Synthesis and characterization of clove/gelatin coated silk sutures for surgical site infection and wound healing

Abstract

Surgical Site infections (SSIs) affect up to 5% of surgical procedures, posing a significant postoperative complication. This study aims to develop and evaluate antibacterial clove/gelatin-coated sutures to reduce SSI infection. Clove extract, known for its antimicrobial properties, was incorporated into a gelatin matrix as a biocompatible coating for silk-braided sutures. Sutures were dip-coated in clove/gelatin solutions at concentrations of 5, 10, 15, and 20%. The surface morphology, chemical composition, and mechanical strength of the coated suture were characterized by using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and tensile testing. Antimicrobial efficacy was assessed via zone-of-inhibition assays against (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecalis). The 20% Clove/gelatin coating exhibited the highest antibacterial activity (17.5 ± 0.875 mm) and demonstrated low cytotoxicity in MTT assay with human primary dermal fibroblast. Hemolytic assays revealed that all composite concentrations resulted in <1% hemolytic activity. Approximately 93% of drug release was observed in 20% formulation within 72 h which is promising results compared with untreated sutures. Based on its superior antibacterial performance, the 20% formulation was selected for in vivo studies. In a rat abdominal incision model, wounds closed with coated sutures exhibited complete healing, while those closed with uncoated sutures remained partially healed. These findings highlight the potential of clove/gelatin-coated sutures for enhanced antimicrobial protection and improved wound healing in surgical applications.

Graphical Abstract

Introduction

Hospital-acquired infections (nosocomial infections) are diseases that patients often experience during hospitalization significantly increasing healthcare costs [1]. Patients are mostly at risk for nosocomial infections due to medical equipment, surgery, immunosuppressive medications, concurrent diseases, and the rise of bacteria that are resistant to several drugs. Surgical site infections (SSIs), primarily brought on by contaminated suture materials used in surgery and medical implants, are the causes of nosocomial infections [2]. SSIs are frequent and often seen as postoperative complications, occurring in as many as 5% of surgical procedures [3]. Since most commercially available absorbable and non-absorbable sutures lack antimicrobial qualities, germs originating from the natural flora of the skin can easily penetrate wounds through capillary action. These microorganisms multiply after adhering to suture surfaces and forming biofilms, which are intrinsically challenging to control [4]. According to the data, the most prevalent bacterial pathogens that are responsible for SSIs are S. aureus, E. coli, K. pneumoniae, and E. faecalis [5].

Currently, triclosan-coated sutures (e.g., PDS Plus, Vicryl, and Monocryl) are the only commercially available antimicrobial sutures [6]. However, concerns over triclosan’s toxicity and the compatibility of metal-based antimicrobial agents with tissues have driven the need for safer, biocompatible, and highly effective alternatives [7]. Numerous studies have addressed these issues and emphasized the advancements being made in the creation of coatings made of clove-herbal extract and clove-coated sutures [8]. In the past, herbal extracts such as curcumin, cinnamon, aloe vera, and moringa have been used in combination with other polymeric coating ingredients to make antimicrobial sutures [8–10].

Clove (Syzygium aromaticum), a spice with diverse health benefits, has been widely used in traditional medicine and culinary practices [11]. Clove is a spice with several health benefits because it is high in bioactive components. The primary bioactive substances included in cloves are caryophyllene, eugenol, and acetyl eugenol. Eugenol, the primary bioactive ingredient in clove essential oil, makes up up to 85% of it [12]. Numerous health benefits, such as analgesic, antioxidant, and anti-inflammatory qualities, have been reported. Eugenol is efficient against a wide variety of germs because of its demonstrated antibacterial and antifungal qualities [13]. Another significant bioactive component of cloves is acetyl eugenol, which can make up as much as 15% of the essential oil [14]. One naturally occurring substance found in clove extract essential oil is caryophyllene. It is a member of a class of substances called sesquiterpenes, which are found naturally in plants and have a variety of biological functions [15].

Numerous bioactivities of clove extract include anti-inflammatory, antibacterial, neuroprotective, and antioxidant properties [16]. Clove extract’s antioxidant action is believed to be caused by phenolic compounds, which scavenge free radicals and reduce oxidative stress [17]. These phenolic compounds also reduce the production of inflammatory cytokines and enzymes, which adds to their anti-inflammatory qualities. Eugenol is the primary component of clove extract that gives it antibacterial efficacy against a range of infections [18]. Additionally, research has demonstrated the anticancer properties of clove extract, which causes cancer cells to die and stop proliferating [19]. Moreover, clove extract has been demonstrated to have neuroprotective properties due to eugenol, which decreases inflammation and oxidative stress in the brain [20].

