C₆₀ fullerene promotes post-traumatic recovery of the rat muscle gastrocnemius

ABSTRACT

Aim

The remarkable antioxidant capabilities of biocompatible and safe C₆₀ fullerenes have extensive applications in biomedicine. This study is the first to present an investigation into the effect of water-soluble C₆₀ fullerenes on post-traumatic recovery of the muscle gastrocnemius in rats.

Methods

Tensometry was used to investigate the biomechanical parameters of muscle contraction, specifically the times of reaching and holding the maximum force response of the muscle, and the return of muscle contraction force to the initial value after cessation of stimulation. Blood biochemical indicators were assessed, including concentrations of c-reactive protein, lactate, creatinine, and reduced glutathione, as well as superoxide dismutase and catalase activities 5, 10, and 15 days after initiating open muscle injury. Histopathological analysis was also performed to examine the rat muscle gastrocnemius damage on day 15 after the onset of injury.

Results

It was found that C₆₀ fullerenes reduced the stiffness of injured skeletal muscle, thereby slowing the development of fibrosis, and inhibiting the inflammatory process due to their antioxidant properties. There was also a reduction in histopathological features of muscle damage.

Conclusion

These findings suggest using of carbon nanoparticles to correct pathological conditions that may occur during the physiological repair of damaged muscle tissues.

1. Background

Open injuries are the most frequent and severe type of skeletal muscle damage [1,2]. Inadequate treatment of such injuries, especially at an early stage, contributes to the development of subsequent pathologies of the injured muscle and even loss of its performance [3,4]. Today, there is no unvarying approach to classifying muscle injuries [5], and there is limited evidence to correlate any of their clinical features with the extent of pathology found [6]. This problem is further complicated by the fact that patients with open muscle injuries sustained in extreme conditions often present with a combination of classic major trauma, including both blunt and penetrating wounds, resulting in an increased level of inflammation [7]. Therefore, a comprehensive management strategy for open muscle injuries should include “mitigation” of their consequences at a systemic level as well as direct restoration of the resulting musculoskeletal dysfunction [8,9]. Moreover, novel methods such as dermal substitutes, external tissue expansion, and regenerative matrices used in the treatment of injured muscles can lead to increased fibrotic formation and consequently increased muscle stiffness [10].

The process of repairing damaged muscle tissue involves the coordinated activity of different cell types in response to local and systemic signals. This process requires the participation of several populations of resident muscle cells, including satellite stem cells, fibroblasts, macrophages, and vascular cells. Infiltrating inflammatory cells, together with resident stem cells, play a critical role in restoring homeostasis to the injured muscle. Abnormal repair mechanisms in dystrophic muscle and the final stage of this process, fibrosis, is a common endpoint for almost all open muscle injuries [11]. At the same time, high-severity injuries cause the simultaneous destruction of many tissue components, surpassing the intrinsic capacity for wound healing without scarring and leading to permanent functional deficits. In this case, the nonproductive inflammatory response and associated fibrosis dominate [12]. This problem is further complicated by the fact that, as part of the inflammatory process, stem cell capacity is significantly inhibited and fibrogenic cells are continuously activated. This ultimately leads to the conversion of muscle tissue into nonfunctional fibrotic tissue. Increased fibrosis of skeletal muscle impairs its function and negatively affects regeneration after injury. Consequently, the formation of fibrosis is considered to be a major contributor to muscle weakness [13]. If muscle injury is not adequately treated, fibrosis can become a self-sustaining process that prevents complete muscle regeneration [14]. In addition, if tissue damage persists over a prolonged period – characteristic of the inflammatory process associated with severe open muscle injury – the muscle regeneration process becomes dysregulated. This leads to the replacement of myofibrils with a nonfunctional mass of fibrous tissue [15].

Antioxidant therapy is used quite effectively to reduce the inflammatory processes in muscle injury [16]. So, resveratrol exhibits a protective effect on contusion induced muscle injury and improves its regeneration compared to conventional treatment with non-steroidal anti-inflammatory drugs [17]. Melittin enhances muscle regeneration factors expression in a mouse model of skeletal muscle contusion [18]. The authors [19] found that a hydroxyl radical scavenger, EPC-K1, reduces reperfusion injury in rat skeletal muscle. Finally, quercetin attenuates adipogenesis and fibrosis in human skeletal muscle [20].

