Superior Tendon Repair and Healing Ability of PLGA Sutures Are Related to the Weaker Regulatory T-cell and Macrophage Responses Compared With PGA Sutures in a Rat Model

Abstract

Background:

Absorbable sutures (such as Vicryl sutures) support tendon rupture repair, but the immune response triggered by the sutures affects tendon healing. A rat Achilles tendon model was used in this study to explore the role of sutures in tendon healing.

Purpose:

To evaluate the physicochemical properties, biomechanical properties, cytotoxicity, heat-induced pain sensitivity, histological alterations, and the responses of regulatory T (Treg) cells and macrophages to polyamide (PA), polyglycolic acid (PGA), and poly(lactic-co-glycolic acid) (PLGA) sutures in Achilles tendon healing.

Study Design:

Controlled laboratory study.

Methods:

A rat Achilles tendon rupture model was established, followed by modified Kessler repair with size 5-0 PA, PGA, and PLGA sutures. The repaired tendons were tested at 2 weeks and 4 weeks after surgery. The ultimate load and failure stress of the repaired tendons were evaluated using a uniaxial loading device. The samples were stained with hematoxylin and eosin (H&E) as well as Sirius red for histological evaluation. Immunohistochemical analysis was conducted to investigate alterations in macrophages and Treg cells.

Results:

All 3 sutures (PA, PGA, and PLGA) maintained structural integrity after repair. However, compared with nonabsorbable PA, absorbable sutures (PGA and PLGA) demonstrated higher failure loads. At 4 weeks after suture, the postsutured tendons in the 3 groups were thicker, and the PGA (78.64 ± 4.69 N) and PLGA (71.19 ± 3.81 N) groups demonstrated significantly greater ultimate loads than the PA (54.76 ± 5.79 N) group; compared with the PLGA group, the PGA group exhibited more adhesion. Histologically, the Bonar scores for the PGA (9.60 ± 0.89) and PLGA (9.80 ± 0.83) groups were significantly lower than that for the PA group (11.20 ± 0.83) at 4 weeks postsuture. Collagen type III predominated in the PA and PLGA groups, whereas collagen type I was predominant in the PGA group at 2 weeks postsuture. Immunohistochemical analysis demonstrated that the proportions of CD86⁺ (M1, proinflammatory) macrophages, CD206⁺ (M2, anti-inflammatory) macrophages, and CD25 and Foxp3 (Treg cell markers) were significantly greater in the PGA group than in the PLGA and PA groups at 2 weeks after surgery.

Conclusion:

The PGA and PLGA groups demonstrated comparable biomechanical properties, whereas compared with the PGA group, the PLGA group exhibited superior healing outcomes (fewer adhesions and more balanced collagen remodeling) and milder Treg cell and macrophage responses.

Clinical Relevance:

These results can help clinicians choose suture materials.

Keywords

Achilles tendon suture materials PLGA regulatory T cell macrophage

Tendon rupture constitutes a severe musculoskeletal injury that leads to significant loss of strength and functional impairment and is particularly prevalent among middle-aged men and athletic individuals.¹⁵ Complete ruptures generally require surgical repair to restore tendon continuity and mechanical strength, especially for patients with high functional demands. Compared with delayed repair, early surgical intervention is often associated with superior outcomes and fewer complications.^4,16 Achilles tendon rupture represents a severe and increasingly prevalent musculoskeletal injury, often resulting in significant strength deficits and long-term functional impairment.²² In recent years, its incidence has risen, particularly among physically active men aged 30 to 59, with the number of sports-related cases showing a sustained increase globally.^21,29 Surgical treatment and nonoperative treatment are 2 strategies for treating Achilles tendon rupture injuries, and surgical repair is recommended for high-level athletes or those who need to recover quickly.²⁵ Treatment decisions should be individualized on the basis of the patient's age, functional requirements, and rehabilitation expectations. Surgical intervention significantly reduces the rerupture rate (approximately 2%-3% vs 9%-12%) and facilitates earlier return to work and sports, although it is associated with a greater risk of wound-related complications.^12,24