The hydrolytic breakdown of collagen protein produces the naturally occurring polymer known as gelatin. Its distinct amino acid makeup offers numerous health advantages [21]. Typically available in pills, granules, or powder form, gelatin often requires dissolution in water before usage [22]. Chemically it is composed of 19 amino acids with glycine (27–35%), proline, and hydroxyproline (20–24%), collectively comprising around (47–59%) of its structure. The remaining 41% includes glutamic acid, alanine, arginine, and aspartic acid [23]. The elemental makeup of gelatin is composed of 17% nitrogen, 50.5% carbon, 25.2% oxygen, and 6.8% hydrogen. Its structure consists of a mix of hydrophilic chains that are both single and double-unfolded [24]. At the molecular level, gelatin is composed of polypeptide chains with molar masses of approximately 90 × 10³ g/mol for α-chains (single polymer chains), 180 × 10³ g/mol for β-chains (two covalently crosslinked α-chains), and 300 × 10³ g/mol for γ-chains (three covalently crosslinked α-chains) [25]. Gelatin exhibits exceptional physical properties, including high dispersibility, low viscosity, strong water retention, and excellent film-forming capabilities. Moreover, studies suggest that gelatin possesses inherent antibacterial activity, likely due to its ability to interact with bacterial cell membranes, inhibit microbial adhesion, or function as a physical barrier against bacterial proliferation. Its antimicrobial effectiveness is particularly enhanced when combined with bioactive agents in gelatin-based films and coatings [26, 27].

The development of antimicrobial sutures is essential in addressing the growing challenge of surgical site infections. These sutures not only act as physical wound closures but also serve as a defense against microbial contamination, reducing postoperative infection risks. To be effective, the coating material must exhibit both antibacterial properties and biocompatibility. Clove extract, known for its potent antimicrobial activity, was incorporated into a gelatin matrix and applied to silk-braided sutures via dip coating. The clove/gelatin-coated sutures were evaluated for their surface morphology, antimicrobial efficacy, biocompatibility, and in vitro drug release kinetics, followed by an in vivo assessment of their wound-healing potential.

Materials and methods

Preparation of coating material

Clove buds were procured from a local supplier, thoroughly washed, and air-dried in the shade for 48 h. Once completely dried, an electric blender was used to grind them into fine powder (Haier 3420). To extract clove bioactive compounds, absolute ethanol (Sigma-Aldrich, USA) was used as a solvent. A total of 0.4 g of ground clove powder was mixed with 40 mL of an 80:20 ethanol-to-deionized water solution (32 mL ethanol, 8 mL deionized water). The mixture was centrifuged (Fast Gene CAT. NO. NG003) for 10 min at 4000 rpm after 30 min of incubation at 65 °C in a shaking water bath. The resultant extract was collected and kept at room temperature for later use after the supernatant was filtered using Whatman No. 1 filter paper. A fresh batch of the extract was prepared for each experimental assay.

Gelatin powder (≥95% deacetylated) was sourced from Shanghai Macklin Biochem, China. A final volume of 62.5 mL was obtained by dissolving 2.5 g of gelatin powder in 60 mL of deionized water to create a 4% (w/v) gelatin solution. The solution was magnetically stirred at 40 °C for 30 min until fully homogenized. Clove extract was then incorporated into the gelatin solution at varying concentrations (5, 10, 15, and 20%) and stirred until a uniform coating solution was achieved. The overall preparation process is illustrated in Fig. 1.

Fig. 1.

Preparation of clove/gelatin composite

Dip coating method

Non-absorbable silk braided sutures (Yancheng Huida, China; manufactured by TRUSILK) were coated using a dip-coating technique. The process was conducted with a PTL-MMB01 OK-M210 dip-coating machine (China) under aseptic conditions, ensured by prior cleaning with 70% ethanol. The sterile clove/gelatin coating solution was placed in the designated reservoir of the machine. Sutures were secured in the sample holder and immersed into the coating solution at a controlled speed of 10 mm s⁻¹ for 15 min. They were then withdrawn at the same speed and dried at 37 °C using the sterile dip coater’s heating function. Once dried, the coated sutures were carefully removed, placed in sterile Petri dishes, and transferred to a laminar flow hood for further processing. The sutures were then cut into 1 cm segments for subsequent characterization and testing. This procedure was repeated for all composite concentrations (5, 10, 15, and 20%).