It has been shown that water-soluble and nontoxic (at least at low doses) C₆₀ fullerenes effectively protect cell membranes from oxidation by capturing free radicals before their interaction with lipids [21,22]. On in vivo models, it was found that C₆₀ fullerene has more potent antioxidant properties compared to such well-known natural antioxidants as vitamins C, E, and carotenoids [23], as well as the exogenous antioxidant N-acetylcysteine [24], which often uses in sports medicine. As a result, its use in modeling ischemia-reperfusion, closed injury, and atrophy of the rat muscle soleus leads to significant positive therapeutic effects [25–27].

Thus, the present work aimed to evaluate the water-soluble C₆₀ fullerenes impact on the post-traumatic recovery of rat muscle gastrocnemius by analyzing the selective biomechanical parameters of its contraction and pathohistological features of damages, as well as the biochemical indicators of animal blood after open muscle injury initiation.

2. Materials & methods

2.1. Preparation of nanofluid & its characterization

C₆₀ fullerene (Sigma-Aldrich, purity > 99.95%) aqueous solution (C₆₀FAS) has been prepared according to the ultrasonic method described in detail in [28,29]. The resulting C₆₀FAS (maximum concentration was 0.15 mg/ml) is stable for 12–18 months at a temperature of + 4–25°C [30].

Structural study of water-soluble C₆₀ fullerene particles was performed by atomic force microscopy (AFM; NT-MDT, Apeldoorn, Netherlands) [30]. A drop of sample was transferred on the atomically smooth substrate for layer deposition. Measurements were carried out after the complete evaporation of the solvent. A freshly cleaved surface of mica (SPI supplies, V-1 grade) was used as a substrate. The AFM measurements were carried out in the amplitude modulation tapping mode using ‘RTESPA–150’ type probes (Bruker, Billerica, Massachusetts, USA).

2.2. Experiments in vivo

The experimental animals (3-month-old male Wistar rats weighing 170 ± 5 g) were kept in an air-filtered and temperature-controlled (21 ± 1°C) room under 12-h light/12-h dark conditions [30]. They received water and a normal diet ad libitum. All procedures complied with the ARRIVE guidelines. The usage of the experimental animals was approved by the Biomedical Ethics Committee of the ESC “Institute of Biology and Medicine” of Taras Shevchenko National University of Kyiv (protocol No. 9 dated 4 September 2023) and performed following the “European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” (Strasbourg, 1986) and Article 26 of the Law of Ukraine “On the Protection of Animals from Cruelty” (No. 3447-IV, 21 February 2006), as well as European Union Directive of 22 September 2010 (2010/63/EU) for the protection of animals used for scientific purposes.

The following groups of animals were used in the research (n = 10 in each): control, open injury, and open injury+С₆₀ (oral daily usage of C₆₀FAS in a dose of 1 mg/kg animal body weight after open muscle injury initiation).

The choice of the above dose of C₆₀FAS is based on its high effectiveness in the treatment of various muscle pathologies in vivo [25–27,30].

Anesthesia of experimental rats was performed by intraperitoneal injection of Nembutal (40 mg/kg). Animals were left under deep surgical anesthesia for at least 2 h [30].

Muscle gastrocnemius was isolated from surrounding tissues in the area of the hamstring fossa. This surgical procedure is described in detail in [30]. Then the muscle gastrocnemius was subjected to transverse dissection with a depth of 1 mm in three equidistant places (Figure 1) [31]. Such an open muscle injury model causes inflammation and necrosis of damaged tissues, as well as leads to significant muscle loss and its prolonged regeneration [32].

Figure 1.

Open muscle injury model: 1 - stimulating electrodes; 2 - three transverse dissections with a depth of 1 mm in three equidistant places.

Electrical stimulation of the muscle gastrocnemius was performed by separate series of rectangular 2 ms pulses with a frequency of 50 hz and a duration of 6 s using a strain gauge generator [30].

2.3. Biomechanical & biochemical analyses

Research of muscle contraction dynamics and blood biochemical indicators of rats was performed 5, 10, and 15 days after open muscle injury initiation.