Surgical sutures are crucial for wound closure and promoting healing, and they are generally classified into absorbable and nonabsorbable types. The choice of suture usually depends on factors such as the specific tendon involved, the extent of the injury, and the desired healing time. Poly(lactic-co-glycolic acid) (PLGA) and polydioxanone sutures are commonly used as absorbable sutures.^1,8 Vicryl (polyglactin 910) is a widely used absorbable suture composed of a copolymer of lactic acid and glycolic acid. It exhibits excellent biocompatibility and superior mechanical properties, including high tensile strength and flexibility, making it particularly suitable for soft tissue repair. The material demonstrates outstanding performance in the reconstruction of tendons and ligaments.³⁵ Although Vicryl elicits minimal tissue reactivity, mild inflammatory responses may occasionally occur at specific anatomic sites.¹¹ PLGA and Vicryl sutures are both composed of a copolymer derived from polyglycolic acid (PGA) and polylactic acid (PLA). As bioabsorbable materials, they degrade in vivo and eliminate the need for suture removal after surgical procedures. The degradation rate and physical properties of PLGA can be customized by adjusting the ratio of PGA to PLA. Owing to its high drug-loading capacity and mechanical strength, PLGA is extensively used in soft tissue regeneration applications.¹⁸

Tendon healing involves 3 overlapping phases: the inflammatory phase, the proliferative (repair) phase, and the remodeling phase.³¹ Immune responses play a critical role throughout the entire process of both tendon repair and functional recovery. Beyond providing essential mechanical stability in the early stages, sutures, as implanted exogenous materials, may also induce varying degrees of localized inflammation and foreign body reactions through their degradation byproducts, thereby influencing the immune microenvironment and ultimately the quality of tissue regeneration. Polyamide (PA) is a nonabsorbable suture, whereas PGA and PLGA are synthetic absorbable materials. Different absorbable sutures exhibit variations in strength retention and degradation behavior, which in turn influence the tissue response. PGA has a high degree of crystallinity and degrades through hydrolysis, and its acidic products may amplify local inflammatory responses. As a copolymer of lactic and glycolic acids, PLGA offers tunable degradation kinetics and mechanical properties through compositional adjustment, thereby enabling more controlled strength retention.¹⁸ In recent years, research has shifted from simple mechanical comparisons to functional modification of materials, with strategies such as surface engineering and drug delivery being used to modulate the peri-implantation microenvironment and optimize both biological and biomechanical repair outcomes.¹⁰ Macrophages are broadly classified into proinflammatory M1 (CD80/CD86⁺) and anti-inflammatory M2 (CD206⁺) phenotypes, the sequential polarization of which critically influences extracellular matrix remodeling and healing outcomes. Systematic reviews have demonstrated a marked upregulation of CD68⁺ macrophages after acute Achilles tendon injuries, with elevated CD86⁺/CD206⁺ expression observed in partial tears. Conversely, complete ruptures exhibit a relative reduction in CD206⁺ levels, suggesting that M2 macrophage involvement may be associated with tissue integrity and reparative status.³³ Regulatory T (Treg) cells (CD4⁺CD25⁺Foxp3⁺) contribute to functional regeneration by attenuating excessive inflammation via the Treg–interleukin (IL)-33 axis, and disruptions in this signaling pathway are linked to failed regeneration.² Hydrolytic byproducts of PGA can initiate the complement cascade and exacerbate acute inflammatory infiltration.⁵ Acidification resulting from PLGA degradation may potentiate M1 polarization and provoke a foreign body response proportional to material dimensions.³⁰ Although previous studies have defined the physicochemical properties, degradation kinetics, and effects of materials such as PGA and PLGA on local inflammation and have confirmed that immune regulation plays a key role in tendon healing, systematic evidence is lacking to clarify the relationships among the physicochemical properties of materials, the dynamic changes in immune cells, and the final quality of tendon repair. The mechanism of the chain of physicochemical properties, immune regulation, and tissue reconstruction still needs to be systematically explored in tendon repair models.

Considering the differences in physicochemical properties, degradation behaviors, and immunomodulatory effects among various suture materials, we hypothesized that different materials would influence tendon repair by differentially regulating the local immune microenvironment, thereby affecting collagen remodeling patterns and adhesion formation. By using a rat Achilles tendon repair model, this study systematically evaluated the physicochemical and biomechanical properties of 3 types of sutures (PA, PGA, and PLGA) under controlled experimental conditions. Special emphasis was placed on analysis of their effects on macrophage polarization and Treg responses, with the goal of providing experimental evidence for optimizing suture material selection in tendon repair.

