Combined Teriparatide and Exercise Effects on Diabetic Skeletal Muscle Ultrastructure

Abstract

Background/Aim

The effects of combined teriparatide (TPTD) administration and exercise therapy on diabetic skeletal muscle remain unclear. This study investigated the effects of combined TPTD and treadmill exercise on skeletal muscle and mitochondrial ultrastructure in Akita mice, a model of type 1 diabetes.

Materials and Methods

Male C57BL/6J-Ins2<Akita>/J mice were randomly assigned to four groups: control, exercise therapy (EX), TPTD administration, and combined exercise plus TPTD (EXTP) (n=6 each). TPTD (80 μg/kg) was administered subcutaneously three times weekly for six weeks, and treadmill exercise was performed five days/week. Body weight, fasting blood glucose, grip strength, wet muscle weight, muscle fibre cross-sectional area (CSA), mitochondrial and sarcomere ultrastructure, and gene expression were evaluated.

Results

Baseline fasting blood glucose levels were higher in the EXTP group than in the EX and TPTD groups. The EXTP group showed a greater reduction in fasting blood glucose than the other groups and higher wet muscle weight normalised to tibial length than the control group. A-band length and A-band length normalised to tibial length were also higher in the EXTP group. Electron microscopy showed larger subsarcolemmal and intermyofibrillar mitochondrial CSA in the EXTP group. Body weight-normalised grip strength, muscle fibre CSA, myofibril CSA, mitochondrial number, aspect ratio, roundness, and most gene expression levels did not differ among groups.

Conclusion

Combined TPTD administration and exercise therapy may induce structural changes in skeletal muscle and mitochondria under diabetic conditions and may contribute to maintenance or remodelling of skeletal muscle ultrastructure and mitochondrial morphology.

Introduction

The prevalence of sarcopenia, a syndrome characterised by the age-related loss of skeletal muscle mass and a decline in muscle strength (1), increases with age (2). Therefore, maintaining and improving skeletal muscle function are important for extending the healthy life expectancy of older adults.

In recent years, diabetes mellitus has attracted attention not only as a cause of reduced bone mineral density but also as an important factor associated with skeletal muscle disorders, including skeletal muscle atrophy and impaired muscle function (3, 4). Diabetic skeletal muscle impairment is believed to be caused by multiple factors, including metabolic abnormalities, chronic inflammation, oxidative stress, and mitochondrial dysfunction (5, 6). Moreover, skeletal muscle abnormalities are not limited to functional impairment but can also be observed as ultrastructural changes at the electron microscopic level. Mitochondria play a central role in skeletal muscle energy metabolism, and mitochondrial dysfunction is closely associated with impaired muscle function. In aged and diabetic skeletal muscles, morphological abnormalities such as mitochondrial fragmentation, swelling, and disruption of cristae structure have been reported, in addition to reduced mitochondrial respiratory capacity and altered expression of molecules related to mitochondrial biogenesis (7, 8).

Interventions for skeletal muscle disorders, including sarcopenia, emphasise the importance of exercise therapy, particularly resistance exercise, and nutritional strategies, such as those focusing on protein intake; however, no established pharmacological treatment is currently available (9-11). In this context, teriparatide (TPTD), a parathyroid hormone preparation used for the treatment of osteoporosis, may affect skeletal muscles, in addition to its effects on bone metabolism. Activation of parathyroid hormone/parathyroid hormone type 1 receptor (PTH/PTH1R) signalling has been reported to promote myogenic differentiation, suggesting that this pathway may affect skeletal muscles (12). However, the effects of TPTD on skeletal muscles and mitochondria in diabetes-associated skeletal muscle impairment have not yet been completely investigated.

Exercise therapy is known to improve skeletal muscle and mitochondrial function, and its combination with pharmacological therapy may produce synergistic effects. We have previously reported that a combination of TPTD administration and treadmill exercise promotes bone formation and improves cortical bone strength (13); however, its effects on skeletal muscles and mitochondria remain unclear. Therefore, the present study aimed to investigate the effects of combined TPTD administration and exercise therapy on skeletal muscles and mitochondria in Akita mice, a mouse model of type 1 diabetes. Clarifying these effects may provide new insight into the structural response of diabetic skeletal muscle to combined pharmacological and exercise-based intervention, particularly from the perspective of mitochondrial and sarcomere ultrastructure.