The following biomechanical parameters [27], which are indicators of the overall performance of a muscle and have a clear tendency to change with an increase in its stiffness, were used:

• - time to reach the maximum force response of the muscle (t_start). The maximum force generation that an active muscle is capable of is an important indicator for fast non-targeted movements. The magnitude of its change indicates the level of physiological dysfunction of the neuromuscular preparation during the realization of maximal force tasks;

• - time of retention of the maximum force response of the muscle (t_max). Its value is an indicator of the adaptability of the muscular system to new states resulting from the development of pathological processes. Changes in this parameter are associated with a decrease in the efficiency of individual muscle fibers, a decrease in their number, and, accordingly, an increase in the ratio of “muscle-fibrous tissue;”

• - time of muscle contraction force return to the initial value after stimulation cessation (t₀). Its value is directly related to the amount of fibrous tissue formed in the injured muscle.

The following biochemical parameters, which are indicators of muscle injury [30], namely the concentrations of c-reactive protein (CRP), creatinine, lactate, and reduced glutathione (GSH) as well as the activity of catalase (CAT) and superoxide dismutase (SOD) in the blood plasma of rats were used. These parameters were measured using the biochemical analyzers RNL-200 and JN-1101-TR2 (Netherlands).

2.4. Histopathological analysis

For general histopathological analysis [33], the muscle gastrocnemius samples were separated and fixed in 10% formalin, embedded in paraffin, cut into 5 µm sections, and stained with hematoxylin and eosin (H&E) [34]. In addition, to detect connective tissue and muscle fibers, sections of the muscle gastrocnemius were stained with hematoxylin and picrofuchsin by van Gieson. Digital microphotographs of stained sections (×100 and × 400 magnification) were taken using an Olympus B×41microscope and an Olympus C-5050 Zoom digital camera.

2.5. Statistical analysis

Statistical evaluation of the results was conducted using the software package Statistica 8.0 (Dell, USA) through the procedure of analysis of variances (ANOVA) with mixed design [30,34]. Two between-group factors were supposed: 1) open muscle injury (10 levels − 10 consecutive non-relaxation contractions of the muscle gastrocnemius when electrical stimulation is applied); 2) C₆₀FAS treatment (two levels – no and use of C₆₀FAS). Time was considered as a within-group factor with three levels (5, 10, and 15 days after initiation of open muscle injury). The Shapiro-Wilk W-test and Levene’s test were used to assess for normality and the equality of variances across groups, respectively. The Bonferroni post-hoc test was used for multiple pairwise comparisons between different groups. The differences between the experimental groups were considered significant at p < 0.05. Each of the experimental force curves is the result of averaging 10 similar tests. Each biochemical measurement was carried out at least three times. Data are expressed as the means ±SEM for each group.

3. Results

3.1. C₆₀FAS characterization by AFM

When observing the process of deposition of C₆₀ fullerene particles from C₆₀FAS on the mica surface, it was noted that the complete evaporation of water varied significantly in duration, ranging from 1 to 60 s within the area covered by the aqueous solution. This variation was accompanied by a significantly non-uniform distribution of the deposited substance over the surface. We carried out AFM measurements at locations with different surface concentrations of C₆₀ fullerene.

The AFM image of the high-concentration area shows many clusters with heights ranging from 20 to 120 nm (Figure 2(a)), characteristic of colloidal C₆₀FAS. Such aggregates were observed rarely or completely absent in low-concentration areas. The AFM image of the low-concentration area reveals surface-spaced objects up to 3 nm in height (Figure 2(b)), which correspond to individual C₆₀ fullerenes (diameter ~0.7 nm) or their nanoaggregates, which typically do not exceed four times the molecular diameter in size.

Figure 2.

AFM images of С₆₀ fullerene layer deposited from C₆₀FAS (0.15 mg/ml). The images were measured within one sample over different surface areas. The arrows indicate the height of the particles.

3.2. Biomechanics of injured muscle contraction

Figure 3 illustrates the results of 10 consecutive non-relaxation contractions of the muscle gastrocnemius of rats. As observed, during prolonged activation and the development of smooth tetanic muscle contraction, a decrease in the value of its force is evident. This is associated with alterations in the level of phosphorylation of myosin light chains, development of fatigue processes, a reduction in the number of active muscle fibers, inflammatory processes, and fibrosis of muscle tissue [13,24–27]. Based on the obtained mechanograms, the selective biomechanical parameters as indicators of muscle dysfunction (see below) were calculated at 5, 10, and 15 days following the open muscle injury initiation.