Methods

Sutures

In this study we used sutures of size 5-0 PA (Bei Yue Shen), size 5-0 PGA (Jinhuan Medical), and size 5-0 PLGA with a ratio of 90:10 of PGA:PLA (Jinhuan Medical). The mechanical properties of the 3 sutures were evaluated using a uniaxial loading device (MTS).

Cell Viability Assay

PLGA and PGA sutures were immersed and extracted in Dulbecco's modified Eagle medium (DMEM) (Invitrogen), and the extracts were added in equal proportions to cultured Achilles tendon cells. A Cell Counting Kit-8 (CCK-8) (Dojindo, Japan) was used to evaluate the viability of the Achilles tendon cells in the presence of different absorbable sutures. Cells were seeded in 96-well plates and cultured with suture extract for 24 hours or 72 hours. Afterward, CCK-8 reagent (10 µL/well) was added to each well and further incubated for 2 hours at 37°C. A microplate reader was used to measure the absorbance of the samples at 450 nm. The experiments were performed in triplicate.

Animal Model

Sprague-Dawley rats (250-300 g) were used for the study. All animal experiments were approved by the Ethical Committee of Laboratory Animals, and all the procedures were carried out following the Guidelines for the Care and Use of Laboratory Animals. The rats were housed in controlled temperature (21 ± 2°C) rooms with a 12-hour light-dark cycle, and food and water were provided.

The rats were randomly assigned to 4 groups: the PA suture group (n = 15), the PGA suture group (n = 15), the PLGA suture group (n = 15), and the normal control group (n = 5). Under sterile conditions, an incision was made along the lateral edge of the left Achilles tendon, and the tendon was then transected in the middle and sutured immediately using a modified Kessler technique with size 5-0 sutures.⁹

Hot Plate Test

Postoperative pain sensitivity after Achilles tendon rupture repair using different suture materials was assessed at 2 and 4 weeks after surgery via the hot plate test, which was conducted before euthanasia. Each group included 5 rats (the normal control group, PA suture group, PGA suture group, and PLGA suture group), and testing was conducted in a blinded manner. All procedures were approved by the Institutional Animal Care and Use Committee and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals. Before testing, the rats were acclimated to the experimental environment for ≥30 minutes. The hot plate was calibrated and pretested to ensure temperature accuracy and uniform heat distribution before each testing session. Each rat was then individually placed on a calibrated hot plate maintained at 55°C. The latency to the first nociceptive response of the hindlimb (licking, lifting, or jumping) was recorded. To prevent thermal injury, a cutoff time of 30 seconds was used, after which the animal was immediately removed if no response occurred. The recorded latencies were used to compare pain sensitivity among the groups.

Macroscopic Analysis

Adhesion formation of the palmar and dorsal surface tendons was assessed on the basis of the length and density of the fibrotic adhesions. On the basis of the density of fibrous tissue, adhesion severity is macroscopically evaluated and classified as follows: (1) no adhesion, defined as the absence of significant adhesions surrounding the tendon with only minimal granulation tissue present; (2) membranous adhesion, characterized by thin, filmy tissue without impairment to tendon gliding; (3) loose adhesion, consisting of soft, thin, fibrous tissue loosely attached to the tendon and easily separable; and (4) moderately dense adhesion, presenting as firm fibrous tissue with moderate density, resulting in partial restriction of tendon movement.

Biomechanical Analysis of the Achilles Tendon

The biomechanical properties of the regenerated Achilles tendon were evaluated using a uniaxial tensile testing system (MTS Systems Corp). After euthanasia, the Achilles tendon was harvested, and the proximal gastrocnemius and soleus muscles were preserved. The samples were kept hydrated with saline-soaked gauze and tested within several hours to minimize dehydration effects. Mechanical testing was performed 1 week after harvesting for both the 2-week postoperative group and the 4-week postoperative group.

