Combined photobiomodulation and exosome therapy for muscle and tendon regeneration: an experimental rabbit study

Abstract

Achilles tendon injuries are common, especially among athletes, and their treatment remains challenging. Reduced muscle strength and immobility can exacerbate tendon stiffness, leading to disability. This study investigated the effects of adipose tissue–derived mesenchymal stem cell exosomes and low-level laser therapy (LLLT) on muscle and Achilles tendon elasticity in a rabbit model. Twelve New Zealand white rabbits underwent ultrasound imaging of Achilles tendons and gastrocnemius muscles with a 40 MHz probe during flexion and dorsiflexion. Foot stress during movement was measured. To create a partial tendon injury, a 5 mm longitudinal incision, 3 mm deep, was made in the Achilles tendon. Male rabbits were randomly divided into four groups: control, LLLT (830 nm, 15 mW, 3 J/cm²), exosome injection (200 µg/mL), and combined LLLT with exosomes. Treatments were applied on days 1, 8, and 15. Ultrasound evaluations were performed before injury, immediately after, and on days 8 and 21. Tendon and muscle samples were analyzed histologically with hematoxylin-eosin staining. On day 21, the force applied to induce flexion and dorsiflexion of the Achilles tendon and gastrocnemius muscle significantly increased in the combined treatment group compared to the others (P < 0.05). The thickness strain of both muscle and tendon improved significantly with combination therapy (P < 0.05). Histology showed compact, parallel collagen fibers and a well-organized extracellular matrix in tendons treated with LLLT and exosomes. No inflammation was observed, and muscle atrophy was reduced. Combining low-level laser therapy with exosomes derived from ADSCs enhanced the histological, ultrasound-derived, and mechanical aspects related to Achilles tendon repair. This included improvements in tendon and gastrocnemius strain, as well as flexion and dorsiflexion force.

Introduction

Achilles tendon injuries, which include acute ruptures and chronic tendinopathy, are among the most common and significant musculoskeletal disorders [1, 2]. They lead to pain, reduced mobility, impaired functional performance, and a significant socioeconomic burden. These injuries are particularly common among athletes and physically active individuals, posing a major challenge in the fields of sports medicine and orthopedics [3]. Current treatment strategies consist of conservative management options such as physical therapy and exercise-based rehabilitation, pharmacological interventions, and, in severe cases, surgical repair. While these approaches can alleviate symptoms and partially restore function, they often fall short in achieving complete structural and biomechanical recovery. Pharmacological treatments, including nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroid injections, primarily provide temporary relief of symptoms without improving tendon structure. Surgical interventions, on the other hand, can be associated with prolonged recovery times, potential complications, and variable clinical outcomes [4]. As a result, there is a crucial need for the development of effective regenerative therapies that can enhance tendon healing and restore tissue quality in orthopedic and sports medicine research.

Photobiomodulation (PBM) or low-level laser therapy (LLLT) has emerged as a promising regenerative treatment for musculoskeletal disorders. PBM involves the application of low-intensity light to stimulate biological processes, which leads to enhanced tissue repair, reduced inflammation, and improved functional recovery. The proposed mechanisms of action for PBM include stimulating mitochondrial cytochrome c oxidase activity, increasing adenosine triphosphate (ATP) production, modulating reactive oxygen species, and activating signaling pathways related to cell proliferation, angiogenesis, and collagen synthesis [5]. Previous experimental and clinical studies have shown that PBM promotes tendon healing by reducing inflammation, stimulating fibroblast proliferation, enhancing collagen deposition, and improving the remodeling of the extracellular matrix. Additionally, other biophysical modalities, such as electrical stimulation, have demonstrated beneficial effects in animal models by promoting fibroblast activation and accelerating tissue repair [6, 7]. These findings further support the regenerative potential of physical interventions for tendon healing. Collectively, this evidence suggests that PBM is a promising therapeutic strategy for enhancing tendon regeneration and improving functional outcomes.

Exosome-based therapy has recently emerged as a novel and promising strategy for tendon regeneration. Exosomes are nanosized extracellular vesicles secreted by various cell types that carry biologically active molecules, including proteins, RNAs, lipids, and growth factors, and play a crucial role in intercellular communication and tissue repair. Recent evidence supports the therapeutic potential of exosomes derived from different cellular sources for tendon healing. For instance, exosomes from adipose tissue-derived mesenchymal stem cells (ADSC-Exos) have been delivered using hydrogel systems, demonstrating the ability to reduce fatty infiltration, improve histological outcomes, and enhance the biomechanical properties of injured tendons in rat models. Similarly, exosomes derived from tenocytes have been shown to increase the expression of collagen types I and III and tenomodulin while promoting the proliferation of mesenchymal stem cells through transforming growth factor-beta (TGF-β)-dependent pathways in rabbit Achilles tendon models. In vitro studies with canine flexor tendon cells have also revealed that plasma-derived exosomes can enhance tenocyte proliferation, maintain migratory capacity, and increase collagen deposition in a dose-dependent manner [8]. Additionally, exosomes derived from bone marrow mesenchymal stem cells, tendon-derived stem cells, and macrophages have shown regenerative effects by promoting cell proliferation, migration, and differentiation, regulating the expression of extracellular matrix-related genes, suppressing inflammation, and improving biomechanical properties of tendons [9]. Despite these encouraging findings, several challenges remain regarding the optimization of exosome isolation and delivery methods, understanding their mechanisms of action, and standardizing therapeutic outcomes.

