antibacterial potential. Cu(O)NPs alter the cell wall's negative charge, which compromises the integrity of the cell wall. According to the alternative theory put forth, the interaction between the copper cations of the nanoparticles and the lipids of the cell wall results in peroxidation and pore opening in the cell membranes.

According to Wang et al., CDs were discovered to facilitate the movement of epithelial cells through a process called epithelial-mesenchymal transition. Furthermore, CDs have been shown to lower inflammation and encourage tissue repair, which helps the wound healing process as a whole (Wang et al. 2020).

Chitosan, also known as Poliglusam, is a polymer that is compatible with living organisms, bestowed with the ability to scavenge free radicals (antioxidant) and has the potential to fight against microorganisms. However, its mechanical strength is limited. Keratin has a lower viscosity and poor mechanical properties, although they can be improved by adding natural (gelatin, chitosan) or synthetic (polyvinyl alcohol, PEO, etc.) polymers. PVA and PEO are both hydrophilic, biocompatible, high molecular weight polymers with superior mechanical properties. The FDA-approved synthetic polymer polyvinyl alcohol (PVA) is non-toxic, biodegradable, and reasonably priced. The amount of secondary alcohol functional groups in its semi-crystalline structure varies depending on the degree of hydrolysis. This in turn controls the polymer's crystalline nature, solubility, and chemical properties.

The spinnability of chitosan can be significantly increased by polyethylene oxide (PEO), and the resulting composite nanofibers are stable and non-cytotoxic to cells. When keratin is blended with PVA and PEO, the solution becomes more viscous, making electrospinning easier. Taurine is used for faster skin tissue regeneration. The FDA-approved cationic polymer poly l-lysine has garnered a lot of interest in tissue engineering due to its biodegradability and biocompatibility. The positively charged poly l-lysine readily interacts with the negatively charged phospholipid surface on a variety of cell types. Among the poly l-lysine family, ε‐Poly‐l‐lysine (ε‐PL) has the strongest antibacterial activity and stops the majority of germs from growing. For antibacterial wound healing, cellulose acetate (CA) has been identified as an appropriate semi-synthetic biomaterial. It has a number of desirable qualities, including mechanical strength, hydrolytic stability, biodegradation, bio-compatibility, insolubility in water, availability, and affordability. Metabolic events in the body create free radicals, which harm cells and tissues. In response, living cells release antioxidants to neutralize these free radicals. The detrimental impacts of excessive free radicals generated in damaged tissues can be counteracted through the application of natural polymers, such as chitosan and keratin, in various wound treatments. The facilitation of endogenous hemostasis, the uptake of inflammatory exudates, and the encouragement of cellular proliferation are the likely mechanisms linked to wound healing by wound dressings. The qualities of wound dressing enhance the strength of newly developed tissues and act as a barrier to prevent infections. In addition, they alleviate pain by obstructing nerve terminals and minimize the creation of cicatrix/scar tissue.

The study's conceptual innovation is the creation of a multipurpose smart wound dressing that uses the electrospinning technology to combine sophisticated nanomaterials, natural polymers, and phytoconstituents produced from plants. While electrospinning is an established method, its application here is reimagined by incorporating piezoelectric electrospun biopolymer nanofibers, which introduce a dynamic, mechano-responsive element to enhance tissue regeneration. The dressing also features biosynthesized carbon dots derived from medicinal plants—an emerging green nanotechnology approach that bridges traditional phytotherapy with modern nanomedicine. In addition, the inclusion of a copper-coated cotton fabric provides antimicrobial protection and potential electrical conductivity, further contributing to wound healing. In order to demonstrate a thorough and translationally meaningful approach to advanced wound care, this composite dressing was tested for 34 days in a full-thickness wound model in Yorkshire pigs, a clinically relevant large animal model. The wound inspections, planimetry and photography were performed on days 4, 11, 18, 25 and 34. The animals were euthanized on day 34 for gross necropsy and wound tissue collection for histological evaluation.

According to Trøstrup et al. (2013), chronic wounds often have co-morbidity, which causes a significant increase in systemic inflammatory indicators including leukocytes, C-reactive protein, or procalcitonin in the early stages. Animals were monitored for morbidity and viability throughout the procedure, three hours after the procedure, and until Day 34, when the study came to an end. No mortality and clinical signs were observed. The animals were active and responsive during the whole study period, with no deviation from normal behavior, posture, movement, and skin condition. The pain score was found to be 2 (moderate) on day 4 (day of procedure), and from day 5 to 8, the pain score was 1 (mild), and then, from day 9 till day 34, the pain score was found to be 0 (no pain). There were no aberrant results on days 4 or 34, and the baseline hematological parameters and clinical biochemistry were both within the normal range (Tables 5, 6).

The wound scarring score of test item WD2 treated wounds revealed a significant color mismatch in the quantitative evaluation of the wounds treated with various treatment groups P1, P2, and P3 (compared to surrounding) on day 4 to slight mismatch or perfect color on necropsy day (day 34). Surface was changed from shiny to matte or shiny with ‘moderate’ to ‘no or mild distortion’. The texture of wounds was changed from hard on day 4 to normal or just palpable on day 34. Day 4 showed no bio-film, whilst Day 34 showed "no or a scanty "bio-film. Wounds treated with test item WD4, routine care, and negative control wounds showed a comparable pattern of wound scarring scores.