Before testing, the maximum and minimum diameters of the tendon cross-section at the repair site were measured using a digital caliper. The cross-sectional area was calculated under the assumption of an elliptical geometry (S = πab). A custom fixture was used to secure the plantar surface of the distal tendon, while the proximal tendon-muscle junction was reinforced with sutures and fixed to the upper grip. After a small preload was applied to eliminate slack, the tendon was loaded under displacement control at a rate of 0.1 mm/s until failure. Ultimate failure load and stress (N/mm²) were calculated. The repair sutures were retained to assess the mechanical properties of the tendon-suture construct.

Histological Analysis

Samples were collected from the rats for histological examination and fixed in 4% paraformaldehyde. The samples were then dehydrated, embedded in paraffin, and sectioned at a thickness of 5 µm. For histological evaluation, the sections were stained with hematoxylin and eosin (H&E) and Sirius red dye. The results were scored in a blinded manner according to the established scoring system; scoring was performed by 3 observers, and interobserver reliability was assessed by kappa statistics or intraclass correlation coefficients.¹³

Immunohistochemical Analysis

Immunohistochemical analysis was conducted to investigate alterations in antigen expression associated with macrophages and Treg cells. Sections were rehydrated after deparaffinization. To eliminate nonspecific background signal staining caused by endogenous peroxidase, the slides were treated with hydrogen peroxide for 10 to 15 minutes, followed by 2 washes with buffer. Heat-induced antigen retrieval was performed with citric acid buffer in a microwave oven, and the sections were incubated with pepsin digestion solution at 37°C for 10 minutes to retrieve the antigens. The sections were incubated with primary antibody overnight at 4°C, followed by secondary antibody at room temperature for 30 minutes. Then, diaminobenzidine color development was performed after the sections were washed with phosphate-buffered saline, and the sections were counterstained with hematoxylin. Recombinant anti-CD86 antibody (1:100 dilution; Cell Signaling), recombinant anti-mannose receptor antibody (1:1000 dilution; Abcam), CD4 monoclonal antibody (1:200 dilution; Immunoway), recombinant anti-IL-2 receptor alpha antibody (CD25) (1:500 dilution; Abcam), and anti-FOXP3 antibody (1:100; Abcam) were used as primary antibodies for the analysis of macrophages and Treg cells. Secondary antibodies included enzymatically labeled goat anti-rabbit immunoglobulin G (IgG) polymer (ZSGB-Bio) and enzymatically labeled goat anti-mouse IgG polymer (ZSGB-Bio). Images were captured with a digital slide scanner (Scanner Nano Zoomer; Hamamatsu). The immune cells were quantitatively evaluated using ImageJ software (https://imagej.nih.gov/ij/index.html). Positive cells were quantified using ImageJ by converting the images to grayscale, applying background subtraction, and setting a threshold for positive staining areas. The “Analyze Particles” function was used to measure positive areas, and the cell density was calculated by normalization to the tissue area. Statistical analysis was used to compare group differences.

Statistics

Each experiment was repeated in triplicate. Statistical analysis among groups was conducted using a t test or 1-way analysis of variance with Tukey post hoc test. All data analyses were performed using SPSS software. A value of P < .05 was considered to indicate statistical significance.

Results

Physicochemical Properties and Cytotoxicity of Suture Materials

Tensile tests revealed that the failure load of the absorbable suture group (PGA and PLGA) was greater than that of the nonabsorbable suture group (PA) (Figure 1A). The effects of PGA and PLGA on proliferation were assessed via CCK-8 assay, and no significant difference in proliferation was detected between 24 and 72 hours after seeding (Figure 1B).

Figure 1.

Physicochemical properties and cytotoxicity of polyamide (PA), polyglycolic acid (PGA), and poly(lactic-co-glycolic acid) (PLGA) sutures. (A) Tensile strength test curves for the PA, PGA, and PLGA sutures. (B) Results of the CCK-8 assay for PGA and PLGA suture degradation products 24 and 72 hours after seeding. The bars indicate standard deviations. Statistical significance was determined using 1-way analysis of variance with Tukey post hoc test. **P < .01.

Biomechanical Properties of the Achilles Tendon

The ultimate load and failure stress were used to evaluate the biomechanical properties of the Achilles tendon at 2 and 4 weeks after suturing with different suture materials (Figure 2A).