Both photobiomodulation (PBM) and exosome therapy individually show significant regenerative potential, but their combined therapeutic effects on Achilles tendon healing have not been extensively studied. Emerging evidence indicates that PBM can influence exosome secretion, alter their cargo composition, and enhance their biological activity, which could improve their regenerative capabilities and create opportunities for synergistic treatment strategies [5]. The rationale for combining these approaches lies in their complementary mechanisms: PBM enhances cellular metabolism, modulates inflammation, stimulates angiogenesis, and promotes collagen synthesis. In contrast, exosomes play a role in regulating intercellular signaling, facilitating extracellular matrix remodeling, and supporting tissue regeneration. Additionally, various therapeutic and preventive strategies such as corticosteroid injections, preventive exercise programs, blood flow restriction training, and neuromuscular electrical stimulation have shown varying levels of effectiveness in tendon management and rehabilitation. However, their ability to restore the native structure and function of tendons remains limited [10–12].

We hypothesized that combining PBM with adipose-derived exosomes would enhance tendon healing and reduce muscle atrophy more effectively than either treatment alone. The primary objective of this preclinical study was to investigate the effects of PBM, exosome therapy, and their combination on the healing of degenerative or partial Achilles tendon injuries, along with associated muscle changes, in a rabbit model. This was assessed using ultrasonographic and histological evaluations.

Materials and methods

The study involved twelve male adult New Zealand White rabbits, aged 5 ± 1 months and weighing 2.8 ± 0.2 kg. Both Achilles tendons of each rabbit were included in the research. The right Achilles tendon of each rabbit remained intact, serving as the healthy contralateral tendon, while the left Achilles tendon was injured. The twelve injured left tendons were divided into four groups: control, laser, exosome, and exosome plus laser, with three tendons in each group. The rabbits were obtained from the Razi Pasteur Institute (Karaj, Iran) and housed at the Laboratory Animal Center Uiversity under controlled environmental conditions. They had unrestricted access to food and water and were maintained under a 12-hour light/12-hour dark cycle with standard room temperature.

This animal model is deemed suitable for investigating the development of tendinitis and the subsequent healing process due to its high anatomical and physiological similarity to human muscle structure [13]. The work has been reported in line with the ARRIVE guidelines 2.0 [14].

Initially, both hind limbs of the animals were shaved and thoroughly cleaned. Anesthesia for the surgical procedures was induced using subcutaneous ketamine (1.2 mg/kg) and xylazine (0.8 mg/kg), sufficient for the 30-minute surgery. Before the surgery, the right limb was disinfected with a 10% sterile iodine solution. Subsequently, an injection of 10 mg/kg of sodium cefazolin, an antibiotic, was administered into the muscle to prevent potential infection.

Next, a longitudinal incision was made in the skin overlying the Achilles tendon. The incision site was approximately 1.5 cm distal to the calcaneus and aligned along the length of the tendon. After exposing the Achilles tendon, a controlled longitudinal incision measuring 5 mm in length and approximately 3 mm in depth was made in each tendon, following a previously established rabbit Achilles tendon injury model [15, 16]. The injured tendon was then repaired using size 0.5 surgical nylon sutures. This procedure was developed to create a standardized partial Achilles tendon injury model for the subsequent evaluation of tendon healing and tissue responses (Fig. 1). To prevent and treat potential infections, Enrofloxacin 10% injectable solution was given subcutaneously at 8 mg/kg to each rabbit after anesthesia recovery, with a second dose 24 h later. Meloxicam (MCC 15 mg) was administered as an analgesic and anti-inflammatory by dissolving half of a 15 mg tablet in 3 mL of water, creating a 2.5 mg/mL solution. This was given orally by gavage immediately after anesthesia and again 24 h later. The same treatment regimen was followed for all animals. Postoperative wound healing was monitored through regular visual inspections and ultrasound examinations during follow-up. All surgical procedures, postoperative care, and interventions were monitored and approved by the supervising veterinarian.

Fig. 1.

Surgery for Achilles tendon injury; (a) A longitudinal incision is made on the skin, followed by the opening of the tendon sheath, (b) The tendon is completely separated from the skin; (c) A 5 mm incision is made on the tendon, (d) The rupture site is sutured to promote healing

After Achilles tendon injury, 12 injured tendons were divided into four groups (n = 3 each): control, low-level laser therapy, exosome therapy, and exosome-laser combination. Three batches of four rabbits were used, with one rabbit sequentially assigned to each group, ensuring three per group for balance. The control group received 1 mL of normal saline at the injury site on days 1, 8, and 15, followed by non-irradiated placement under the laser probe. The low-level laser therapy group received 830-nm laser irradiation on the same days [15]. The exosome therapy group received 200 µg/mL exosomes on days 1, 8, and 15 [8]. The exosome–laser combination group received the same exosome concentration and time points, along with simultaneous low-level laser irradiation.