Characterizations

Fourier transform infrared spectroscopy

Fourier-transform infrared (FTIR) spectroscopy was used to determine functional groups and molecular interactions between the clove extract and gelatin matrix. FTIR analysis was conducted using a Bruker Vertex 70 spectrometer on clove/gelatin-coated sutures, with a scan range of 4000–400 cm⁻¹.

Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM) was employed to characterize the surface morphology of the coated sutures. SEM (JSM-6490A-JEOL Japan) was used on coated sutures.

Tensile strength

Tensile strength is a critical parameter in suture construction, influencing the knotting efficiency and mechanical stability of the material. A suture with insufficient tensile strength may fail under the repetitive forces exerted during knotting, leading to premature breakage. Therefore, assessing the tensile strength of coated sutures is essential to ensure their mechanical reliability.

In this study, the tensile strength (TS) of coated sutures was evaluated using a Universal Tensile Testing Machine (Shimadzu AUTOGRAPH AG-X plus) equipped with a load cell. A coated suture measuring 75 cm in length was secured between two grips positioned 15 cm apart. The mechanical performance of the sutures was assessed to determine the structural integrity of the coating and its resistance to tensile forces.

Clove release profile

The drug release kinetics of clove/gelatin-coated sutures were assessed in phosphate-buffered saline (PBS, pH 7.4) to assess their release behavior in physiological conditions. Coated sutures were cut into 3 cm segments and soaked in 5 mL of PBS. At 10-min intervals, the PBS was taken out and refilled with 3 mL of fresh PBS. Prior to immersion, the sutures were weighed, and post-exposure measurements were recorded while still damp. The clove release profile was quantified using a UV-Vis spectrophotometer at an absorbance of 292 nm, analyzing the collected PBS solutions. All experiments were performed in triplicate, and average values were taken out.

Kinetics of clove release

Five kinetic models were applied to the experimental data to ascertain the release mechanism of clove compounds: zero-order, first-order, Korsmeyer–Peppas, Higuchi, and Hixson–Crowell. Among these models, the Hixson-Crowell model displayed the highest correlation coefficient (R²), indicating the best fit for the observed release profile. The details of this model are described below.

Hixson and Crowell model

The Hixson–Crowell model identifies the release from systems where the surface area and the diameter of particles changes [28]. Hixson–Crowell obtained the equation that defines the rate of dissolution in terms of the cube root of the particle weight, and the radius is not taken to be constant, which can be expressed by the given equation:

$${\mathit{Mo}}^{\frac{1}{3}}-{\mathit{Mt}}^{\frac{1}{3}}=\mathit{Kt}$$ 1

where M$o$ is the initial amount of drug, Mt is the remaining amount of drug in the pharmaceutical dosage form at time “t” and the proportionality constant is denoted by κ.

Antimicrobial testing

The antimicrobial activity of clove extract-coated nonabsorbable silk sutures were evaluated via a standard disc diffusion method. The formation of an inhibition zone around the sutures confirmed their antibacterial efficacy.

Luria-Bertani (LB) broth (1 L) and LB agar were prepared according to the manufacturer’s instructions and sterilized via an autoclave at 121 °C along with Petri plates. Bacterial strains, including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, and Enterococcus faecalis, were inoculated into fresh sterile broth in sterile Falcon tubes and incubated at 37 °C in a shaking incubator. The optical density (OD) of the cultures was measured via a spectrophotometer to ensure bacterial growth was in the log phase.

A 0.1 mL bacterial inoculum was evenly spread onto LB agar plates. Suture threads were prepared by cutting them into 1 cm lengths and placing them on Petri dishes inoculated with bacteria. Each plate was divided into six sections: four sections contained sutures coated with clove/gelatin composites at concentrations of 5, 10, 15, and 20%, while the remaining two sections contained sutures coated with clove extract and gelatin, respectively. The inoculated plates were incubated at 37 °C overnight, and the zones of inhibition were measured to assess antibacterial activity.

Hemolysis assay

A hemolysis assay was used to determine the hemolytic effect of Clove/gelatin composite. The coated sutures were immersed in a PBS solution. The fresh human blood sample was extracted via IV injection by a medical nurse and placed in an EDTA tube for the experiment, and 3 mL of it was used. After this, they were centrifuged at 10,000 rpm for 10 min to get the supernatant by transferring them to two micro centrifuge tubes each of 1.5 mL. To conduct this process, 1 mL PBS was added to 500 μL of blood, and afterward, the mixture was centrifuged at 6000 rpm for 10 min to isolate the RBCs. Following five iterations of this process using PBS at a pH of 7.4, the sample was found to contain a pure pellet.