Figure 3.

The force of 10 consecutive non-relaxation contractions of muscle gastrocnemius of rats (1–10) during application of 50 hz stimulation of 6 s duration: control, open injury and open injury+C₆₀ - mechanograms of a control group, groups of injured rats and rats who daily consumed orally C₆₀FAS at a dose of 1 mg/kg after the open muscle injury initiation; t_start - time to reach the maximum force response of the muscle; t_max - time of retention of the maximum force response of the muscle; t₀ - time of muscle contraction force return to the initial value after stimulation cessation; 5, 10 and 15 days − 5, 10 and 15 days after the open muscle injury initiation, respectively.

Figure 4 shows changes in the time to reach the maximum force response of the muscle gastrocnemius (t_start) during 10 consecutive non-relaxation contractions. Five days following the initiation of an open muscle injury, this index was 387 ± 8 ms at the 1^st contraction (control − 97 ± 4 ms). At the 10^th contraction, its value increased to 563 ± 11 ms. The application of C₆₀FAS significantly reduced this index, which was 143 ± 7 and 319 ± 9 ms at the 1^st and 10^th contraction, respectively. Consequently, the positive effect of C₆₀FAS was about 270% and 176% at the 1^st and 10^th contractions, respectively, compared to the open injury group.

Figure 4.

Time to reach the maximum force response of muscle gastrocnemius of rats (t_start) during 10 consecutive non-relaxation contractions (1–10) in the case of 50 hz stimulation of 6 s duration: control, open injury, and open injury+C₆₀ - control group, group of injured rats and group of rats that daily consumed orally C₆₀FAS at a dose of 1 mg/kg after the open muscle injury initiation; 5, 10 and 15 days − 5, 10 and 15 days after the open muscle injury initiation, respectively; *p < 0.05 relative to the control group; ^#p < 0.05 relative to the open injury group.

On the 10^th day after the open muscle injury initiation, the time to achieve the maximum force response of muscle gastrocnemius significantly increased and was 440 ± 12 and 680 ± 21 ms on the 1^st and 10^th contraction, respectively, which indicates an increase in connective tissue components in the studied muscle compared to the control group. In contrast, when C₆₀FAS was applied, this index remained practically at the same level as on the 5^th day of the experiment, viz: 146 ± 5 and 331 ± 9 ms for the 1^st and 10^th contractions, respectively.

Mechanograms obtained on day 15 following the initiation of an open muscle injury demonstrate a decrease in the time to reach the maximal force response of the muscle gastrocnemius to 202 ± 7 and 401 ± 9 ms at the 1^st and 10^th contraction, respectively. However, these values exceeded the control ones by ~ 200% and ~ 400%, respectively. The application of C₆₀FAS reduced this value, which was 109 ± 3 (~12% relative to control) and 205 ± 5 (~112% relative to control) ms at the 1^st and 10^th contraction, respectively.

Figure 5 shows the results of changes in the retention time of the maximum force response of the muscle gastrocnemius (t_max) during 10 consecutive non-relaxation contractions. In the control group, the value of this parameter was maintained in all time frames of the experiment, with a value of 6 s. Five days after the open muscle injury initiation, this parameter was 2843 ± 12 ms at the 1^st contraction and fell to almost zero at the 10^th contraction. The application of C₆₀FAS increased it to 4720 ± 23 and 1090 ± 15 ms at the 1^st and 10^th contraction, respectively.

Figure 5.

Time of retention of the maximum force response of the muscle gastrocnemius (t_max) during 10 consecutive non-relaxation contractions (1–10) in the case of 50 hz stimulation of 6 s duration: control, open injury, and open injury+C₆₀ - control group, group of injured rats and group of rats that daily consumed orally C₆₀FAS at a dose of 1 mg/kg after the open muscle injury initiation; 5, 10 and 15 days − 5, 10 and 15 days after the open muscle injury initiation, respectively; *p < 0.05 relative to the control group; ^#p < 0.05 relative to the open injury group.

