For biomechanical testing, 14 samples of canine abdominal fascia muscles and skin were prepared. They were collected and frozen for a period over several months from dogs weighing 5–15 kg. Within 12 h of the initiation of the thawing process, the samples were divided and measured at room temperature. The original samples were cut into tissue blocks measuring 3 × 10 cm, with 3 cm parallel to the midline of the abdomen and 5 cm on each side of the midline. To prevent drying, the test samples were covered with moistened cloths. Prior to conducting biomechanical testing, the tissue blocks underwent incisions along the midline of the abdomen, then the incision was opposed randomly only with (a) simple interrupted suture pattern with USP 2-0 suture, (b) simple continuous suture pattern with USP 2-0 suture, (c) simple interrupted suture pattern with USP 3-0 suture, (d) simple continuous suture pattern with USP 3-0 suture, (e) simple interrupted suture pattern with USP 4-0 suture, (f) simple continuous suture pattern with USP 4-0 suture. Each suture combination treatment was performed for four times. All incisions were created and sutured by the same veterinary surgeon. The suturing technique utilized a small stitch (3–5 mm) with a suture length to incision length (SL:WL) ratio of 4:1. The control group samples did not undergo midline incision and suturing prior to the biomechanical testing.

The biomechanical experiments utilized an LLOYD LR10K plus mechanical testing machine equipped with a 1,000 N load cell. The test samples were secured to the tensile tester following the depiction in Figure 1B. In their initial position, the samples were kept in their natural extension, without the addition of extra tension. Sample thickness, clamping length, and width were measured using a vernier caliper. The measurement software applied a pre-load stress of 0.1 N/mm and recorded the corresponding stress values at 10%, 20%, 30%, 40%, and 50% strain, along with the stress and strain values at maximum tensile force. The stretching process involved documenting endpoint types, encompassing suture breakage, rupture of the sutured tissues, and other instances of tissue tearing.

This experiment involved a total of 27 adult beagles with an average weight of 13.4 ± 1.48 kg. All animal experiments were conducted at the Experimental Animal Center of Nanjing Agricultural University. The Animal Ethics Committee of Nanjing Agricultural University approved the experiment (Approval number: NJAU No. 20230123014), ensuring compliance with all relevant ethical regulations for animal testing and research. A pre-experimental period of 2 weeks was provided to allow the animals to acclimate to the environment. The animals were individually housed in cages under a 12 h light/12 h dark cycle within the animal facility. Each experimental dog received a complete commercial diet, and access to clean drinking water was available at all times. Caretakers monitored the dogs’ overall health and activity on a daily basis. Throughout the experiment, no unexpected events occurred that compromised the safety and well-being of the dogs. At the designated endpoints, the dogs were euthanized by intravenous injection of 200 mg/kg pentobarbital sodium solution in accordance with the 2020 AVMA Guidelines for the Euthanasia of Animals (30).

To investigate whether there are significant differences in tissue reactions after suturing canine abdominal wall surgical incisions with different suture sizes, three types of sutures (USP 2-0, 3-0, and 4-0 PGA) produced by China HaiDiKe Company were selected for the experiment. The incisions in dogs were closed randomly only with (a) simple interrupted suture pattern with USP 2-0 suture, (b) simple continuous suture pattern with USP 2-0 suture, (c) simple interrupted suture pattern with USP 3-0 suture, (d) simple continuous suture pattern with USP 3-0 suture, (e) simple interrupted suture pattern with USP 4-0 suture, (f) simple continuous suture pattern with USP 4-0 suture. Each suture combination treatment was performed for four times.