The subsequent step involved the addition of clove extract, gelatin, and composite concentrations (5, 10, 15, and 20%) to the blood sample while simultaneously adjusting the final volume using PBS. After that, the samples were subsequently put in a shaking incubator set at 80 rpm for 4 h. After that, the samples were centrifuged for 10 min at 5000 rpm, and the absorbance of the collected supernatant was measured at 540 nm in triplicate using a microplate reader. To establish positive and negative controls, blood samples in PBS and Triton-X-100 were used, respectively. The following equation was used to determine the percentage of the hemolytic activity:

$$\mathrm{Hemolysis}\left(\%\right)=\frac{\mathrm{Absorption}\mathrm{Sample}\text{-}\mathrm{Absorption}\mathrm{Negative}\mathrm{Control}}{\mathrm{Absorption}\mathrm{Positive}\mathrm{Control}-\mathrm{Negative}\mathrm{Control}}\times 100$$ 2

Cytocompatibility of coated sutures

The cytocompatibility of the clove/gelatin composite was assessed using human primary dermal fibroblast (HDFa) cell culture. The HDFa were cultured in liquid media containing DMEM (89%), Fetal Bovine Serum (10%), and Penicillin/Streptomycin (1%) were all obtained from Gibco. The composite was cultured with 5 × 10⁴ HDFa cells (counted using a hemocytometer) along with a control well containing only HDFa cells. Cultivation was performed at 37 °C in a 5% CO₂ incubator for 48 h. The cells were then allowed to grow on both the control and composite solutions for 48 h in a 5% CO₂ incubator under the above-stated conditions. The culture medium was then added in a sterile 96 well plates in triplicates for each sample and control and incubated again for 12 h to recover from handling. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) Reagent was added to each well and cell cultures were incubated again for 2 h. Cells were periodically viewed under inverted microscopes for presence of intracellular punctate purple precipitate. Upon the clear visibility of precipitate under the microscope, 100 µl of Detergent agent was added to all the wells and well plate was swirled. Plate was placed in dark for 2 h and the absorbance was measured at 450 nm via a microplate reader (Accuris, Smart Reader 96 – MR 9600). The values obtained were added computed in excel using the Eq. (3):

$$\mathrm{Cell}\mathrm{viability}\left(\%\right)=\frac{\mathrm{Absorbance}\mathrm{test}}{\mathrm{Absorbance}\mathrm{control}}\times 100$$ 3

In vivo investigation

The efficacy of clove/gelatin-coated sutures to cure wounds was examined using 6 Wistar rats (60–65 g). These rats were sourced from the Atta-Ur-Rahman School of Applied Biosciences, NUST, Islamabad, Pakistan. The rats were acclimatized for at least 7 days under a 12-h light/dark phase with unlimited food and water access. The rats were put on a fasting period of 8 h before surgery. Anesthesia was induced using Ketamine (40–60 mg/kg) and Xylazine (5–10 mg/kg). Following the removal of hair from the dorsal region, the spine was cut 1 cm on both sides. A coated suture was used for one side’s stitching, while an uncoated suture was used as a control on the other. Post operates; the rats received a subcutaneous injection of tramadol (0.03 mg) for pain management. For 14 days, daily photos of the wound region were obtained to evaluate infection control and wound healing. Regular measurements of the wound’s length, width, and area were taken along with an evaluation of its color, appearance, and signs of infection. The analysis focused on the variations in wound healing and infection control between the left (uncoated sutures) and right (coated sutures) sides, which relied on macroscopic observations and images.

Histological evaluation

On the 0th, 7th, and 14th days after surgery, abdominal skin pieces were obtained. The specimens were placed in a 10% neutral-buffered formalin for 48 h and sectioned into 4 μm slices. These specimens were processed and stained with hematoxylin and eosin (H&E). The stained samples were evaluated by a senior pathologist (blinded to the experimental groups) to compare the histological differences between the coated and uncoated groups.

Statistical analysis

The analysis was accomplished in triplicate, and analysis of variance (ANOVA) and Student’s t test. A p value of less than 0.05 was considered statistically significant.