On day 10 of the experiment, the value of this index decreased significantly to 3281 ± 14 ms at the 1^st contraction and nearly reached zero at the 10^th contraction. Application of C₆₀FAS increased in the retention time of the maximum force response of the muscle gastrocnemius to 5002 ± 3 and 1605 ± 5 ms at the 1^st and 10^th contraction, respectively.

On day 15 of the experiment, this indicator was 3943 ± 26 and 240 ± 12 ms at the 1^st and 10^th contractions, respectively. The application of C₆₀FAS in this case did not show significant changes compared with day 10 of the experiment, which was 4961 ± 24 and 1893 ± 17 ms at the 1^st and 10^th contractions, respectively.

Figure 6 shows the results of changes in the time of muscle contraction force return to its initial value after stimulation cessation (t₀) during 10 consecutive non-relaxation contractions of the muscle gastrocnemius of rats. 5 days after the open muscle injury initiation, this parameter was 1.32 ± 0.10 and 1.83 ± 0.10 s at the 1^st and 10^th contraction, respectively, with unchanged control values of 0.6 ± 0.1 s. The application of C₆₀FAS significantly reduced this parameter: 0.91 ± 0.10 and 1.09 ± 0.10 s at the 1^st and 10^th contraction, respectively.

Figure 6.

Time of muscle contraction force return to the initial value after stimulation cessation (t₀) during 10 consecutive non-relaxation contractions (1–10) in case of application of 50 hz stimulation of 6 s duration: control, open injury and open injury+C₆₀ - control group, group of injured rats and group of rats that daily consumed orally C₆₀FAS at a dose of 1 mg/kg after the open muscle injury initiation; 5, 10 and 15 days − 5, 10 and 15 days after the open muscle injury initiation, respectively; *p < 0.05 relative to the control group; ^#p < 0.05 relative to the open injury group.

On day 10 of the experiment, this parameter was 2.07 ± 0.20 and 2.81 ± 0.20 s at the 1^st and 10^th contractions, respectively, which was several hundred percent higher than the control value. Application of C₆₀FAS decreased this parameter, which was 1.24 ± 0.10 and 1.59 ± 0.10 s at the 1^st and 10^th contractions, respectively.

On the 15^th day of the experiment, the time for the return of muscle contraction force to the initial value after stimulation cessation was 1.27 ± 0.10 and 1.69 ± 0.10 s at the 1^st and 10^th contraction, respectively, which was much higher than the control value. Application of C₆₀FAS reduced this parameter, which was 0.72 ± 0.10 (~43% relative to the open injury group) and 1.08 ± 0.10 s (~36% relative to the open injury group) at the 1^st and 10^th contraction, respectively.

3.3. Blood biochemical indicators of rats with injured muscle

CRP is produced by the liver and is a marker of inflammatory reactions in the body. The blood test for CRP shows the level of over-inflammatory processes in damaged muscle tissue, the concentration of which can increase 1000-fold in this case [35]. The increase of CRP concentration from 0.50 ± 0.04 mg/l in the control to 5.78 ± 0.20 mg/l after 5 days of the experiment is evidence of acute inflammatory processes in the damaged muscle (Figure 7). The slight decrease in its concentration to 5.23 ± 0.40 and 4.0 5 ± 0.30 mg/l on the 10^th and 15^th day of the experiment, respectively, indicates the presence of intense inflammatory processes occurring after the open muscle injury initiation throughout the experiment. The application of C₆₀FAS significantly reduced the value of this index, which was 3.95 ± 0.20, 2.07 ± 0.20, and 1.22 ± 0.10 mg/l on the 5^th, 10^th, and 15^th day of the experiment, respectively.

Figure 7.

Concentrations of CRP, creatinine and lactate in the blood plasma of rats: control, open injury and open injury+C₆₀ - control group, group of injured rats and group of rats that daily orally administered C₆₀FAS at a dose of 1 mg/kg after the open muscle injury initiation; 5, 10 and 15 days − 5, 10 and 15 days after the open muscle injury initiation, respectively; *p < 0.05 relative to the control group; ^#p < 0.05 relative to the open injury group.

The change in the concentration of creatinine, which is formed during the destruction of intramuscular structures, makes it possible to assess the degree of myocyte damage. This index increased from 50 ± 2 µM in the control to 225 ± 6, 197 ± 4, and 185 ± 5 µM on the 5^th, 10^th, and 15^th day of the experiment, respectively. Application of C₆₀FAS decreased its value to 188 ± 3, 149 ± 5, and 79 ± 2 µM on the 5^th, 10^th, and 15^th days of the experiment, respectively (Figure 7).