Due to the animal’s immobility due to tendon injury, the gastrocnemius muscle is expected to atrophy. Therefore, ultrasound imaging of the muscle and tendon will be performed to extract thickness and thickness strain. B-mode ultrasound imaging was conducted using the Sonix TOUCH Ultrasound System (Ultrasonix Medical, Richmond, Canada) with a frequency of 40 MHz for the Achilles tendon and 14 MHz for the gastrocnemius muscle in rabbits. The thickness of both the Achilles tendon and gastrocnemius muscle was measured before and on days 1, 15, and 21 after the induction of injury, in both flexion and dorsiflexion positions. Images were recorded at a sampling rate of 30 frames per second, with a pixel size of 0.05 mm. B-mode sequential images were acquired to assess mechanical parameters for each sample during flexion and dorsiflexion of the Achilles tendon and gastrocnemius muscle (Fig. 2).

Fig. 2.

Ultrasound images at a frequency of 40 MHz and a depth of 1 cm: (a) Inflammation of the Achilles tendon at the injury site, and (b) A visible rupture of the Achilles tendon

The ultrasonic images reveal signs of inflammation and structural changes at the site of the Achilles tendon injury. Notably, these images depicted swollen and edematous regions within the tendon, which are typically associated with increased thickness, and microstructural alterations. Such changes are crucial for diagnosing inflammation and evaluating the tissue healing and repair processes [17]. The ultrasound image of the Achilles tendon injury shows distorted, opaque, and irregular areas, indicating possible rupture and structural instability (Fig. 2b). Analyzing these images helps assess the injury’s severity and monitor healing after treatment.

A low-level laser therapy (LLLT) device is Gallium-Aluminum-Arsenide (Ga-Al-As, MedArt. Uni-laser 201, Asah Medico Co., Hvidovre, Denmark), with a wavelength of 830 nm, a power output of 15 mW, and a spot area of 0.90 cm², was utilized. Calibration of the laser was performed to ensure stability and consistency of the output throughout the experimental procedures. Continuous-wave irradiation was applied in a non-contact mode at a distance of 0.5 cm from the tendon surface. Irradiation was performed at three treatment points on each side of the tendon for a total duration three minutes per tendon, corresponding to an energy density of 3 J/cm² and a total energy of 2.7 J per tendon. Control animals underwent the same positioning and handling procedures as a sham exposure without active laser irradiation. These parameters were established to evaluate the efficacy and reliability of the laser for both therapeutic and research applications [18].

Human adipose-derived mesenchymal stem cell (ADSC)-derived exosomes were commercially obtained from GENOCELL Co., Tehran, Iran. The purchased preparation was subsequently characterized in our laboratory. The exosome preparation was characterized by scanning electron microscopy (SEM) for morphological assessment, dynamic light scattering (DLS) for particle-size distribution, and zeta-potential analysis. DLS showed a particle size range of 40–170 nm, with a mean size of 112.6 ± 59.7 nm. The measured zeta potential values renged from − 30 to + 35 mV. Additionally, surface markers of the exosomes-specifically CD9, CD63, and CD81-were assessed using flow cytometry. The concentration of the isolated exosomes from adipose-derived MSCs was quantified using a bicinchoninic acid (BCA) protein assay. The results showed that the exosome concentration was approximately 600 µg per 3 mL. The levels of the important growth factors in exosomes have been quantified, which include VEGF (2000–2500 pg/mL), IGF-1 (50–55 pg/mL), PDGF (350–400 pg/mL), and KGF (350–400 pg/mL). The SEM images demonstrated predominantly rounded/vesicular structures with an average size of approximately 100 ± 10 nm, supporting the morphological characteristics of the extracellular vesicle preparation.

To assess the impact of exosomes, a dosage of 200 µg/mL was injected subcutaneously at a 45° angle using an insulin syringe. This procedure was performed on the first, eighth, and fifteenth days following surgery, directly into both sides of the Achilles tendon sheath during each session.

The preparation contains 1 × 10⁹ particles/3 mL, equating to 3.33 × 10⁸ particles per tendon. No leakage was observed during injection.

To evaluate how the Achilles tendon and gastrocnemius muscle respond to longitudinal loading, consecutive ultrasound images were captured during ankle flexion (initial length, l₀) and dorsiflexion (length, l). A digital force gauge (Lutron, Taipei, Taiwan; ±0.01 N) recorded the applied force. The ankle was evaluated in two standardized positions: at 0° flexion and 90° dorsiflexion in relation to the tibial axis. A digital force gauge (Lutron, Taiwan; ±0.01 N) was used to apply force to the plantar surface, employing a 2.5-cm lever arm and starting with an initial preload of 0 N. All measurements were taken manually by the same operator, ensuring consistent positioning and force application procedures throughout the evaluation.Anatomical landmarks were used to identify the tendon and muscle, and their thickness was measured at the same anatomical location in both ankle positions. The change in tissue thickness due to longitudinal loading was calculated from the ultrasound images using the following formula for thickness strain: [(l₀ − l)/l₀] × 100, where l₀ represents the tissue thickness before loading and l represents the thickness during loading. Additionally, the displacement of the tendon and muscle structures was assessed by tracking their positions in consecutive ultrasound images (see Fig. 3). This displacement was considered an ultrasound-derived measure of transverse tissue deformation in response to longitudinal loading.

Fig. 3.

Ultrasound images: (a) Achilles tendon in flexion and dorsiflexion, (b) Gastrocnemius muscle in flexion and dorsiflexion

To confirm the results of the physical parameters of the gastrocnemius muscle and Achilles tendon, a histological study was performed. On day 21 post-injury, tissue sampling was conducted in the experimental groups to assess tendon healing. Rabbits were deeply anesthetized with intramuscular ketamine (35–50 mg/kg) and xylazine (5–7 mg/kg) for tissue harvesting, confirmed by the absence of withdrawal and corneal reflexes. Death was confirmed by cessation of respiration and heartbeat. All procedures were performed in accordance with the ARRIVE guidelines [14].