Figure 2.

Biomechanical analysis of the Achilles tendon and hot plate test of Sprague-Dawley rats. (A) Flowchart of the construction process of the animal Achilles tendon rupture model. (B) Biomechanical properties of the rat Achilles tendon at 2 weeks and 4 weeks after suturing. (C) Pain response time of Sprague-Dawley rats placed on a 55°C hot plate. The bars indicate standard deviations. Statistical significance was determined using 1-way analysis of variance with Tukey post hoc test. *P < .05. **P < .01. ***P < .001. PA, polyamide; PGA, polyglycolic acid; PLGA, poly(lactic-co-glycolic acid).

As shown in Figure 2B, the failure stress of the experimental groups (PA, PGA, and PLGA) was significantly lower than that of the normal control group at 2 weeks, but no significant difference in the ultimate load was found between the experimental groups and control group at 2 weeks. At 4 weeks after suturing, the failure stress of the experimental groups (PA, PGA, and PLGA) was significantly lower than that of the normal control group, whereas the ultimate load of the experimental groups was significantly greater than that of the control group, and the ultimate loads of the PGA (mean ± SD, 78.64 ± 4.69 N) and PLGA (71.19 ± 3.81 N) groups were significantly greater than that of the PA group (54.76 ± 5.79 N).

Hot Plate Test

The hot plate test was used to assess the sensitivity of the rats in the 4 groups to heat-induced pain (Figure 2A). As shown in Figure 2C, the pain response times of the PGA group (6.50 ± 0.48 seconds) were significantly shorter than those of the PA group (10.43 ± 1.99 seconds) at 2 weeks, and no difference was found in the pain response time between the PGA group and the PLGA group at 2 weeks postoperation. The pain response times of the PGA (7.63 ± 0.368 seconds) and PLGA (7.58 ± 1.21 seconds) groups were significantly shorter than those of the PA (9.53 ± 0.56 seconds) group at 4 weeks after tendon surgical suturing. The results revealed that the PA, PGA, and PLGA treatment groups exhibited reduced heat-induced pain sensitivity in rats with tendon rupture.

Histological Analysis

Compared with animals of the control group, the Achilles tendons of the animals in the experimental groups (PA, PGA, and PLGA) were thicker, and the Achilles tendons of the animals in the experimental groups at 2 weeks were thicker than those of the corresponding animals in the experimental groups at 4 weeks after suturing (Figure 3A). As shown in Figure 3B, adhesion was observed in both the PGA and PLGA groups, and the adhesions in the PGA group were more severe than those in the PLGA group at 4 weeks after suturing. In the PGA group, the adhesion was loose, whereas in the PLGA group, only membrane adhesion was observed.

Figure 3.

Macroscopic and histological analysis of the rat Achilles tendon rupture model with different suture materials at 2 weeks and 4 weeks after suturing. (A) Representative images of the macroscopic appearance. (B) Representative images of the macroscopic adhesion appearance at 4 weeks after suturing. (C) Representative images of hematoxylin and eosin (H&E) and Sirius red staining. (D) Bonar scores of Achilles tendons stained with H&E. The bars indicate standard deviations. Statistical significance was determined using 1-way analysis of variance with Tukey post hoc test. *P < .05. **P < .01. ***P < .001. PA, polyamide; PGA, polyglycolic acid; PLGA, poly(lactic-co-glycolic acid).

Sirius red staining can distinguish between type I and type III collagen, and H&E staining and Sirius red staining were used to assess the histological changes in the Achilles tendons of the Sprague-Dawley (SD) rats (Figure 3C). A Bonar scoring system was used to evaluate pathological changes in the tendon tissue. The scores of the PA, PGA, and PLGA suture materials were significantly greater than those of the control group at 2 and 4 weeks after tendon surgical suturing; in addition, the scores of the PGA (9.60 ± 0.89) and PLGA (9.80 ± 0.83) groups were significantly lower than that of the PA (11.20 ± 0.83) group at 4 weeks after tendon surgical suturing (Figure 3D).