In control, the lactate level was 9.3 ± 0.3 mm. After the initiation of open muscle injury, its value increased to 17.4 ± 0.7, 15.8 ± 0.8, and 14.7 ± 0.6 mm on the 5^th, 10^th, and 15^th day of the experiment, respectively. The application of C₆₀FAS decreased the lactate level to 12.1 ± 0.4, 11.3 ± 0.5, and 9.7 ± 0.3 mm on the 5^th, 10^th, and 15^th day of the experiment, respectively (Figure 7).

SOD catalyzes the transformation reaction of free superoxide radicals into oxygen and hydrogen peroxide and is a key enzyme of the antioxidant system. The activity of SOD on days 5, 10, and 15 of the experiment was 8.7 ± 0.3, 7.8 ± 0.4, and 5.9 ± 0.3 Units/ml, respectively, with a control value of 1.9 ± 0.1 Units/ml. When C₆₀FAS was applied, these values decreased significantly and were 7.1 ± 0.3, 4.4 ± 0.2, and 2.9 ± 0.2 Units/ml on the 5^th, 10^th, and 15^th day of the experiment, respectively (Figure 8).

Figure 8.

Indices of pro- and antioxidant balance (SOD, CAT and GSH) in the blood plasma of rats: control, open injury and open injury+C₆₀ - control group, group of injured rats and group of rats that daily consumed orally C₆₀FAS at a dose of 1 mg/kg after the open muscle injury initiation; 5, 10 and 15 days − 5, 10 and 15 days after the open muscle injury initiation, respectively; *p < 0.05 relative to the control group; ^#p < 0.05 relative to the open injury group.

CAT is a ferment that catalyzes the decomposition of hydrogen peroxide formed in the process of biological oxidation into water and molecular oxygen. CAT activity after the open muscle injury initiation increased from 0.8 ± 0.1 mm/min in the control to 5.4 ± 0.4, 4.9 ± 0.4, and 3.4 ± 0.2 mm/min on days 5, 10 and 15 of the experiment, respectively. Application of C₆₀FAS reduced this index to 4.5 ± 0.4, 2.5 ± 0.2, and 1.5 ± 0.1 mm/min on the 5^th, 10^th, and 15^th day of the experiment, respectively (Figure 8).

Cellular mechanisms of antioxidant defense are also associated with the functioning of a powerful glutathione link [36]. The protective functions of GSH under oxidative stress are determined by its ability to catalyze the cleavage of hydrogen peroxide and fatty acid hydroperoxides. After the initiation of open muscle injury, the GSH concentration was 10.7 ± 1.0, 9.2 ± 0.7, and 7.9 ± 0.5 μM on the 5^th, 10^th and 15^th day of the experiment, respectively, while the control was 1.9 ± 0.2 μM. After the application of C₆₀FAS, these values were 8.9 ± 0.5, 6.8 ± 0.6, and 3.7 ± 0.4 μM on the 5^th, 10^th and 15^th day of the experiment, respectively (Figure 8).

3.4. Histopathological analysis of the injured muscles

The muscle gastrocnemius of the control group has a normal histological structure. Muscle fibers are grouped in bundles, which are separated by thin layers of connective tissue. The connective tissue contains ground substance and collagen fibers (Figure 9(a)).

Figure 9.

Histological images of the muscle gastrocnemius: a – control group; b – open muscle injury; c – open muscle injury+C₆₀. Hematoxylin and picrofuchsin staining by van Gieson. Scale bars −25 μm.

In animals with muscle injury, partially destroyed fibers are observed on the 15^th day after initiation of the open muscle injury. Some of them are slightly altered, hypochromatic, with signs of edema, and hyperchromatic, with signs of destruction. Muscle fibers decrease in diameter, and their transverse striation is disrupted. The volume of connective tissue increases significantly. Fibrosis is observed. The content of the ground substance of connective tissue is also increased (Figure 9(b)).