The gastrocnemius muscle and Achilles tendon were carefully dissected and fixed in 10% formalin for two 24-hour periods. After fixation, samples were embedded in paraffin blocks, and 5 μm longitudinal sections were prepared and stained with hematoxylin-eosin (H&E) for microscopic evaluation, conducted by an expert pathologist. Histological images were acquired at standardized magnifications of ×250 and ×400, with 200-µm and 20-µm scale bars, respectively. Three histological sections per tendon were evaluated, examining five microscopic fields from each section, totaling 15 fields per tendon. Fields were selected using a standardized procedure under consistent imaging conditions, and the assessment was done blinded to treatment allocation. In order to evaluate the repair process, criteria related to myofibril structures and tissues [19] were introduced and examined. The scoring ranges for the histological parameters were as follows: inflammation (0–2), tissue structure (0–2), blood vessels (0–1), organization (0–2), cellular density (0–2), and cellular matrix (0–2). The total histological score was determined by adding the scores from these six parameters, resulting in a possible total score ranging from 0 to 11. Higher scores indicate better overall histological healing. Muscle fibers from three animals per group were measured by a blinded pathologist. The mean cross-sectional area of 20 fibers was calculated for each animal, and data were expressed as mean ± SD (n = 3). Individual fibers were not considered independent biological replicates.

All statistical analyses were conducted using IBM SPSS Statistics. Extracted parameters (thickness strain, flexion-dorsiflexion force difference, tendon and muscle thicknesses) were reported as mean and standard deviation. Repeated-measures ANOVA was used to compare means between groups, factoring in treatment group, time, and mechanical parameters. The time × treatment-group interaction was assessed for differences among groups. Mauchly’s test evaluated sphericity. Pairwise comparisons were conducted with Bonferroni adjustment for multiple comparisons. Effect sizes were calculated using eta squared (η²). A P-value < 0.05 was deemed statistically significant. A pilot assessment with three samples per group was conducted to obtain preliminary estimates of the mean and standard deviation. These data informed the required sample size, assuming a significance level of 0.05 (95% confidence) and 80% power, which determined the final sample size.

Results

The assessment of the repair process of the Achilles tendon and the gastrocnemius muscle in 1, 15, and 21 days after injury, focusing on structural changes through ultrasound imaging of flexion and dorsiflexion of the Achilles tendons, and histopathology, was conducted. The quantitative results of Achilles tendon thickness (mm) in flexion (F) and dorsiflexion (DF) in control, laser, exosome, and exosome-laser groups at 1, 15, and 21 days of treatment are shown in Table 1 as mean and standard deviation.

Table 1.

Mean and standard deviation of Achilles tendon thickness (mm) in flexion (F) and dorsiflexion (DF) in control, laser, exosome, and exosome-laser groups at 1, 15, and 21 days of treatment

Group		Control	Laser	Exosome	Exosome-laser	P-value
Day 1	F	1.25 ± 0.08	1.35 ± 0.01	1.25 ± 0.08	1.16 ± 0.04	0.063
	DF	2.09 ± 0.19	2.19 ± 0.06	1.62 ± 0.13	2.41 ± 0.73	0.214
P-value		0.130	0.001	0.022	0.171
Day 15	F	2.00 ± 0.03	1.95 ± 0.21	1.56 ± 0.26	1.82 ± 0.09	0.213
	DF	2.25 ± 0.10	2.44 ± 0.32	2.26 ± 0.30	2.63 ± 0.09	0.232
P-value		0.007	0.050	0.096	0.164
Day 21	F	1.62 ± 0.62	2.06 ± 0.24	1.40 ± 0.33	1.83 ± 0.05	0.050
	DF	2.05 ± 0.77	2.85 ± 0.15	2.14 ± 0.45	2.97 ± 0.03	0.013
P-value		0.133	0.002	0.027	0.001

Before any surgery (Health tendon), the tendon thickness values were approximately similar across all groups (1.94 ± 0.08 mm in flexion and 2.06 ± 0.14 mm in dorsiflexion). There are no significant differences between groups on 1 day after surgery in flexion and dorsiflexion states (P > 0.05). In every group, between two states of flexion and dorsiflexion, there are significant increases in laser, exosome, and exosome-laser groups (P < 0.05). There is no significant difference between the two flexion and dorsiflexion states in the control, which could be due to inflammation caused by surgery 1 day after injury.

On day 15, Achilles tendon thickness significantly increased in all groups compared to day 1 (P = 0.005). However, there was no significant difference between the groups in Achilles tendon thickness at day 15 in both flexion and dorsiflexion (P > 0.05). In every group, there’s a significant increase in the control group between flexion and dorsiflexion (P < 0.05), with no significant differences in other groups (P > 0.05).

On day 21, a downward trend in tendon thickness was observed across all groups. The control group displayed minimal change (P > 0.05). In contrast, the laser and exosome groups showed a significant reduction in thickness (P < 0.05). In this group, the tendon thickness nearly returned to levels observed in healthy, uninjured tissue. Significant differences were observed between the tendon thicknesses of the groups on day 21 in dorsiflexion.