As shown in Figure 3C, the tendon fibers were more disorganized at 2 weeks than at 4 weeks after suture, and the thickness of the tendon fibers varied at 2 weeks and became uniform at 4 weeks after surgical suturing of the tendon. At 2 weeks postsuture, a large quantity of type III collagen was observed in the repaired tissue in the PA group and PLGA group, whereas the tendon fibers in the PGA group were primarily composed of type I collagen. Most of the type III collagen in the repaired tissue in the PA group and PLGA group was replaced by type I collagen after 4 weeks after suture, whereas a gradual increase in type III collagen was observed in the PGA group at the same postoperative time point. These results indicated that the tissue repair process of the PGA suture material was delayed.

Immunohistochemistry Analysis

Macrophage subtypes were identified by labeling specific surface markers such as CD86 and CD206, followed by enumerating positively stained cells to calculate their percentage within the total cell population. Moreover, the M2:M1 ratio was conventionally derived from the quantitative ratio of M2 macrophages (CD206⁺) to M1 macrophages (CD86⁺).

At 2 and 4 weeks after the operation, the proportions of CD86 (M1 macrophages) and CD206 (M2 macrophages) were significantly greater in the PGA group than in the PLGA and PA groups. At 2 weeks postoperation, compared with the PA (1.82 ± 0.33) and PLGA (1.69 ± 0.43) groups, the PGA group had significantly higher CD86 expression (3.28 ± 0.45). Similarly, compared with the PA (0.75 ± 0.12) and PLGA (0.89 ± 0.11) groups, the PGA group exhibited significantly higher CD206 expression (1.13 ± 0.11). At 4 weeks postoperation, compared with the PA (1.70 ± 0.26) and PLGA (1.34 ± 0.19) groups, the PGA group continued to have significantly higher CD86 expression (2.96 ± 0.40). Additionally, compared with the PA (2.14 ± 0.34) and PLGA (1.55 ± 0.25) groups, the PGA group had significantly higher CD206 expression (3.07 ± 0.26). These findings highlight that the PGA group exhibited significantly greater polarization of macrophages toward both the M1 phenotype and the M2 phenotype. The ratio of CD206 to CD86 in the PA, PGA, and PLGA groups increased from 0.43, 0.35, and 0.57 at 2 weeks to 1.28, 1.06, and 1.19 at 4 weeks after surgery, respectively (Figure 4B).

Figure 4.

Results of immunohistochemical staining of Achilles tendon tissue at 2 weeks and 4 weeks after surgical suturing of the tendon with different suture materials. (A) Representative images of CD86, CD206, CD4, CD25, and Foxp3 immunohistochemical staining. (B) Quantification of the area positive for CD86 and CD206 as a percentage of the total area, and the ratio of CD206-positive cells to CD86-positive cells. (C) Quantification of the area positive for CD4, CD25, and Foxp3 as a percentage of the total area. The bars indicate standard deviations. Statistical significance was determined using 1-way analysis of variance with Tukey post hoc test. *P < .05. **P < .01. ***P < .001. PA, polyamide; PGA, polyglycolic acid; PLGA, poly(lactic-co-glycolic acid).

CD4, CD25, and Foxp3 are markers of Treg cells. At 2 weeks after surgery, the proportions of CD4, CD25, and Foxp3 cells in the PGA group were significantly greater than those in the PLGA and PA groups. Specifically, compared with the expression of CD25 in the PA (0.09 ± 0.01) and PLGA (0.08 ± 0.01) groups, that in the PGA group was significantly greater (0.16 ± 0.01). Similarly, Foxp3 expression was significantly greater in the PGA group (0.13 ± 0.01) than in the PA (0.10 ± 0.01) and PLGA (0.08 ± 0.01) groups. At 4 weeks after surgery, the proportion of Foxp3 in the PGA group was significantly greater than that in the PLGA and PA groups. Specifically, the Foxp3 expression in the PGA group was 0.12 ± 0.01, which was significantly greater than that in the PA group (0.06 ± 0.01) and the PLGA group (0.05 ± 0.01).