Results

Fourier transform infrared spectroscopy (FTIR)

The FTIR spectra were recorded in the range of 4000 cm⁻¹ to 400 cm⁻¹ to analyze the functional groups present in the coated sutures. In Fig. 2A, the peak at 3427 cm⁻¹ corresponds to the OH group, indicating the presence of phenol. The peak at 2932 cm⁻¹ is attributed to the alkyl C-H stretch (sp³), the peak at 1723 cm⁻¹ signifies the presence of an ester (C=O) group, and the peak at 1617 cm⁻¹ corresponds to aliphatic alkenes, whereas the sharp peak at 1512 cm⁻¹ indicates the presence of an aromatic group. Additionally, the peak at 1451 cm⁻¹ is associated with methylene (CH₂) vibrations, and the peaks at 1358 cm⁻¹, 1043 cm⁻¹, 916 cm⁻¹, and 760 cm⁻¹ correspond to methyl (CH₃), C-O methylene, and C=C bonds, respectively [29].

Fig. 2.

FTIR spectrum of A clove extract, B gelatin solution, C clove/gelatin composite at different concentrations

In Fig. 2B, the peak at 3433 cm⁻¹ indicates the presence of hydrogen bond and Amide A group. The peak at 1630 cm⁻¹ corresponds to the presence of the Amide I group, while the peak at 1565 cm⁻¹ indicates the presence of the Amide II group. The peaks at 1460 cm⁻¹ and 1380 indicate the presence of a methyl group (CH₃), and the peak at 1240 cm⁻¹ corresponds to the presence of the Amide III group [30].

In Fig. 2C, the peaks range from 3328 cm⁻¹ to 3532 cm⁻¹, indicating the stretching of the OH group in all samples with changing intensities and positions due to hydrogen bonding, suggesting the presence of phenol [29]. The CH stretching range of 2800 cm⁻¹ to 3000 cm⁻¹ confirms the presence of a methyl group. The Amide I peak at 1638 cm⁻¹, primarily associated with C=O stretching in the peptide backbone, reflects gelatin’s structural integrity. The presence of clove extract influences the conformation and arrangement of polypeptide chains, leading to variations in the Amide I region [31]. The Amide II band arises from the C-N stretching and N-H bending, while C-O stretching occurs due to the interaction of eugenol with alcohols, esters, and ethers. Additionally, out-of-plane C-H bending vibrations confirm the presence of eugenol [29].

Scanning electron microscopy (SEM)

SEM images of uncoated Silk braided non-absorbable sutures are depicted in Fig. 3A, B illustrating that the Silk braided non-absorbable suture is completely coated with the clove extract. Looking at the coated images of the silk braided nonabsorbable suture, one gets the impression that coated sutures are homogenous. We place a Clove/gelatin-coated suture in a vacuum to capture a scanning electron image, turning our suture sample into a dried-up one. Thus, these little micro-cracks would be present in the SEM images due to stress when the samples are dried. Our coated sample appears uniform before the process of drying.

Fig. 3.

SEM of uncoated (A) and coated suture (B)

Tensile strength

The statistical analysis of a sample revealed that the tensile strength of silk-braided suture was slightly lower than that of coated suture, sustaining 47 N. It shows that the analytical mechanical properties of clove particle-coated sutures exhibit higher average tensile strength than the uncoated silk braided sutures. This can be explained by the fact that fibers in the silk-braided suture have enhanced binding with the aid of gelatin, which implies that the combination presents a higher resistance to the axial load, as illustrated in Fig. 4. Sutures that have a high coefficient of friction encounter the problem of tissue drag when being passed through the biological tissue and thus experience even a high value of tissue injury. Thus, one has to be acquainted with the frictional properties that are inherent to a suture material [32, 33]. Therefore, the results of his study reveal that the coefficient of friction of the treated silk braided suture is less as compared to the untreated silk braided suture; the treated silk braided suture’s knot is not weakened as easily as compared to the untreated silk braided knot.

Fig. 4.

Tensile strength of clove/gelatin composite

Clove release profile

The drug release behavior of the Clove/gelatin composite was evaluated, as shown in Fig. 5. All release profiles followed first-order kinetics, suggesting a diffusion-controlled mechanism [34].

Fig. 5.

Clove release profile at different concentrations

The release profiles for different concentrations of Clove/gelatin-coated sutures (5, 10, 15, and 20%) are shown in Fig. 5. An initial burst release occurred within the first 25 h, followed by a gradual decline in the release rate after 50 h. Beyond this point, the release transitioned into a slow, nearly linear phase, continuing up to 72 h.