Since immobility of the animal can cause atrophy of the gastrocnemius muscle, the thickness of the gastrocnemius muscle was measured in two positions: flexion and dorsiflexion using ultrasound imaging. The results of the gastrocnemius muscle thickness, as mean and standard deviation, in each group are given in Table 2.

Table 2.

Mean and standard deviation of gastrocnemius muscle thickness (mm) in flexion (F) and dorsiflexion (DF) in control, laser, exosome, and exosome-laser groups at 1, 15, and 21 days of treatment

Group		Control	Laser	Exosome	Exosome-laser	P-value
Day 1	F	13.11 ± 0.62	14.15 ± 0.65	13.60 ± 0.76	14.09 ± 0.67	0.285
	DF	12.67 ± 0.61	13.83 ± 0.52	12.92 ± 0.72	13.69 ± 0.88	0.200
P-value		0.179	0.054	0.007	0.121
Day 15	F	16.53 ± 0.36	16.04 ± 0.59	17.09 ± 0.24	16.51 ± 0.91	0.255
	DF	13.90 ± 0.76	15.01 ± 0.66	16.56 ± 0.28	15.21 ± 1.79	0.079
P-value		0.040	0.002	0.014	0.134
Day 21	F	14.64 ± 0.34	16.09 ± 2.04	16.02 ± 0.99	16.33 ± 1.99	0.552
	DF	13.85 ± 0.67	14.90 ± 0.48	12.69 ± 1.87	13.15 ± 1.50	0.239
P-value		0.051	0.376	0.047	0.030

Qualitative analysis of ultrasound images shows that tendon injuries lead to muscle atrophy from inactivity. Treatment protocols help restore muscle thickness. Both the control and laser treatment groups show increased muscle thickness post-treatment, indicating the repair process has started. Notably, laser-exosome therapy significantly improved muscle structure and condition. Gastrocnemius muscle thickness was measured as 14.27 ± 0.67 mm in flexion and 13.62 ± 0.97 mm in dorsiflexion via ultrasound imaging before injury. Increased tendon repair brings the muscle closer to health in flexion, but inflammation remains in dorsiflexion (Table 2).

On day 1, muscle thickness across all groups was similar and within the normal range. Following this, changes in muscle thickness were notably observed in treatment groups. As shown in Table 2, the thickness of the gastrocnemius muscle increased during the healing process. Although the muscle thickness increased over time, there was no significant difference between the muscle thicknesses on days 1, 15, and 21 after injury (P > 0.05). On day 21, there was a significant difference between the flexion and dorsiflexion states of the exosome and exosome-laser groups (P < 0.05). A repeated-measures ANOVA analysis showed that there was a significant increase in muscle thickness measured in all groups from day 15 compared to day 21 (P = 0.043).

Table 3 presents the mean and standard deviation of the force change (N) for flexion (F) and dorsiflexion (DF) of the Achilles tendon and gastrocnemius muscle in the control, laser, exosome, and exosome-laser groups at 1, 15, and 21 days of treatment.

Table 3.

Mean and standard deviation of the force change applied (N) to create flexion (F) and dorsiflexion (DF) of the Achilles tendon and gastrocnemius muscle in control, laser, exosome, and exosome-laser groups at 1, 15, and 21 days of treatment

Group	Control	Laser	Exosome	Exosome-laser	P-value
Day 1	1.89 ± 1.57	1.14 ± 0.47	1.79 ± 1.25	1.44 ± 0.16	0.802
Day 15	0.83 ± 0.14	1.11 ± 0.48	2.02 ± 0.35	1.05 ± 0.95	0.125
Day 21	0.74 ± 0.43	0.68 ± 0.31	1.64 ± 0.97	2.06 ± 0.25	0.045

On day 1, no significant differences were observed among the groups in the mean force required to induce flexion and dorsiflexion of the Achilles tendon and gastrocnemius muscle (P = 0.802). On day 15, although the exosome group showed the highest mean value compared to the control, laser, and exosome–laser groups, the difference did not reach statistical significance (P = 0.125). By day 21, a statistically significant difference among the groups was detected (P = 0.045). The exosome–laser group demonstrated the greatest force relative to the other groups. Between-group analysis shows that there is a significant difference between the control group and the combined group (P = 0.022) and the laser node and combined group (P = 0.018).

In Table 4, the mean and the standard deviation of the thickness strain of the Achilles tendon and gastrocnemius muscle in the control, laser, exosome, and exosome-laser groups at 1, 15, and 21 days of treatment are shown.

Table 4.

Mean and standard deviation of the thickness strain of the Achilles tendon and gastrocnemius muscle in control, laser, exosome, and exosome-laser groups at 1, 15, and 21 days of treatment

Group		Control	Laser	Exosome	Exosome-laser	P-value	Effect size (η2)
Achilles tendon	Day 1	0.09 ± 0.06	0.13 ± 0.05	0.24 ± 0.02	0.17 ± 0.10	0.088	0.52
	Day 15	0.13 ± 0.04	0.26 ± 0.07	0.45 ± 0.09	0.44 ± 0.05	0.001	0.87
	Day 21	0.26 ± 0.09	0.39 ± 0.13	0.54 ± 0.04	0.63 ± 0.06	0.004	0.79
Gastrocnemius muscle	Day 1	0.04 ± 0.03	0.02 ± 0.01	0.05 ± 0.01	0.03 ± 0.02	0.345	0.45
	Day 15	0.19 ± 0.07	0.007 ± 0.06	0.07 ± 0.03	0.09 ± 0.07	0.034	0.76
	Day 21	0.12 ± 0.05	0.11 ± 0.07	0.27 ± 0.12	0.24 ± 0.06	0.049	0.75