The proportions of CD4+ and CD25+ cells were significantly greater in the PGA group than in the PLGA group. The CD25 expression in the PGA group was 0.10 ± 0.01, which was significantly greater than that in the PLGA group (0.07 ± 0.01), whereas the CD25 expression in the PA group was 0.09 ± 0.01 (Figure 4C). These results indicated that compared with the PLGA and PA groups, the PGA group had significantly greater proportions of Treg cells at both 2 and 4 weeks after surgery, as indicated by increased CD25 and Foxp3 expression.

Discussion

In this study, we used a rat Achilles tendon model to evaluate the efficacy of PA, PGA, and PLGA sutures for tendon repair. At 4 weeks after tendon surgical repair, the PGA and PLGA groups exhibited significantly higher ultimate load values than did the PA group, and the adhesion of the PGA group was more severe than that of the PLGA group. In the PLGA group, abundant type III collagen was observed in the repair tissue at 2 weeks after suturing, which was largely replaced by type I collagen by 4 weeks. In contrast, in the PGA group, the tendon fibers were primarily composed of type I collagen at 2 weeks after surgery, whereas the amount of type III collagen progressively increased by 4 weeks. At the 2-week postoperative interval, the proportions of CD86 (M1 macrophages), CD206 (M2 macrophages), and CD25 and Foxp3 (Treg cell markers) were significantly greater in the PGA group than in the PLGA and PA groups. Compared with that at the 2-week time point, the M2:M1 macrophage ratio significantly increased at 4 weeks after surgery in the PA, PGA, and PLGA groups.

A thorough understanding of the properties of different suture materials is beneficial for selecting suture material and improving patient treatment outcomes. Suture materials, infection, inflammatory responses, and other factors affect the efficacy of surgical wound healing. Despite the efficacy and many advantages of absorbable PGA, its application in suturing is limited by a relatively high incidence of inflammatory reactions.^14,17 Our findings indicated that the proportion of macrophages was greater in the PGA group than in the PLGA group, and an increase in the number of macrophages was also reported by Bryan et al.³ In the PGA group, the simultaneous increase in M1 (CD86⁺) and M2 (CD206⁺) macrophages indicated a complex immune response. At 2 weeks after surgery, both M1 and M2 macrophages were in a transitional polarization state, whereas the progressive increase in type III collagen at 4 weeks reflected a delayed shift toward tissue repair. This mixed immune response may account for the slower tissue healing rate observed in the PGA group than in the PLGA and PA groups. Various strategies have been used to prevent adhesion, reduce inflammation, and promote wound healing. Our group recently reported that combined treatment with hydrogel microspheres and hyaluronic acid methacrylate (HAMA) hydrogels significantly enhanced endogenous tendon repair and effectively inhibited the formation of exogenous fibroblast adhesion.³⁴ PLGA is synthesized through the copolymerization of cyclic dimers of lactic acid and glycolic acid at specific ratios. No significant difference in ultimate load was detected between the PLGA and PGA groups in this study, but the M1 macrophage responses of the PLGA group were more moderate than those of the PGA group, and the severity of adhesion in the PLGA group was lower than that in the PGA group, which provides valuable information for the selection of suture materials.

Macrophages play a pivotal role in regulating tissue repair processes by both promoting and suppressing inflammation. M1 macrophages exacerbate inflammatory responses, whereas M2 macrophages attenuate inflammation and facilitate tissue repair and proliferation. Understanding the phenotypic transition of macrophages during tendon repair can provide valuable information for the future treatment of tendon injuries. Our findings indicated that compared with ratio of CD206:CD86 at 2 weeks after surgery, that at 4 weeks after surgery was elevated in terms of PGA- and PLGA-induced responses. During the early tissue repair process, the immune environment typically involves both inflammatory and prohealing signals, resulting in a mixed macrophage phenotype that concurrently expresses M1 and M2 markers. This phenomenon reflects partial polarization and transitional activation rather than a strict binary state, suggesting a delayed polarization shift or a heterogeneous inflammatory environment wherein macrophages perform both proinflammatory and reparative functions.^20,28 Targeting the molecular mechanisms underlying macrophage polarization and modulating macrophages are promising therapeutic strategies for treating tendon repair disorders. Chen et al⁷ demonstrated that conditioned medium from human bone marrow–derived mesenchymal stem cells suppressed the M1 phenotype but promoted the M2 phenotype, conferring beneficial effects on tendon-bone healing. In the current study, M1 (CD86⁺) and M2 (CD206⁺) macrophages, as well as Treg cells, which are closely associated with refracture risk and healing quality, were significantly increased in the PGA group.²⁶ Although PGA materials elicited a more pronounced inflammatory response, the immune reaction failed to transition effectively to the reparative phase, thereby increasing the risk of refracture. Modulating macrophage and Treg polarization could enhance repair outcomes and reduce refracture risk.