Materials and Methods

Animals. Male C57BL/6J-Ins2<Akita>/J mice (Japan SLC, Shizuoka, Japan), a mouse model of type 1 diabetes, were used in this study. All animals were housed under controlled conditions at 23±2°C and 40±20% humidity with a 12-h light/dark cycle and were allowed free access to standard chow and water. The mice were acclimatised until 14 weeks of age. This study was approved by the Animal Research Committee of the Akita University (approval no. a-1-0542).

Experimental design and group allocation. At 14 weeks of age, the mice were randomly allocated to the following four groups: control (control, n=6), exercise therapy (EX, n=6), TPTD administration (TPTD, n=6), and combined exercise therapy and TPTD administration (EXTP, n=6). The intervention period was six weeks, and various assessments were performed after euthanasia at 20 weeks of age.

Exercise therapy protocol. In the EX and EXTP groups, treadmill exercise were performed using a treadmill apparatus (MK-680; Muromachi Kikai, Tokyo, Japan) according to a previously reported treadmill exercise protocol in diabetic mice (14). The running speed was set to 10 m/min. The exercise duration was progressively increased from 10 min on day 1 to 20 min on day 2. From day 6 onwards, the mice were exercised for 60 min/day, 5 days/week, for six weeks.

TPTD administration. TPTD (h-PTH; Asahi Kasei Pharma Corp., Tokyo, Japan) was administered subcutaneously at a dose of 80 μg/kg three times per week for six weeks (15). The control and EX groups received the same volume of vehicle.

Body weight and fasting blood glucose measurements. Body weight was measured twice: at the start and end of the intervention period under the same conditions and at the same time in the morning. After a 6-h fast, blood samples were collected from the tail vein, and blood glucose levels were measured using a Glutest Mint II device (Sanwa Kagaku Kenkyusho Co., Ltd., Nagoya, Japan). Measurements were performed before and after the intervention at the same time of the day.

Grip strength measurement. After the 6-week intervention period, grip strength was measured using a digital force gauge (DST-2N; Imada Co. Ltd., Japan). Forelimb grip strength was measured while the mouse grasped the mesh with both forelimbs, and four-limb grip strength was measured while the mouse grasped the mesh with all four limbs. Forelimb and four-limb grip strengths were measured five times, and the mean value was used for analysis. Sufficient rest was provided between the measurements.

Tissue collection. Under anaesthesia induced by intraperitoneal administration of a three-drug mixture of medetomidine hydrochloride (0.3 mg/kg; Domitor; Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), midazolam hydrochloride (4.0 mg/kg; Dormicum; Astellas Pharma Inc., Tokyo, Japan), and butorphanol tartrate (5.0 mg/kg; Vetorphale; Meiji Seika Pharma Co., Ltd., Tokyo, Japan), the right hindlimb was ligated proximally and perfusion fixation was performed, according to previous reports on hindlimb blood flow occlusion models (16). The tibialis anterior muscles were harvested. Left hindlimb muscles were used for electron microscopic analysis, whereas right hindlimb muscles not subjected to perfusion fixation, including the tibialis anterior, gastrocnemius, extensor digitorum longus, and soleus muscles, were used for wet muscle weight measurement.

Wet muscle weight measurement. The wet weight of each muscle was measured using an electronic balance (TE214S; Sartorius AG, Göttingen, Germany) and the total weight of the four muscles was defined as the lower leg wet muscle weight. Tibial length was measured using calipers (Shinwa Rules Co., Ltd., Niigata, Japan), and wet muscle weight was normalised to tibial length. Wet muscle weight was also normalised to body weight measured at the end of the intervention period and expressed as a percentage.

Muscle fibre cross-sectional area (CSA). After fixation, muscle tissues were embedded in frozen embedding medium, and serial sections were prepared. The sections were stained with toluidine blue solution, rinsed with distilled water, dehydrated, cleared, and mounted. 10 randomly selected fields per animal were imaged under a light microscope (Axioscope 5; Carl Zeiss, Jena, Germany). The acquired images were analysed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). The CSA of at least 50 muscle fibres per animal was measured, and the mean CSA for each animal was calculated and used for statistical analysis (17).