On day 1, there was no significant difference in Achilles tendon thickness strain between the groups (P = 0.088). By day 15, tendon thickness strain had increased (P = 0.001). However, on day 21, tendon thickness strain was highest in the combined exosome-laser group and lowest in the control group. At this point, the difference between the groups became significant (P = 0.004), indicating that the treatments had an effect on tendon elasticity. On day 21, Achilles tendon thickness strain in the group receiving combined exosome-laser treatment was significantly different from that of the other groups. The healthy tendon thickness strain was calculated to be 0.32 ± 0.12, which is close to the result of the combination group after day 15 of treatment. Repeated-measures ANOVA showed a significant effect of time on tendon thickness strain (P < 0.001), with Mauchly’s test confirming the sphericity assumption (P = 0.427). A significant time × treatment-group interaction was observed (P < 0.05), indicating differences in tendon-thickness strain among treatment groups. Post-hoc LSD analysis revealed significant differences between groups 1 and 3 (P = 0.001), groups 1 and 4 (P = 0.001), groups 2 and 3 (P = 0.012), and groups 2 and 4 (P = 0.012). Significant differences were also noted between days 1 and 15, 0 and 21, and 15 and 21 (P < 0.001 for all comparisons). Positive correlations were found for days 1 and 15 (r = 0.617, P = 0.033), days 1 and 21 (r = 0.580, P = 0.049), and days 15 and 21 (r = 0.935, P < 0.001). Significant differences were observed in flexion and dorsiflexion forces at days 15 and 21 (P = 0.033 and 0.045, respectively). The effect sizes for tendon strain between groups were large at all assessment time points, with η² values of 0.52, 0.87, and 0.79 on days 0, 15, and 21, respectively.

In contrast, gastrocnemius muscle thickness strain increased in the exosome and exosome-laser (0.24 ± 0.06) groups compared with the control and laser groups, with significant intergroup differences (P = 0.049). These findings indicate that the combined treatment had divergent effects on tendon and muscle tissues.

A superficial scar was observed in all four groups during the early healing stage. By day 15, the scar had become smaller and softer, and by day 21, it had nearly disappeared, restoring the skin’s surface. Notably, in the exosome and exosome-laser groups, the scar was smaller compared to the control and laser groups, and almost fully resolved by day 15. Histological analysis of tendon samples after treatment showed varying degrees of healing depending on the intervention. In the control group, the tendon tissue exhibited poor structure, with sparse, abnormally shaped cells and ongoing inflammation. Collagen fibers were disorganized, and there were fewer fibroblasts, along with lower levels of fibrosis and neo-vascular formation, indicating a weak regenerative response (Fig. 4a). In the laser-treated group, samples showed increased density of type I collagen fibers and improved organization compared to controls. Fibroblasts appeared more active and well-structured, with a reduction in inflammatory tissue. The presence of a cohesive fibrillar network and signs of early active regeneration were clearly observable (Fig. 4b). In the exosome-treated group, tissue exhibited notable structural improvements, including an increase in collagen fiber density and better cellular interaction with the surrounding environment. Fibroblasts were active and organized, and collagen structures were in the early stages of healing. Evidence of neo-vascular structures forming and a reduction in chronic inflammation were also observed (Fig. 4c). The group that received both exosome and laser treatment showed the most improvements in tissue architecture. The collagen fibers, particularly type I, were well-organized and densely packed. Fibroblasts were actively dividing, and a strong, coherent fibrillar network was visible. Furthermore, inflammation and fibrosis were greatly reduced, indicating active and uniform tissue regeneration (Fig. 4d). The treatment groups demonstrated increased collagen fiber density and better organization of the extracellular matrix compared to the injured control tendons, suggesting enhanced collagen deposition and remodeling during tendon repair. While the histological appearance indicated a type I collagen-rich matrix, definitive identification of collagen subtypes was not achievable with H&E staining alone.

Fig. 4.

Histological images of Achilles tendon sections in the studied groups with hematoxylin-eosin staining, examined with scale bars of 20 and 200 μm. (a) Control group, (b) Laser group, (c) Exosome group, (d) Exosome-laser group

Figure 5. Histological images of gastrocnemius muscle sections in the studied groups with hematoxylin-eosin staining, examined with scale bars of 20 and 200 μm. (a) Control group, (b) Laser group, (c) Exosome group, (d) Exosome-laser group.

Fig. 5.

shows histological images of the gastrocnemius muscle in the studied groups, including control, laser, exosome, and exosome-laser, with hematoxylin and eosin staining

The results of the Achilles tendon scoring [19] of the studied groups, based on the extraction of the parameters of cellular matrix, cell density, vascularization, inflammation, organization, and tissue structure, as well as the degree of inflammation of the gastrocnemius muscle, are shown in Table 5.

Table 5.