In animals with muscle injury, that received C₆₀FAS, the degree of pathohistological changes in the muscle gastrocnemius is lower: most of the fibers are slightly altered and some of them are hypochromatic, with the signs of edema. The connective tissue is increased, but less than in the previous experimental group. The increase in connective tissue occurred mainly due to the ground substance. Fibrosis is weakly expressed (Figure 9(c)).

4. Discussion

Since the biomedical applications of nanoparticles are highly dependent on their size [37], we performed a structural study of C₆₀FAS by AFM technique. It turned out that the studied C₆₀FAS is a polydisperse fluid, containing large C₆₀ fullerene aggregates with a size of ~ 20–120 nm, and also individual C₆₀ molecules (~0.7 nm) and C₆₀ fullerene nanoaggregates with a size of ~ 1.4–2.8 nm, which is in good agreement with theoretical data [28]. Note that the zeta potential value of the resulting C₆₀FAS was −25 ± 2 mV [22]. A high negative charge of the colloidal particles indicates a low degree of aggregation over time. In summary, the detected predominantly nanoformation of the C₆₀ fullerenes detected in an aqueous solution indicates their suitability for in vivo tests.

The dynamics of the beginning of the muscle contractile process are determined by subtle mechanisms of interaction between motoneuron pools with subsequent activation of the interaction between actin and myosin filaments. It is the stiffness of the muscle that determines its force of contraction. In other words, the dependence on “stimulation-contraction force” in the regeneration of damaged muscle is related to the level of fibrosis. Even small changes in the structure of passive elastic properties of muscle fibers lead to changes in the time of reaching the maximum muscle force response [13]. Note that the difference in the levels of the described positive effects of C₆₀FAS at the early and late stages of muscle gastrocnemius regeneration may be because, at the early stage (5 days after the initiation of muscle injury), the post-traumatic inflammatory reaction with progressive necrosis of damaged muscle fibers is intensively occurring in muscle tissue [38]. Further use of C₆₀FAS promotes the inhibition of muscle fibrosis, which is consistent with the results [20] on the use of antioxidants against fibrosis formation in active muscle.

The time of retention of the maximum force response of the muscle or the stationary state of an active muscle is a temporary section of its force-contractile activity without a significant deviation to one side or the other during its activation. This parameter indicates the level of force production of the muscle, which corresponds to the physiological state of the neuromuscular preparation at a given time [39] and is an indicator of the course of repair processes in it. As can be seen, the effect of water-soluble C₆₀ fullerenes on this index has a multifactorial character, in particular, the reduction of muscle stiffness and, as a consequence, fibrosis due to their antioxidant properties [16,20] and suppression of the course of the inflammatory process in closed muscle injuries [40].

The change in the time taken for the muscle contraction force to return to its initial value after stimulation cessation is directly influenced by the increase in the stiffness components of the active muscle [25]. While the results of the first two tests (on the 5^th and 10^th day of the experiment) can be explained by the suppression of inflammatory processes by the antioxidant properties of C₆₀ fullerenes, on the 15^th day of the experiment inflammatory processes do not play a key role in the increase of muscle stiffness [11,41]. The results obtained suggest a potential relationship between C₆₀ fullerenes and the reduction of fibrosis and, as a consequence, muscle stiffness in the process of its post-traumatic repair. The interaction of different cell types during muscle injury repair is supported by numerous factors including proteolytic enzymes, growth factors, angiogenic factors, and fibrogenic cytokines, which together stimulate connective tissue formation. However, there are still many unknown initiators and fibrotic pathway participants, and significant differences between tissue systems. At the same time, experimental evidence suggests that post-traumatic fibrosis is associated with redox imbalance [42], and NADPH oxidase may be the most important link between redox imbalance, free radical production, and fibrosis [43]. It has also been shown that maintaining redox balance may be an effective therapeutic strategy for fibrotic disorders [20,44], and that reducing muscle stiffness may influence its post-traumatic biomechanics.

In summary, many studies have addressed the problem of finding effective therapeutic methods to reduce fibrosis formation. For example, analysis of quantitative muscle stiffness using shear wave elastography shows that fibrosis is reduced in a porcine model of volumetric muscle loss injury by applying nintedanib as an antifibrotic agent [45]. Losartan administration reduces fibrosis but hinders the functional recovery of rat muscle after injury [46]. Finally, quercetin attenuates fibrosis in human skeletal muscle [20].