Electron microscopy. After fixation, the tibialis anterior muscle samples were embedded in Quetol 812 epoxy resin (Nisshin EM Co., Ltd., Tokyo, Japan), and ultrathin sections of approximately 70 nm were prepared. Two types of ultrathin sections, transverse and longitudinal sections of muscle fibres, were prepared. The samples were observed using a transmission electron microscope (HT7700; Hitachi, Tokyo, Japan) at a magnification of 3,000×. For each animal, 10 fields were randomly selected from both longitudinal and transverse sections. Mitochondria were classified as intermyofibrillar or subsarcolemmal and 50 mitochondria in each compartment were analysed per animal. The parameters analysed were the area, number, roundness, and aspect ratio. The aspect ratio was calculated as the long-axis diameter divided by the short-axis diameter, with the long axis defined as the maximum mitochondrial diameter and the short axis as the maximum diameter perpendicular to the long axis. Sarcomere length and A-band length were measured in 50 myofibrils from the longitudinal sections of each animal. All measurements were averaged for each animal and used for statistical analyses.

Gene expression analysis. Total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was synthesised using the First-Strand cDNA Synthesis Kit (Cytiva, Tokyo, Japan) according to the manufacturer’s instructions. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed using TaqMan Fast Advanced master mix (Thermo Fisher Scientific) and TaqMan Gene Expression Assays on a LightCycler 480 Instrument II (Roche Diagnostics GmbH, Mannheim, Germany). Rplp0 (Mm00725448_s1) and Hprt1 (Mm01545399_m1) were used as endogenous control genes, and relative expression levels were calculated using the ΔΔCt method. The target genes encoded Myogenin (Myogenin; Mm00446194_m1), Atrogin-1/F-box protein 32 (Atrogin-1; Mm01207878_m1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a; Mm00447183_m1), and parathyroid hormone 1 receptor (PTH1R; Mm00441046_m1).

Statistical analysis. Data are presented as mean±standard deviation. Normality was assessed using the Shapiro-Wilk test. Values more than two standard deviations from the overall mean were excluded from the corresponding analyses. Group comparisons were performed using one-way analysis of variance, followed by Tukey’s multiple comparison test. A p-value of <0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Boston, MA, USA).

Results

Body weight change. Before treatment, body weight differed significantly among the groups (p=0.038), with the EXTP group showing a significantly lower body weight than the EX group (p=0.029). Body weight changes also differed significantly among the groups (p=0.026), with the EXTP group showing a significantly greater increase than the EX group (p=0.015) (Table I).

Table I. Changes in body weight, decrease in blood glucose levels, and grip strength.

Data are presented as mean±standard deviation. *One animal in the EX group was excluded from all blood glucose analyses because its pre-treatment blood glucose value met the exclusion criterion described in the Statistical Analysis section (EX, n=5 for blood glucose analyses). EX: Exercise therapy group; TPTD: teriparatide administration group; EXTP: combined exercise therapy and TPTD administration group. ^ap<0.05 vs. EX group; ^bp<0.05 vs. TPTD group; ^cp<0.05 vs. Control group using Tukey’s multiple comparison test.

Fasting blood glucose levels and changes in blood glucose levels. Before treatment, fasting blood glucose levels differed significantly among the groups (p=0.019), with significantly higher levels in the EXTP group than in the EX (p=0.046) and TPTD groups (p=0.029). After treatment, fasting blood glucose levels did not differ significantly among the groups. The reduction in blood glucose levels differed significantly among the groups (p=0.003), and the EXTP group showed a significantly greater reduction than the control (p=0.016), EX (p=0.006), and TPTD groups (p=0.009) (Table I). One value in the EX group was excluded from the analysis of changes in blood glucose levels according to the exclusion criterion described in the Statistical Analysis section.

Grip strength. Body-weight-normalised forelimb and four-limb grip strengths did not differ significantly between the groups (Table I).

Wet muscle weight. The wet muscle weight normalised to tibial length differed significantly among the groups (p=0.013), with the EXTP group showing a significantly higher value than the control group (p=0.008). In contrast, tibial length, wet muscle weight, and wet muscle weight normalised to body weight did not differ significantly among the groups (Table II).

Table II. Wet muscle weight.

Data are presented as mean±standard deviation. Wet muscle weight was normalised to body weight measured at the end of the intervention period and expressed as a percentage. For wet muscle weight analyses, the sample sizes were control, n=5; EX, n=5; TPTD, n=4; and EXTP, n=6 after application of the exclusion criterion. For the analysis of wet muscle weight normalised to tibial length, the sample sizes were control, n=5; EX, n=4; TPTD, n=4; and EXTP, n=6 because tibial length was unavailable for one mouse in the EX group. EX: Exercise therapy group; TPTD: teriparatide administration group; EXTP: combined exercise therapy and TPTD administration group. ^ap<0.05 vs. Control group using Tukey’s multiple comparison test.