Gelatin-based matrices not only facilitate the sustained release of bioactive compounds but also protect encapsulated biomolecules from degradation. This characteristic makes them suitable carriers for controlled drug delivery, depending on the specific biological activity of the loaded molecules [35].

Clove release kinetics

The R² values for the Hixson–Crowell time equation model are shown in Table 1. The drug release kinetics were evaluated by various models based on the % of drug release at pH 7.4 over specified time intervals (Fig. 6).

Table 1.

The Hixson–Crowell kinetic model on drug release

Composite	Hixson–Crowell
Sr. No (%)	R2	K1
Clove/gelatin composite 5%	0.9877	−0.0219
Clove/gelatin composite 10%	0.9877	−0.0219
Clove/gelatin composite 15%	0.9877	−0.0219
Clove/gelatin composite 20%	0.9877	−0.0219

Fig. 6.

The Hixson–Crowell kinetic model of drug release

Antimicrobial activity

The antimicrobial efficacy of Clove/gelatin-coated sutures was evaluated against various bacterial strains, as illustrated in Fig. 7. The results demonstrated significant antibacterial activity, primarily attributed to the clove extract, while gelatin functioned as an encapsulating and stabilizing agent, enhancing bioactivity through sustained release.

Fig. 7.

Bar graph of the antimicrobial activity of clove/gelatin suture

The antibacterial effectiveness varied with clove concentration, with the highest inhibition observed at 20% clove extract. Among the tested strains, Staphylococcus aureus exhibited the largest zone of inhibition (17.5 mm), followed by Escherichia coli (17 mm), Klebsiella pneumoniae (16.5 mm), and Enterococcus faecalis (15 mm). These findings underscore the potential of Clove/gelatin-coated sutures for antimicrobial applications in surgical settings (Fig. 8).

Fig. 8.

The figure shows antimicrobial activity of clove/gelatin suture in A S. aureus, B E. coli, C K. pneumoniae, D E. faecalis

Hemolytic activity

The hemolytic assay evaluated the potential of the Clove/gelatin composite to induce red blood cell (RBC) lysis. This process involves the rupture of RBC membranes, leading to the release of intracellular content. As a positive control, 0.1% Triton X-100 induced complete hemolysis, whereas phosphate-buffered saline (PBS) served as a negative control, exhibiting minimal hemolysis. The assay was conducted using four different concentrations of the Clove/gelatin composite (5, 10, 15, and 20%), as shown in Fig. 9.

Fig. 9.

Hemolytic activity of clove/gelatin composite at different concentrations

The results demonstrated that hemolytic activity increased with higher clove extract concentrations. At the highest concentration (20%), the composite exhibited approximately 0.54% hemolysis, whereas the lowest concentration (5%) resulted in only 0.075% hemolysis. Since a hemolytic rate below 1% is generally considered non-hemolytic, these findings suggest that the Clove/gelatin composite is biocompatible and safe for use as a drug carrier.

Cytocompatibility

The cytocompatibility of the Clove/gelatin composite was assessed using the MTT assay, evaluating the percentage of viable human primary dermal fibroblasts (HDFa) after 48 h of incubation. The results, shown in Fig. 10, indicate that the Clove/gelatin composite exhibited minimal cytotoxicity. Cell viability after a post-incubation period was recorded at 111.2%, 103.7%, 95.2%, and 87.7% for 5%, 10%, 15%, and 20% composite concentrations, respectively, compared to 100% viability in the control group.

Fig. 10.

MTT assay shows cell viability of clove/gelatin coated at various concentrations

Interestingly, the 5% and 10% composite concentrations resulted in enhanced cell viability (>100%), likely due to the presence of eugenol, the primary bioactive compound in clove extract. Eugenol has been reported to promote wound healing by stimulating angiogenesis and upregulating key growth factors, including vascular endothelial growth factor (VEGF), and transforming growth factor beta (TGF-β). These findings confirm the biocompatibility of the Clove/gelatin composite, supporting its potential for biomedical applications [36, 37].

In vivo evaluation

Silk braided sutures coated with a Clove/gelatin composite at a 20% clove concentration were selected for in vivo evaluation due to their superior antibacterial activity, as evidenced by a larger zone of inhibition compared to 5, 10, and 15% concentrations. Additionally, the hemolytic activity of the 20% Clove/gelatin composite was <1%, confirming its safety for biomedical applications.