The results of Achilles tendon scoring, the degree of inflammation and muscle myofibril area (µm²) in the gastrocnemius muscle for the studied groups: control, laser, exosome, and exosome-laser, based on histopathological analysis

Group	Atrophy	Cross-sectional area (µm2)	Inflammation	Tissue Structure	Blood Vessels	Organization	Cellular Density	Cellular Matrix	Total
Control	+++	141.482 (35.234)	0	1	0	1	1	0	3
Laser	++	178.516 (44.629)	1	1	0	1	1	1	5
Exosome	-	181.699 (23.621)	2	1	1	1	2	1	8
Exosome-Laser	-	183.415 (25.678)	1	2	1	2	2	2	10

Microscopic examinations of the control group revealed a lack of organization in the extracellular matrix, negatively impacting tissue function. While there was a local increase in cell density indicating initial repair attempts, these efforts did not reach normal physiological levels. The tissue integrity was inefficient, with no signs of vascularization observed (3 score). Additionally, muscle atrophy was noted at a level of 3 plus. Severe inflammation and atrophy of the gastrocnemius muscle indicate potential future issues with tissue and vascular structure and function, negatively impacting repair and regeneration.

The Achilles tendon results of the laser group showed a total score of 5 and 2 plus for atrophy. There was a less favorable organization of the extracellular matrix, characterized by loose filaments and granulation fibers. Compressibility was slightly increased, indicating an unstable tissue repair state, with heterogeneous areas and no signs of vascularization. Mild inflammation and atrophy were observed in the muscle. Overall, these results suggest an unstable state in comparison to the control group, impacting the repair process.

In the exosome group, the Achilles tendon score was 8, with observed negative atrophy. The extracellular matrix showed minimal organization, and compact, parallel collagen fibers were not clearly present—only loose fibers with granulation tissue. Cell density was physiological, indicating improved cellular activity for repair. However, abnormalities in the tissue scaffold integrity and weak vessels were noted, and inflammation was absent. Overall, there was improved cell density and reduced atrophy, but further enhancement of the extracellular matrix organization is needed for better tissue integrity. The exosome group is progressing towards tissue repair.

In the results of the exosome-laser group, the tendon score was 10, and muscle atrophy was negative. The results showed the presence of compact and parallel collagen fibers, which are a strong and orderly structure in the extracellular matrix and help in better tissue repair. Cell density was observed at a physiological level. This result shows an improvement in cellular activities and helps in the repair processes. The presence of homogeneity in tissue integrity indicated an effective structural adaptation to normal tissue. Organization is clearly evident in this group. No inflammation was observed, and the muscle did not atrophy.

Next, the cross-sectional area of the gastrocnemius muscle myofibril was measured based on tissue images using Image J software (Table 5). These changes mainly showed as increased muscle myofibril area (µm²), indicating tissue regeneration and remodeling. In this context, a thicker muscle myofibril area reflects better muscle condition and reduced atrophy. Muscle thickness increased, indicating reduced atrophy and improved condition after tendon function restoration.

Discussion

This study showed that photobiomodulation (PBM) and adipose tissue-derived stem cell exosomes (ADSC-Exos), especially when used together, significantly improved healing of degenerative partial Achilles tendon injuries and reduced gastrocnemius muscle atrophy in rabbits. Key findings included restored tendon thickness, improved ultrasound-derived tendon characteristics and flexion–dorsiflexion force measurements, reduced muscle atrophy, and better histological organization in the combined treatment group. This research explores the effects of PBM and exosome therapy on tendon healing, suggesting that PBM may enhance tissue regeneration through extracellular vesicles [5, 20–22].

Ultrasound evaluation showed changes in tendon morphology during healing. On day 1, there were no significant differences in tendon thickness among groups, indicating the acute inflammatory phase marked by edema and tissue swelling [23]. By day 15, thickness increased in all groups, reflecting the proliferative phase with fibroblast activation and extracellular matrix deposition [6, 8, 9]. However, by day 21, tendon thickness decreased significantly in the laser, exosome, and combined treatment groups, while the control group showed minimal remodeling. The reduced tendon thickness aligned with decreased postoperative swelling and better tendon morphology, as histopathological findings indicated. Additionally, gastrocnemius muscle assessments revealed increased thickness in treatment groups, particularly with combined therapy, which likely reflects improved tendon function and the direct regenerative effects of PBM and exosomes [24–26]. Notably, differences in flexion and dorsiflexion measurements on day 21 suggest that functional recovery aids in muscle remodeling.

Biomechanical analysis showed the treatments’ regenerative effects. By day 21, treatment groups had significantly greater applied force for flexion and dorsiflexion compared to controls, indicating better functional restoration of the tendon-muscle unit. The exosome-treated group had the highest force values, suggesting enhanced tissue maturation. Tendon thickness strain was reduced in the combined treatment group, approaching healthy tendon values, which indicates improved stiffness and structural integrity due to better collagen alignment and cross-linking [26, 27]. Conversely, increased gastrocnemius muscle thickness strain in the exosome and combined groups implies improved muscle elasticity and functional recovery. These findings suggest that regenerative therapies can enhance tendon stiffness while maintaining muscle flexibility, leading to better functional outcomes during healing. Histopathological evaluation confirmed that the combined PBM-exosome treatment led to a superior regenerative response compared to the control group, which showed poor tissue repair characterized by inflammation, limited vascularization, and muscle atrophy. In contrast, PBM treatment enhanced collagen type I deposition, fibroblast organization, and reduced inflammation. Exosome therapy further boosted cellular activity and neovascularization while minimizing inflammation. The combined therapy achieved the highest histological score, featuring organized collagen fibers, optimal cell density, and complete prevention of muscle atrophy. These findings aligned with improved ultrasonographic and biomechanical results, supporting the effectiveness of this treatment for tissue restoration, similar to previous studies [8, 9, 28] on tendon repair. The mechanisms of photobiomodulation (PBM) and exosome therapy are not fully understood but may involve complementary biological pathways.