Muscle fibre CSA. Representative toluidine blue-stained histological images are shown in Figure 1A. The muscle fibre CSA did not differ significantly between the groups (Figure 1B).

Figure 1.

Histological evaluation of muscle fibre cross-sectional area. (A) Representative toluidine blue-stained images of tibialis anterior muscle fibres. (B) Quantitative analysis of muscle fibre cross-sectional area (CSA). Control: Control group (n=6), EX: exercise therapy group (n=6); TP: teriparatide administration group (n=6); EXTP: combined exercise therapy and TPTD administration group (n=6). Scale bars=100 μm. Data are presented as mean±standard deviation.

Myofibril CSA. Representative electron microscopy images are shown in Figure 2A. The myofibril CSA did not differ significantly between the groups (Figure 2B).

Figure 2.

Ultrastructural evaluation of myofibril cross-sectional area. (A) Representative transmission electron microscopy images of myofibrils. (B) Quantitative analysis of myofibril cross-sectional area (CSA). Control: Control group (n=6); EX: exercise therapy group (n=6); TP: teriparatide administration group (n=6); EXTP: combined exercise therapy and TPTD administration group (n=6). Scale bars=1 μm. Data are presented as mean±standard deviation.

A-Band length. Representative electron microscopy images of the sarcomere structure and A-band are shown in Figure 3A. The A-band length differed significantly between the groups, with the EXTP group showing a significantly higher value than the EX group (p=0.039) (Figure 3B). The A-band length normalised to the tibial length was also significantly higher in the EXTP group than in the control group (p=0.022) (Figure 3C).

Figure 3.

Ultrastructural evaluation of A-band length. (A) Representative transmission electron microscopy images. (B) Quantitative analysis of A-band length and (C) quantitative analysis of A-band length normalized to tibia length. Control: Control group (n=6); EX: exercise therapy group (n=6); TP: teriparatide administration group (n=6); EXTP: combined exercise therapy and TPTD administration group (n=6). Scale bars=1 μm. Data are presented as mean±standard deviation. *p<0.05 using Tukey’s multiple comparison test.

Mitochondrial analysis

Subsarcolemmal mitochondria. Representative electron microscopy images of subsarcolemmal mitochondria are shown in Figure 4A. The cross-sectional area of the subsarcolemmal mitochondria was larger in the EXTP group than in the other groups. Quantitative analysis showed that the mitochondrial area in the EXTP group was significantly greater than that in the control group (p=0.023) (Figure 4B). In contrast, the mitochondrial number, aspect ratio, and roundness did not differ significantly among the groups (Figure 4C-E).

Figure 4.

Ultrastructural analysis of subsarcolemmal mitochondria. (A) Representative transmission electron microscopy images of subsarcolemmal mitochondria. (B-E) Quantitative analysis of the subsarcolemmal mitochondrial (B) area, (C) number, (D) aspect ratio, and (E) roundness. EX: exercise therapy group (n=6); TP: teriparatide administration group (n=6); EXTP: combined exercise therapy and TPTD administration group (n=6). Scale bars=1 μm. Data are presented as mean±standard deviation. *p<0.05 using Tukey’s multiple comparison test.

Intermyofibrillar mitochondria. Representative electron microscopy images of the intermyofibrillar mitochondria are shown in Figure 5A. The CSA of the intermyofibrillar mitochondria in the EXTP group was larger than that in the other groups. Quantitative analysis showed that the mitochondrial area in the EXTP group was significantly higher than that in the control (p=0.001), EX (p=0.026), and TPTD groups (p=0.006) (Figure 5B). In contrast, the mitochondrial number, aspect ratio, and roundness did not differ significantly among the groups (Figure 5C-E).

Figure 5.

Ultrastructural analysis of intermyofibrillar mitochondria. (A) Representative transmission electron microscopy images of intermyofibrillar mitochondria. (B-E) Quantitative analysis of the intermyofibrillar mitochondrial (B) area, (C) number, (D) aspect ratio, and (E) roundness. Control: Control group; EX: exercise therapy group (n=6); TP: teriparatide administration group (n=6); EXTP: combined exercise therapy and TPTD administration group (n=6). Data are presented as mean±standard deviation. Scale bars=1 μm. *p<0.05 and **p<0.01 using Tukey’s multiple comparison test.