Preoperative analgesia and sedation were managed by subcutaneous injection of meloxicam (0.2 mg/kg), intramuscular injection of bupropion (0.2 mg/kg), and dexmedetomidine (15 mcg/kg). Propofol (5 mg/kg) was injected intravenously to induce anesthesia in dogs. Airway access was established and anesthesia was maintained with isoflurane. Local anesthesia was performed by subcutaneous injection of 0.5% lidocaine at the surgical site. After anesthesia, sterile techniques were employed, and three intermittent incisions with a length of 3 cm were made along the midline of the abdomen, starting 3 cm posterior to the xiphoid process, with a spacing of 1–2 cm between each pair of incisions. Each incision was ensured to enter the abdominal cavity after dissecting the linea alba. These intermittent incisions aimed to avoid mutual interference of inflammation caused by different suture materials in different incisions after closure. Each experimental animal’s three incisions were sutured by the same surgeon using either simple interrupted or simple continuous suturing. Suturing followed a small stitch (3–5 mm) technique with an SL:WL of 4:1. Different suture specifications were alternated among different animals for incisions in different locations to minimize the impact of wound position on healing. Meloxicam (0.1 mg/kg) was injected subcutaneously daily for 5 consecutive days after surgery.

During the experiment, the experimental animals were observed daily for signs of wound dehiscence, incisional hernia, wound infection, and the appearance of the skin after suturing. Skin samples were collected at 1 day, 7 days, and 14 days after suturing. Linea alba samples were collected at 1 day, 7 days, and 3 months after suturing. Each sample was divided into aliquots and stored in freezing tubes and tissue fixatives for the detection of inflammatory factor expression levels and histological evaluation, respectively.

Samples were collected and immediately frozen in liquid nitrogen and homogenized in TRIzol reagent (Angle Gene) to obtain mRNA. RNA was extracted and purified from abdominal wall wounds using chloroform, isopropanol, and ethanol. RNA purity and quantity were assessed using the NANODROP ONE system (Thermo). In accordance with the manufacturer’s protocol, the total RNA (100 μg) was reverse transcribed into cDNA using a reverse transcription enzyme kit (TransGen Biotech). By using the PerfectStart Uni RT&qPCR Kit (TransGen Biotech) and the 7300 Real-Time PCR System, the absorbance of the resulting cDNA was analyzed by qPCR. The primer sequences for real-time fluorescence quantitative PCR were designed using PrimerBLAST software (NCBI), and their sequences are shown in Table 1. Comparative Ct (∆∆Ct) analysis of the tissues’ mRNA expression was performed with normalization based on the average expression of the GAPDH reference mRNA in the tissues.

Tissue samples for protein content analysis were rapidly frozen in liquid nitrogen immediately after harvesting. The frozen tissues were ground into powder under liquid nitrogen cooling. A appropriate amount of PBS was added to the tissue powder and thoroughly homogenized. After homogenization, the suspension was centrifuged (20 min, 3,000 rpm), and the supernatant was transferred to a new tube. The protein concentration was determined using the Bradford protein assay (BioRad). The concentration of IL-1β was measured using the Canine IL-1β ELISA kit (Angle Gene). The concentration of IL-6 was measured using the Canine IL-6 ELISA kit (Angle Gene). The concentration of TNF-α was measured using the Canine TNF-α ELISA kit (Angle Gene). A total protein amount of 10 μg per well was used for the detection, following the manufacturer’s protocol. Normalization was performed for each protein concentration.

After sampling, the specimens intended for histological examination were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 mm. The sections were stained with hematoxylin and eosin (H&E) and then dehydrated and coverslipped. The overall condition of the sections and the position of the sutures were observed using an optical microscope (LEICA DM500) at a magnification of 100×. Photographs were taken by a pathologist unaware of the experimental groups at four different locations (upper, lower, left, and right) around the suture holes on each slide using a magnification of 400×. According to the relevant provisions in ISO10993, the degree of inflammation and tissue reactivity were evaluated. The degree of inflammation scoring included counts of polymorphonuclear cells, lymphocytes, plasma cells, macrophages, and giant cells, as well as the degree of necrosis. The tissue reactivity score was calculated by adding the inflammation score (multiplied by 2), neovascularization score, fibrosis score, and fat infiltration score. Cell counts were scored based on the number of cells per high-power field at 400× magnification: absent (0), 1–5/HPF (1), 5–10/HPF (2), severe infiltration (3), and full field (4). Scores for necrosis, neovascularization, fibrosis, and fat infiltration were categorized as absent (0), mild (1), moderate (2), moderate-to-severe (3), and severe (4).