Two parallel incisions were made, with the right incision closed using Clove/gelatin-coated sutures and the left incision closed using uncoated sutures. Wound healing and signs of infection were assessed over 14 days, with erythema (redness) serving as the primary indicator of an inflammatory response. Representative images from days 1, 7, and 14 are shown in Fig. 11.

Fig. 11.

In vivo imaging of skin with coated and uncoated suture. a–c From days 0, 7, and 14

By day 7, the coated suture site exhibited reduced inflammation in comparison to the uncoated suture site. By day 14, the sutures were removed, and the uncoated wound side still displayed signs of inflammation and redness, while the coated suture wound side was completely healed. Additionally, noticeable hair regrowth at the coated wound site indicated an absence of inflammation, further supporting the enhanced wound healing properties of the Clove/gelatin-coated sutures.

Hematoxylin and eosin staining

Hematoxylin and eosin (H&E) staining was performed to evaluate the histological progression of wound healing at different time points as shown in Fig. 12.

Fig. 12.

Hematoxylin and eosin staining stained images of skin tissues for coated and uncoated suture. A–F From days 0, 7, and 14

Early inflammatory phase (Day 0): Fig. 12A, B depicts the initial inflammatory response, characterized by fibrin deposition and the presence of blood clots. This phase marks the body’s immediate reaction to injury, with immune cell infiltration to prevent infection and initiate repair.

Intermediate healing phase (Day 7): Fig. 12C, D shows significant tissue remodeling. Inflammatory infiltrates are reduced, and early granulation tissue formation is evident. Fibroblast proliferation and collagen deposition have begun, contributing to extracellular matrix development. Mild vascularization (capillary formation), a critical tissue regeneration factor, is also observed.

Late healing phase (Day 14): uncoated suture (Fig. 12E): granulation tissue appears more mature, with partially organized collagen fibers. However, residual inflammatory cells are still present, and re-epithelialization remains incomplete or slower than expected for optimal healing.

Coated suture (Fig. 12F): compared to the uncoated suture, the clove/gelatin-coated suture demonstrates superior wound healing. The collagen fibers are denser and more aligned, indicating enhanced tissue remodeling. Re-epithelialization appears more complete, with a significantly lower presence of inflammatory cells, suggesting reduced inflammation and improved healing dynamics. These findings confirm the beneficial effects of clove/gelatin-coated sutures in promoting faster and more efficient wound healing compared to uncoated sutures.

Discussion

Surgical site infections (SSIs) remain a significant clinical challenge due to biofilm formation and multidrug-resistant pathogens. The development of antimicrobial sutures is crucial in mitigating these infections. This study demonstrates that Clove/gelatin-coated silk sutures offer a promising solution by combining antimicrobial activity, sustained drug release, and biocompatibility.

FTIR spectroscopy confirmed the successful incorporation of clove bioactive compounds within the gelatin matrix. Characteristic peaks corresponding to clove extract (e.g., phenolic –OH at 3427 cm⁻¹, ester C–O at 1723 cm⁻¹) and gelatin (amide I at 1630 cm⁻¹, amide II at 1565 cm⁻¹) were observed. The overlapping aromatic C=C peak at 1451 cm⁻¹ suggests hydrogen bonding between eugenol and gelatin, which likely enhances coating stability and prolongs drug release. Additionally, SEM analysis revealed a uniform morphology and strong adhesion of the Clove/gelatin composite to the suture surface, which is critical for sustained antimicrobial activity and mechanical integrity.

The coated sutures exhibited enhanced tensile strength (47 N) compared to uncoated sutures, which was attributed to gelatin’s role in reinforcing fiber binding. Furthermore, the reduced coefficient of friction of coated sutures improves knot security and reduces tissue drag during suturing, addressing key limitations of conventional sutures. Hemolysis assays indicated that all tested concentrations exhibited hemolytic activity below 1%, which aligns with safety thresholds and underscores the biocompatibility of the material.

Clove/gelatin-coated sutures demonstrated a concentration-dependent antibacterial effect. The 20% formulation exhibited the highest antibacterial activity, achieving inhibition zones of 17.5 mm against S. aureus, 17 mm against E. coli, 16.5 mm against K. pneumoniae, and 15 mm against E. faecalis. These inhibition zones surpass those reported for commercial triclosan-coated sutures (e.g., PDS Plus, ~12–15 mm against gram-positive bacteria). The superior efficacy against S. aureus compared to E. coli may be due to differences in cell wall permeability, with the lipophilic nature of eugenol facilitating penetration of gram-positive bacteria.