Gene expression. Myogenin expression was significantly higher in the EX group than in the control group (p=0.011) and TPTD group (p=0.023) (Figure 6A). In contrast, the expression levels of Atrogin1, PGC1a, and PTH1R did not differ significantly among the groups (Figure 6B-D).

Figure 6.

Gene expression analysis in skeletal muscle. Relative mRNA expression levels of (A) Myogenin, (B) Atrogin-1, (C) PGC1a, and (D) PTH1R. PGC1a: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PTH1R: parathyroid hormone/parathyroid hormone type 1 receptor; EX: exercise therapy group (n=6); TP: teriparatide administration group (n=6); EXTP: combined exercise therapy and TPTD administration group (n=6). Data are presented as mean±standard deviation. *p<0.05 using Tukey’s multiple comparison test.

Discussion

In this study, we investigated the effects of combined TPTD administration and treadmill exercise on skeletal muscles and mitochondria in Akita mice, a mouse model of type 1 diabetes. Although baseline fasting blood glucose levels differed significantly among the groups, with higher levels in the EXTP group than in the EX and TPTD groups, the EXTP group showed a significantly greater reduction in fasting blood glucose levels than the other groups. The EXTP group also showed increased wet muscle weight normalised to tibial length, A-band length, A-band length normalised to tibial length, and CSA of both subsarcolemmal and intermyofibrillar mitochondria. In contrast, body weight-normalised grip strength, muscle fibre CSA, myofibril CSA, mitochondrial number, aspect ratio, roundness, and expression levels of most genes did not differ significantly between the groups. These results suggest that combined TPTD administration and exercise therapy may affect skeletal muscle ultrastructure and mitochondrial morphology under diabetic conditions, although they do not produce clear improvements in muscle strength or muscle fibre hypertrophy.

The novelty of this study lies in its detailed evaluation of the effects of combined TPTD administration and exercise therapy on skeletal muscle and mitochondrial ultrastructure in a mouse model of type 1 diabetes. Although previous studies have reported the effects of combined TPTD and exercise therapy on bone and muscle mass (13), few studies have focused on mitochondrial morphology and sarcomere structure under diabetic conditions. In the present study, the EXTP group showed increased wet muscle weight normalised to tibial length as well as increased A-band length and mitochondrial area. These findings suggest that combination therapy may be involved in not only inducing muscle mass changes but also structural alterations at the myofibrillar and mitochondrial levels in diabetes-associated skeletal muscle impairment.

The EXTP group showed a significantly greater reduction in fasting blood glucose levels than the other groups. However, baseline fasting blood glucose levels were significantly higher in the EXTP group than in the EX and TPTD groups. Therefore, the greater reduction in blood glucose levels observed in the EXTP group should be interpreted cautiously, as baseline differences may have influenced the magnitude of the observed change. Under diabetic conditions, hyperglycaemia-related metabolic abnormalities, oxidative stress, inflammation, and mitochondrial dysfunction are believed to contribute to skeletal muscle atrophy and impaired muscle function (5, 6). Although the morphological changes in skeletal muscle and mitochondria observed in this study may be associated with changes in glucose metabolism, the present findings do not establish a causal relationship between the reduction in blood glucose levels and these structural changes. Exercise therapy influences skeletal muscle glucose uptake and mitochondrial function (18, 19), but whether TPTD modifies these exercise-related metabolic effects remains unclear. Furthermore, as insulin levels, glucose uptake capacity, oxidative stress markers, inflammatory cytokines, and mitochondrial function were not directly evaluated in this study, further investigations are required to clarify these relationships.

Wet muscle weight normalised to tibial length was significantly higher in the EXTP group than in the control group; however, wet muscle weight, muscle fibre CSA, myofibril CSA, and body-weight-normalised grip strength did not differ significantly among the groups. These findings suggest that the combined therapy may have exerted some influence on skeletal muscle mass, although the changes were not sufficient to detect clear muscle fibre hypertrophy or functional improvement. Wet muscle weight can be affected not only by muscle fibre size, but also by intramuscular water content and interstitial components; therefore, it does not necessarily correspond directly to muscle fibre CSA. In addition, grip strength is influenced by neuromuscular coordination and peripheral neuropathy, which may explain why morphological changes were not reflected as functional improvements within a short intervention period.