PBM is known to stimulate mitochondrial activity, increase ATP production, modulate reactive oxygen species, enhance angiogenesis, and promote collagen synthesis [5]. Meanwhile, exosomes facilitate intercellular communication by transferring proteins, growth factors, microRNAs, and other bioactive molecules that regulate cellular proliferation, extracellular matrix remodeling, and tissue regeneration [8, 9]. Emerging evidence suggests that PBM may also influence exosome secretion, cargo composition, and biological activity [29, 30]. Functional measurements, including the deformation of relative tendon thickness, showed variations in the mechanical response of the tendon after treatment [31, 32]. This indicates that exosome-laser therapy may be safe and effective, although the mechanisms, including exosomal miRNAs, need further study. Thus, PBM might not only have direct regenerative effects but could also enhance the therapeutic efficacy of exosomes by promoting their production, uptake, or biological function. The superior structural, biomechanical, and histological outcomes observed in the group receiving combined treatment support the hypothesis that PBM and exosomes work together to accelerate tendon remodeling and muscle recovery.

Tendon injury triggers a biological repair response that includes inflammatory cell recruitment and the activation of tendon cells and fibroblasts. Initially, collagen type III is predominant, but as remodeling occurs, it transitions to collagen type I, accompanied by improved fibril organization and cross-linking. These processes are crucial for restoring tendon structure and function [33, 34]. Tendon injury triggers a biological repair response that includes inflammatory cell recruitment and the activation of tendon cells and fibroblasts. Initially, collagen type III is predominant, but as remodeling occurs, it transitions to collagen type I, accompanied by improved fibril organization and cross-linking. These processes are crucial for restoring tendon structure and function [35].

This study has several limitations. First, the small sample size (n = 3 per group) may have reduced statistical power and generalizability. Second, the 21-day follow-up limited the assessment of long-term tissue maturation and functional recovery. While effects were observed, the underlying molecular mechanisms of PBM and exosome therapy were not directly investigated, including changes in exosomal cargo and cellular signaling. Future research should involve larger cohorts, extended follow-up, and molecular analyses to enhance understanding and treatment protocols. Additionally, factors such as exosome concentration, sterility, and batch variation were not assessed. Lastly, using human adipose tissue-derived exosomes in rabbits may have introduced species-specific immune responses, despite their cell-free nature. Human-derived exosomes were selected for their relevance in evaluating a human-derived biological product. Additional limitations include possible pseudoreplication, multiple-comparison issues, nonspecific thickness measurements, and the lack of ability to show synergistic interactions without factorial analysis., A limitation of this study is the reliance on H&E staining for histological assessment, which limited the evaluation of collagen organization. Future research should include collagen-specific staining, such as Van Gieson staining, and quantitative analysis to better assess tendon remodeling.

The current study demonstrated that photobiomodulation (PBM), when used in combination with exosomes derived from adipose tissue stem cells, significantly improved the healing of degenerative and partial Achilles tendon injuries while also reducing associated atrophy in the gastrocnemius muscle in a rabbit model. This combined therapy led to better tendon remodeling, restoration of tendon morphology, enhanced improved ultrasound-derived tendon characteristics and flexion–dorsiflexion force measurements, improved collagen organization, and accelerated muscle recovery compared to either treatment alone. Histological analysis further confirmed superior tissue regeneration and remodeling of the extracellular matrix in the group receiving the combined treatment. H&E staining does not specifically identify collagen subtypes; the findings should not be seen as definitive evidence of increased type I collagen. Confirmation requires collagen-specific techniques, like type I collagen immunohistochemistry. These findings suggest that PBM may enhance the regenerative effects of exosomes through complementary biological mechanisms, making it a promising minimally invasive approach for tendon regeneration.

Conclusion

Low-level laser therapy combined with exosomes derived from adipose-derived stem cells (ADSCs) demonstrated significant improvements in both histological and ultrasound parameters related to Achilles tendon repair. This combined treatment also resulted in notable changes in the mechanical behavior of the tendon-muscle unit. Specifically, the strain in the Achilles tendon increased significantly at days 15 and 21, with the highest values observed after the combined exosome and laser treatment at day 21. Additionally, the strain in the gastrocnemius muscle showed significant differences among treatment groups at days 15 and 21, indicating treatment-related changes in muscle mechanical behavior during the healing process. Moreover, by day 21, significant differences in flexion/dorsiflexion force were noted, suggesting alterations in the functional mechanical properties of the tendon-muscle unit. Overall, these findings indicate that the combined approach may promote structural remodeling, with changes in tissue mechanical behavior and functional recovery following an Achilles tendon injury. These preliminary results warrant further investigation into the use of ADSC-derived exosomes in conjunction with low-level laser therapy as a promising strategy for tendon repair.

Author contributions

ASM: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing – original draft. MMD: Conceptualization; Data curation; Formal analysis; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing – original draft; Writing – review & editing. ZHM, SGK, AMG: Conceptualization; Methodology; Project administration; Software; Validation; Visualization; Writing – original draft.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Clinical trial number

not applicable.

Competing interests

The authors declare no competing interests.