Treg cells are a subset of CD4+ T cells with immunosuppressive functions that release mediators to affect other immune cells, including macrophages, dendritic cells, and T cells. In the current study, the proportions of CD25 and Foxp3 (Treg cell markers) in the PGA group were significantly greater than those in the PLGA group and the PA group at 2 weeks after surgery, which indicates that suture composition modulates the local immune microenvironment during the early stages of tendon healing. Regulatory T cells are known to modulate inflammation and tissue regeneration, and the elevated levels observed in this study may reflect a compensatory response to material-induced inflammation. Proto et al²³ demonstrated that Treg cells enhanced the capacity of macrophages to phagocytose apoptotic cells and thereby facilitated the resolution of inflammation. In contrast to their immunosuppressive functions, Treg cells generate reparative mediators that act upon resident parenchymal and structural cells to facilitate tissue repair and regeneration.¹⁹ The interactions between Treg cells, structural cells, and tissue-specific parenchymal cells, as well as their role in Achilles tendon repair and regeneration, will be investigated further.

Biodegradable PLGA is widely used in drug delivery systems because of its excellent biocompatibility; it enhances therapeutic efficacy and has been extensively applied in the treatment of various pathological conditions, including inflammatory diseases and tissue regeneration processes. The immunomodulatory protein CD200 has been demonstrated to suppress the inflammatory activation of macrophages in vitro and reduce local immune cell infiltration around biomedical material implants, and PLGA particles coated with CD200 exhibited anti-inflammatory effects and enhanced phagocytic activity.⁶ Monascin exhibited potent anti-inflammatory effects, and an intranasal drug delivery system using PLGA-loaded monascin enabled sustained release of the medication, leading to an increased population of Treg cells and markedly improving the therapeutic effect on allergic rhinitis.³² Future research will optimize the physicochemical properties of PLGA and explore innovative strategies for targeted drug delivery and controlled release, thereby further expanding the clinical application potential of PLGA-based drug delivery systems.

Limitations

The present study has certain limitations. First, polydioxanone is an absorbable polymer commonly used in equine surgical procedures, and implantation of 3D-printed polydioxanone devices elicited a transient inflammatory response in subcutaneous tissues, with complete absorption occurring within 4 to 7 months.²⁷ We investigated the biological characteristics and immune responses induced by different suture materials at 2 and 4 weeks after Achilles tendon surgery, and extended observation periods enabled a more comprehensive evaluation of the long-term effects of various suture materials on wound closure and subsequent healing processes. Second, the Achilles tendon contains a relatively sparse cellular population; in this study, immunohistochemistry rather than flow cytometry was used to detect changes in Treg cell proportions. Other limitations include a small sample size without power analysis and potential operator bias.

Conclusion

We characterized the biomechanical and other properties of 3 suture materials commonly used in Achilles tendon repair at 2 and 4 weeks after surgery and investigated suture-induced Treg cell and macrophage responses. The PGA and PLGA absorbable suture groups demonstrated comparable biomechanical properties; however, compared with the PGA group, the PLGA group exhibited superior tendon repair and healing ability and more moderate Treg cell and macrophage responses at 2 weeks after operation. Our findings may be helpful for the application of suture materials and Achilles tendon repair.

Footnotes

Final revision submitted February 24, 2026; accepted March 7, 2026.

One or more of the authors has declared the following potential conflict of interest or source of funding: Funding was received from the National Natural Science Foundation of China (82372418) and Peking University Clinical Scientist Training Program (BMU2025PYJH045) supported by the Fundamental Research Funds for the Central Universities.

Ethical approval for this study was obtained from the Ethical Committee of Laboratory Animals of Peking University Medical School (No. DLASBD0007), and all procedures were carried out following the Guidelines for the Care and Use of Laboratory Animals.

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