The analysis of blood biochemical parameters at muscle injury and its further post-traumatic recovery provides an opportunity to evaluate the effectiveness of the applied therapeutic drug. In the present study, we used biochemical indices of inflammation levels (CRP) and fatigue process development (creatinine and lactate), which serve as markers of physiological disturbances in active muscle due to injury. It was found that the application of C₆₀FAS significantly decreased the CRP value and the creatinine fraction, indicating the promise of using antioxidants in the therapy of muscle inflammation [47].

In active muscle, many metabolic processes take place in anaerobic conditions, so it uses a large number of mitochondrial ferments and, as a result, it accumulates a large amount of lactate, which does not have time to be oxidized during its prolonged stimulation. Thus, the proposed antioxidant therapy led to an increase in lactate oxidation, and at the end of the experiment, its concentration approached the control.

Pathological inflammatory processes that occur immediately following muscle injury are a source of free oxygen radicals and contribute to the intensification of lipid peroxidation processes [48]. Therefore, in the present study, we evaluated changes in the blood of experimental animals in biochemical parameters – markers of peroxidation and oxidative stress (SOD, CAT, and GSH) during exposure to C₆₀FAS. It was shown that the long-term application of water-soluble C₆₀ fullerenes, as powerful antioxidants, helps mitigate oxidative processes in damaged muscles. This was achieved by maintaining the balance between prooxidants and the antioxidant defense system, thereby preventing the negative effect of free radicals on cellular and subcellular structures during the post-traumatic recovery of rat skeletal muscles.

Finally, the biomechanical and biochemical results obtained were confirmed by the histopathological analysis: in animals, that received C₆₀FAS (open muscle injury+C₆₀ group), the degree of pathohistological changes in the muscle gastrocnemius was less than in the open muscle injury group; specifically, fibrosis was weakly expressed.

5. Conclusions

Based on the above biomechanical and biochemical data, we can conclude that the application of nanoantioxidants C₆₀ fullerenes within 15 days (daily oral dose 1 mg/kg) after the onset of open injury of the muscle gastrocnemius reduces its stiffness and, as a consequence, inhibits the development of muscle fibrosis, as well as reduces the intensity of inflammatory processes in the body. Histopathological data confirm these results. By influencing the activity of endogenous antioxidants, C₆₀ fullerenes can suppress the formation of significant destruction in injured muscles. Therefore, they can be considered potent nanoagents for the correction of muscle damage that occurs during its physiological repair, which requires further clinical studies.

Funding Statement

This research was supported by the National Research Foundation of Ukraine [2022.01/0004].

Article highlights

• A C₆₀ fullerene aqueous solution (C₆₀FAS) contains mainly individual C₆₀ molecules and their nanoaggregates

• C₆₀FAS improves biomechanical parameters of rat muscle gastrocnemius contraction after open injury

• C₆₀FAS attenuates fibrosis in rat muscle gastrocnemius after open injury

• C₆₀FAS improves rat blood biochemical indicators after open muscle injury

• C₆₀FAS reduces in histopathological features of rat muscle gastrocnemius damages

CRediT authorship contribution statement

Yuriy Prylutskyy: Conceptualization, Writing – review & editing, Supervision. Dmytro Nozdrenko: Investigation, Methodology, Writing – review & editing. Olexandr Motuziuk: Investigation, Methodology, Formal analysis. Svitlana Prylutska: Investigation, Methodology. Igor Vareniuk: Investigation. Natalia Nurishchenko: Investigation. Daria Franskevych: Investigation. Vasil Soroсa: Investigation. Kateryna Bogutska: Formal analysis, Writing – original draft preparation. Uwe Ritter: Writing – review and editing, Project administration.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethics Disclosure

All of the in vivo experiments were approved by the Bioethics Committee of the ESC “Institute of Biology and Medicine” of Taras Shevchenko National University of Kyiv (protocol No. 9 dated September 4, 2023) in accordance with the norms of biomedical ethics in accordance with the Law of Ukraine No. 3447-IV of 21.02.2006, Kyiv, “On the Protection of Animals from Cruelty” as well as the European Union Directive (2010/63/EU) “On the protection of animals used for scientific purposes”.

Supplemental material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17435889.2025.2461988