The experimental groups (WD2 and WD4) outperformed normal care and the negative control group, indicating significant improvements in wound healing throughout the course of the trial. The mean epithelialization score of wounds treated with normal care, WD2, and WD4 was higher than the negative control in the histological study. When comparing wounds treated with routine care, WD2, and WD4, to the negative control, there were no appreciable differences in the mean restoration of rete-ridges score (Epidermis).

The mean total score of collagen fiber orientation, density, and maturity (Dermis) of wound treated with Standard Care, WD2 and WD4 was decreased, as compared to negative control. The mean neovascularization score (Dermis) of wound treated with standard care and WD4 was decreased, as compared to negative control, and comparable in WD2. The mean inflammation and scab scores (Dermis) of wounds treated with standard care, WD2 and WD4 decreased, when compared with negative control (Table 7).

The increase in the mean epithelialization score in WD2, WD4, and standard care was a sign that the wound was healing. Under routine care, the mean restoration of rete ridges, collagen fiber (abnormal orientation, density, and maturity), neovascularization, inflammation, and scab decreased in WD2 and WD4, which was a good sign of wound healing. Student’s ‘T’ test was performed on the different data sets to understand whether the variations in the scores were statistically significant. Standard care and WD4 dressing showed significant difference from the negative control in collagen fiber organization and inflammation; WD2 showed significant difference in collagen fiber maturity and inflammation. These findings suggest that both WD4 and WD2 dressings can enhance wound healing by improving tissue structure and reducing inflammatory responses. Further research is needed to explore the underlying mechanisms driving these effects. A comparison of the test items WD2 and WD4 with the standard care indicated a significant difference in the epidermis in the wounds treated with WD2. A comparison between WD2 and WD4 showed no statistically significant differences except for a decline in collagen fiber organization in wounds treated with WD4.

The primary goal of animal studies is to track the development of granulation tissue, the reduction in wound size, and ultimately the re-epithelialization process. When assessing the efficacy of a given treatment, the kind of sample and the method used to collect the data are essential considerations. Meaningful indicators for evaluating the overall health of the animal include regular weight measurements, monitoring body temperature, and observing traditional signs of inflammation. Advancements in molecular techniques now allow for the detection of genetic material from various infections in wound biopsies or waste material during revisions. Monitoring wound closure is a crucial clinical criterion for assessing patients with wounds that are not healing. Our smart-dressing-material, which has outperformed the standard care provided in the wound-healing of the porcine model, is due for human clinical studies. Efforts are underway to further improve the dressing for exclusive benefits in diabetic and burn-wounds, in addition to general wounds of all types.

Conclusion

Due to either the surface charges and permanent polarization of piezoelectric materials or the temporary deformation brought on by the contraction and protrusion of the associated cells, piezoelectric scaffolds have demonstrated advantageous protein adsorption, cellular attachment, and proliferation. The next generation of scaffolds for tissue engineering may be made of piezoelectric materials. Knowing that advanced wound care solutions are necessary to handle the various issues that wound-inflicted subjects experience, the effort was made to develop a multicomponent nonalayered piezoelectricity-conferred wound dressings with variations in the concentrations of poly l-lysine (PLL) and polyethylene oxide (PEO). Out of the four dressings fabricated with low and high concentrations of PLL and PEO and characterized, two dressings were chosen for the animal study.

The large animal-porcine-model-study data obtained with the two dressing materials, viz. WD2 (poly l-lysine) and WD4 (polyethylene oxide), indicated substantial advancements in wound healing over the observed period and their out performance over standard care and the negative control group. There was no scarring found in all the wounds. In the histopathological evaluation of wound tissues, the test item WD2, WD4 and standard wound care have shown better histopathological, epithelialization and wound healing scores. These indicate that WD2 and WD4 test items have a better wound healing potential as compared to standard wound care employed in the study. These fabrications are a significant advancement over the current commercial nanofibrous dressings because of their unique natural composition of biopolymers and plant derived bioactives, that provide adequate stimuli to the injured cells to repair and regenerate at a faster pace, and more importantly warding off the infections with little or minimal antibiotic administration, and thus address the persistent problem of multidrug-resistant infectives by a careful crafting of the abundant and proven natural bioactives.

The results of the current porcine study are translatable to humans in the case of acute wounds. However, in the specific cases of chronic wounds like the diabetic/burn wounds, pressure ulcers, and others, the reported observations are limited. A detailed investigation into the role of the phytoconstituents and biopolymers should be undertaken, and the necessary inclusions/exclusions should be effected for improved efficacy. This deeper exploration could lead to more effective treatment options for patients suffering from these complex physiological and wound environment conditions. By identifying the most beneficial compounds, researchers can tailor therapies that enhance healing processes and potentially reduce recovery time.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors have actively contributed to the conception and study design, data collection, analyses, and interpretation. The manuscript was drafted, edited and finally approved by all the contributors.

Funding

SK and RS acknowledge the receipt of funding from the AMT division of the Department of Science and Technology, New Delhi, India (No: DST/TDT/AM/2021/02).

Declarations

Conflict of interest

Authors declare no conflicts of interest.

Ethics

The test facility at Palamur Biosciences Pvt. Ltd., Hyderabad, and Telangana, India was certified by the Committee for the Control and Supervision of Experiments on Animals (CCSEA) for Experimentation (Certification No. 1312/PO/RcBiBt-S/RcBiBt-L/09/CPCSEA). All the protocols adopted in the study were approved by the ICMR, New Delhi.