In this study, the A-band length and A-band length normalised to the tibial length increased in the EXTP group. The A-band contains myosin filaments and is an important component of the sarcomere structure (20). Therefore, these findings suggest that the combination therapy may be involved in maintaining or remodelling the myofibrillar structure under diabetic conditions. However, the effect of changes in the A-band length on contractile function remains unclear and should be investigated in future studies.

Mitochondrial analysis revealed a significant increase in the CSA of both the subsarcolemmal and intermyofibrillar mitochondria in the EXTP group. In contrast, the mitochondrial number and shape indices, including aspect ratio and roundness, did not differ significantly among the groups. These results suggest that combination therapy may affect the size of individual mitochondria rather than the mitochondrial number or overall mitochondrial shape. Recent evidence indicates that skeletal muscle mitochondrial characteristics in type 1 diabetes are closely associated with modifiable factors, including cardiorespiratory fitness and glycaemic control (21). In addition, exercise has been shown to induce mitochondrial remodelling through dynamic processes involving mitochondrial biogenesis, mitophagy, fission, and fusion (22). These recent findings support the possibility that the mitochondrial morphological changes observed in the present study may reflect adaptive remodelling associated with exercise and improved glucose metabolism. However, an increase in mitochondrial area based on two-dimensional electron microscopy does not necessarily reflect an increase in total mitochondrial content or functional improvement. Therefore, the present findings should be interpreted cautiously as evidence of mitochondrial structural changes, and their functional significance requires further investigation.

In addition, Myogenin expression increased in the EX group but not in the EXTP group. This finding suggests that the activation of myogenic differentiation signalling induced by exercise stimulation may be differentially regulated by concomitant TPTD administration. However, gene expression changes are time dependent, and a single time-point assessment, as performed in this study, may not fully capture these dynamics. Future studies should include time-course analyses and evaluation of protein expression levels.

Study limitations. First, the sample size was small and the statistical power may have been insufficient. Second, only male mice were included; therefore, sex-related differences were not examined. Third, because this study used a mouse model of type 1 diabetes, our findings did not directly reflect age-related sarcopenia. Fourth, as a wild type control group was not included, the extent of diabetes-induced skeletal muscle alterations could not be directly evaluated. Fifth, baseline fasting blood glucose levels differed among the groups, which may have influenced the magnitude of the changes in blood glucose levels observed during the intervention.

Conclusion

In this study, combined TPTD administration and exercise therapy in Akita mice, a model of type 1 diabetes, reduced blood glucose levels, increased wet muscle weight normalised to tibial length, elongated the A-band, and enlarged the mitochondrial area. These results suggest that the combined therapy may be involved in structural changes in the skeletal muscle and mitochondria under diabetic conditions. However, the improvements in muscle function and the underlying molecular mechanisms remain unclear and require further investigation.

Conflicts of Interest

The Authors declare that they have no competing financial interests or personal relationships that may have influenced the work reported in this study.

Authors’ Contributions

Yasuhito Asaka: Conceptualisation, Methodology, Investigation, Formal analysis, Data curation, Visualisation, and Writing of the original draft. Yuji Kasukawa: Conceptualization, Methodology, Supervision, Writing – review & editing. Koji Nozaka: Methodology, Validation, Writing – review & editing. Hiroyuki Tsuchie: Methodology, Validation, Writing – review & editing. Yuichi Ono: Investigation, Validation, Writing – review & editing. Manabu Akagawa: Investigation, Validation, Writing – review & editing. Yusuke Yuasa: Investigation, Validation, Writing – review & editing. Yo Morishita: Formal analysis, Investigation, Writing – review & editing. Manami Nakanishi: Formal analysis, Investigation, Writing – review & editing. Yuki Ishigaki: Data curation, Investigation, Writing – review & editing. Akari Hisada: Formal analysis, data curation, Investigation, Writing, reviewing, and editing. Hiroki Fujita: Conceptualization, Methodology, Supervision, Writing – review & editing. Naohisa Miyakoshi: Conceptualization, Methodology, Supervision, Writing – review & editing.

Acknowledgements

The Authors thank all the members of the Department of Orthopedic Surgery, Akita University Graduate School of Medicine, for their support and helpful discussions. The authors are grateful to Ms. Midorikawa and Ms. Kato for their technical assistance with the experiments.

Artificial Intelligence (AI) Disclosure

During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning-based image enhancement tools.