The data were presented as mean ± SD. Fisher’s exact test was used to analyze the differences in the occurrence rates of the biomechanical endpoints. Mann–Whitney U test and Wilcoxon test of the stepwise method were used for the remaining data. Statistical significance was defined as p < 0.05. In the figures, two groups without any same letters indicate that those have significant differences. All the statistical analyses mentioned above were performed using SPSS (version 26.0.0.2, IBM), and the figures were generated using GraphPad Prism 9 (version 9.5.1).

Skin biomechanical tests showed no significant stress differences between the experimental and control groups below 30% strain. However, at 40% strain, the USP 4-0 PGA suture group showed significantly lower stress compared to the other experimental groups and the control group, indicating suture support failure in the tissue. The USP 3-0 and 2-0 sutures lost their effective support at a 50% strain (Figure 1A), suggesting suture failure in small-diameter sutures with low tissue deformation. But it is important to note that a 30% abdominal wall deformation is uncommon in clinical practice. The strain in the USP 4-0 suture group was lower at maximum tension, but no significant differences were observed, nor between interrupted and continuous sutures (Figure 1E). Furthermore, the USP 4-0 sutured skin had significantly lower maximum tensile strength with a mean value of 92.5 N (Figure 1G). However, regardless of suture diameter, no significant difference in maximum stress was found between continuous and interrupted sutures (Figure 1F), suggesting similar tissue support capabilities. Tensile testing after suturing identified two endpoints: suture breakage (Figure 1C) and tissue rupture at the suture site (Figure 1D). Larger-diameter sutures caused tissue tearing under high tension, while smaller-diameter sutures were prone to suture breakage (Figure 1H). Interestingly, under high-tension conditions, continuous sutures had a higher likelihood of suture breakage, while interrupted sutures resulted in more tissue rupture (Figure 1I).

Muscle biomechanical test showed no significant stress differences between experimental and control groups below 50% strain (Figure 2A). Maximum tensile strength (Figure 2G), corresponding stress (Figure 2F), and strain (Figure 2E) during muscle elongation were also similar between groups. Similar to the skin tissue, muscle elongation testing revealed suture breakage (Figure 2C) and tissue rupture at the suture site (Figure 2D). However, unlike the skin tissue, half of the muscle tests resulted in non-sutured fascial tearing (Figure 2B). Regardless of suture diameter and technique, there were no significant differences in the occurrence rates of test endpoints (Figures 2H,I). These findings suggest no significant differences in mechanical properties of the abdominal wall after canine linea alba suturing using USP 2-0, 3-0, and 4-0 PGA sutures. The sutured abdominal wall exhibited similar mechanical properties to the intact wall under extreme tension conditions.

No animals showed signs of infection, wound dehiscence, or incisional hernia throughout the experiment. Figure 3A shows the postoperative appearance of the skin incision. Mild redness and exudation were observed in incisions during the initial week, regardless of suture diameter. However, at 1–2 weeks after closure, the USP 2-0 and 3-0 suture groups had a higher incidence of redness and scab formation, while the USP 4-0 group had milder redness and less frequent scab formation.

mRNA expression levels of IL-1β and IL-6 gradually decreased after suturing but remained significantly higher than the control group (Supplementary Figures S1A,B). TNF-α mRNA expression showed no significant differences at 7 and 14 days after suturing (Supplementary Figure S1C). Figures 3B–D show variations in mRNA levels among different suture diameter groups. Except for IL-6 expression at 14 days, which had no significant difference between USP 2-0 and 4-0 groups, inflammatory factor levels were consistently higher in the USP 2-0 group at other time points. Additionally, IL-1β mRNA expression in the USP 2-0 group at 14 days remained significantly higher than the USP 4-0 group at 7 days. The USP 3-0 group had lower IL-1β expression than USP 2-0 and higher than USP 4-0 at 14 days. Suture methods did not significantly affect inflammatory factor expression (Supplementary Figures S1D–F) since simple interrupted sutures position knots outside the skin.