The sustained drug release profile showed a burst release within the first 25 h, followed by a gradual and controlled release up to 72 h, with 93% cumulative clove release. The release kinetics followed the Hixson–Crowell model (R² = 0.9877), indicating an erosion-controlled mechanism that ensures prolonged antimicrobial activity and prevents biofilm formation over time.

MTT assay results confirmed that Clove/gelatin-coated sutures are non-toxic, with cell viability exceeding 87.7% even at the highest (20%) clove concentration. Interestingly, the 5% and 10% formulations exhibited >100% cell viability, likely due to the pro-healing effects of eugenol. Eugenol has been reported to stimulate fibroblast proliferation and upregulate growth factors such as VEGF and TGF-β, which promote angiogenesis and collagen synthesis.

The histological analysis supported these findings: wounds closed with coated sutures exhibited denser collagen alignment, reduced inflammatory cell infiltration, and complete re-epithelialization by day 14, while uncoated suture wounds remained inflamed. These results highlight the synergistic effect of Clove/gelatin-coated sutures in preventing infection while accelerating wound healing.

The in vivo study demonstrated the dual functionality of Clove/gelatin-coated sutures in preventing infections and enhancing tissue regeneration. Rats were incised on both sides of the spine, with the right incision stitched using coated sutures and the left with uncoated sutures. After 14 days, the wound side with coated sutures showed complete healing with no signs of infection, whereas wounds with uncoated sutures exhibited persistent inflammation. This highlights the efficacy of Clove/gelatin-coated sutures in mitigating SSIs and promoting faster recovery.

Conclusion

This study demonstrates the potential of Syzygium aromaticum (clove) as a natural antimicrobial agent for mitigating surgical site infections (SSIs). Silk sutures functionalized with a clove/gelatin composite via dip-coating exhibited potent antibacterial activity, sustained drug release, and excellent biocompatibility. The 20% clove/gelatin formulation achieved optimal antimicrobial efficacy, with inhibition zones of 17.5 mm (Staphylococcus aureus), 17 mm (Escherichia coli), 16.5 mm (Klebsiella pneumoniae), and 15 mm (Enterococcus faecalis), surpassing conventional triclosan-coated sutures. Hemolysis remained below 1%, ensuring clinical safety.

Cytocompatibility was confirmed through MTT assays, with cell viability exceeding 87.7% at the highest concentration and lower concentrations (5%–10%) promoting fibroblast proliferation (>100% viability), likely due to eugenol-mediated angiogenesis and upregulation of VEGF and TGF-β. In vivo studies further validated these findings, demonstrating that coated sutures facilitated complete wound healing within 14 days, with enhanced collagen deposition, reduced inflammation, and accelerated re-epithelialization compared to uncoated sutures.

The composite’s sustained drug release profile, best described by the Hixson-Crowell model (R² = 0.9877), ensures prolonged antimicrobial action while preserving mechanical integrity. These findings position clove/gelatin-coated sutures as a promising dual-action approach for infection prevention and wound healing. However, further studies are needed to assess long-term biocompatibility in large animal models, efficacy against polymicrobial infections, and scalability for clinical translation.

Abbreviations

SSI

Surgical site infections

E. coli

Escherichia coli

S. aureus

Staphylococcus aureus

K. pneumoniae

Klebsiella pneumoniae

E. faecalis

Enterococcus faecalis

FTIR

Fourier transform infrared spectroscopy

SEM

Scanning electron microscopy

PBS

Phosphate-buffered saline

DMEM

Dulbecco’s modified Eagle medium

HDFa

Human primary dermal fibroblast

MTT

3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide

Author contributions

Muhammad Shoaib Butt: conceptualization, supervision, resources, project administration, funding acquisition, writing—review & editing. Hamza Ghafoor: investigation, methodology, visualization, validation, data curation, writing—original draft.

Funding

This work was supported by the Pakistan Science Foundation (PSF) G-5/2, Islamabad-Pakistan under a PSF grant (PSF/Res/C-NUST/Med 521).

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Ethical approval

Ethical approval for the study was obtained from the Institutional Animal Ethical Review Committee at ASAB, ensuring compliance with ethical standards. The IRB approval number [08-2023-ASAB-02/02] was granted before the study.

Supplementary information

The online version contains supplementary material available at 10.1007/s10856-025-06886-3.