Protein expression levels of IL-1β, IL-6, and TNF-α in the skin followed a comparable pattern to mRNA expression. Over time, inflammatory cytokine levels gradually decreased but remained significantly higher than the control group (Supplementary Figures S2A–C). At each time point after suturing, the USP 4-0 PGA suture group showed significantly lower protein expression levels of IL-1β, IL-6, and TNF-α compared to the USP 2-0 PGA suture group (Figures 3E–G). Additionally, both simple continuous and simple interrupted suturing had no significant impact on protein expression levels of these inflammatory cytokines at any time point after skin suturing (Supplementary Figures S2D–F).

Under 100× magnification, the intense purple-colored inflammatory infiltrate was observed around larger suture needle holes on the first-day post-surgery. At 400× magnification, numerous polymorphonuclear cells were visible. On the 7th day post-surgery, the deep staining area around suture holes in the USP 4-0 group was relatively smaller. Polymorphonuclear cell infiltration decreased, but severe infiltration remained in the USP 2-0 group. At this point, an increase in macrophages and giant cells was observed. Mild neovascularization was observed in the skin tissue. After 14 days, the USP 2-0 group showed prominent fibrous capsule formation around suture holes (Figures 4A,B). All groups had mild to moderate polymorphonuclear cell infiltration, with macrophages predominantly around suture holes. Inflammation and tissue reactivity scores decreased with time, with significantly higher scores in the USP 2-0 group compared to the USP 4-0 group (Figures 4C,D).

Three months after linea alba sutures, complete suture absorption was observed, and scar examination revealed no significant differences in mRNA expression levels of IL-1β, IL-6, and TNF-α compared to the control group (Supplementary Figures S3A–C). Figures 5A–C illustrate variations in mRNA levels among experimental groups. At 3 months, no significant differences were observed among suture line groups. However, on postoperative day 1 and day 7, USP 2-0 sutures showed significantly higher mRNA expression levels of IL-1β, IL-6, and TNF-α compared to USP 4-0 sutures. Unlike skin suturing, simple interrupted suturing exhibited higher IL-1β mRNA expression levels compared to simple continuous suturing at 7 days after linea alba closure (Figure 5D).

Three months after suturing, no significant differences in protein expression levels were found compared to the control group (Supplementary Figures S3D–F). On postoperative day 1 and day 7, the USP 4-0 PGA suture group showed significantly lower protein expression levels of IL-1β, IL-6, and TNF-α compared to the USP 2-0 PGA suture group (Figures 5G–I). Simple continuous suturing resulted in lower IL-1β protein expression on the first day after suturing (Figure 5J). However, at 7 days, simple interrupted suturing led to higher levels of IL-1β and TNF-α protein expression (Figures 5J,L).

Under 100× magnification, HE-stained sections of the fascia muscle tissue showed pronounced deep purple inflammatory infiltrates surrounding larger suture needle holes on the first day after the operation. At 400× magnification, numerous polymorphonuclear cells and lymphocytes were observed infiltrating the field of view. Muscle tissue displayed necrotic tissue, neovascularization, and varying effects of different suture diameters on the surrounding tissue on the seventh day. After 3 months, no significant differences were observed among the suture groups in the tissue sections (Figures 6A,B). Inflammatory and tissue reaction scores decreased over time, with significantly higher scores for the USP 2-0 suture group compared to the USP 4-0 suture group (Figures 6C,D). No significant differences in fascia muscle tissue fibrosis caused by different suture diameters were observed during the early post-suturing phase.