Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: Comparison with changes due to jump exercise.

OBJECTIVES: The preventive effects of Korean red ginseng (KRG) on bone loss and microarchitectural deterioration have been extensively studied in animal models. However, few results have been reported for the effects of KRG on the trabecular microarchitecture as compared to changes resulting from physiological stimuli such as exercise load. We compared the effects of KRG and jump exercise on improvements in trabecular microarchitecture and strength of the distal femoral metaphysis in rats. METHODS AND MATERIALS: Eleven-week-old male Wistar rats were divided into sedentary (CON), KRG-administered (KRG), and jump-exercised (JUM) groups. Rats were orally administered KRG extract (200 mg/kg body weight/day) once a day for 6 weeks. The jump exercise protocol comprised 10 jumps/day, 5 days/week at a jump height of 40 cm. We used microcomputed tomography to assess the microarchitecture, volumetric bone mineral density (vBMD), and fracture load as predicted by finite element analysis at the right distal femoral metaphysis. The left femur was used for the quantitative bone histomorphometry measurements.
RESULTS: Although KRG produced significantly higher trabecular bone volume (BV/TV) than CON, BV/TV was even higher in JUM than in KRG, and differences in vBMD and fracture load were only significant between JUM and CON. In terms of trabecular microarchitecture, KRG increased trabecular number and connectivity, whereas the JUM group showed increased trabecular thickness. Bone resorption showed significant decrease by JUM and KRG group. In contrast, bone formation showed significant increase by JUM group.
CONCLUSIONS: These data show that KRG has weak but significant positive effects on bone mass and suggest that the effects on trabecular microarchitecture differ from those of jump exercise. The effects of combined KRG and jump exercise on trabecular bone mass and strength should be investigated.

Introduction

The achievement of as maximal a bone mass and bone strength as possible during growth is considered to be the best protection against age-related bone loss or osteoporosis and the subsequent risk of osteoporotic fractures [1]. Physical activity, nutrition diet, and other lifestyle factors can greatly impact bone health in an adolescent. Regular exercise and a balanced diet during childhood and adolescence, in general, is known to be the major factor affecting maximum achievement of bone mass. Thus, exercise and healthy nutrition during the early stage of life may be an effective countermeaseure to prevent bone fracture by osteoporosis and age-related osteopenia.

The ginseng plant (Panax ginseng Meyer) has been considered a medicinal plant in Asian countries including Korea, China and Japan for thousands of years. This plant is now in wide use as an alternative medicine for the treatment of disease and the promotion of health. Numerous research studies have confirmed the biological activities of ginseng, such as anti-inflammatory [2], anti-oxidative [3, 4], anti-obesity [5], anti-diabetic [6], anti-stress [7], and anti-cancer properties [8, 9]. In general, the commercially available ginseng in South Korea can be broadly categorized as fresh ginseng, white ginseng, or Korean red ginseng (KRG). KRG is made using a process of repeatedly steaming and air-drying fresh raw ginseng without peeling, while white ginseng is produced by sun-drying. KRG is known to have more pharmacological effects and stability compared with fresh and white ginseng, because of the changes to the chemical components (e.g., ginsenosides Rg2, Rg3, Rh1, and Rh2) produced in the steaming process [10]. KRG is an important component in medicine and various health supplements, providing antiaging, antioxidant, and immunomodulatory effects with a low rate of side effects [11, 12]. However, compared with the widespread research on the biological activity of KRG, studies on the responses of bone to KRG remain limited. Several recent studies have shown that KRG and ginsenoside has the ability to inhibit bone loss in the ovariectomy-induced osteoporotic rodent model [13, 14], and to inhibit bone resorption in cell cultures [15-18]. However, little information has been accumulated regarding the effects of KRG on bone microarchitecture and strength. In addition to low bone mass, deterioration of bone microarchitecture is an important determinant of bone fragility in osteoporosis. Evaluation of changes in bone microarchitecture by KRG and determination of its significance in terms of improving bone strength are therefore important.

Physical exercise has been considered suitable methods to help increase in bone mass during growth and to prevent age-related bone loss. However, not all types of exercise exert the same beneficial effects on the skeleton. Among various types of exercise in rat models, jump exercise is one of the most effective methods for increasing bone mass and strength [19-21]. In a previous study, we confirmed that jump exercise can have positive effects on trabecular bone strength via changes to the trabecular thickness (microarchitecture) of the distal femoral metaphysis in growing rats [22]. In contrast, previous research has suggested that administration of KRG extract to irradiated mice prevented loss of trabecular bone mass of the proximal tibia, primarily by significant alterations in trabecular number [23]. KRG administration and jump exercise may thus show different effects on trabecular microarchitecture. To the best of our knowledge, no previous studies have directly compared such effects between KRG administration and exercise, to assess the types and degrees of difference. The objective of this study was thus to compare the effects of exercise and KRG on trabecular bone architecture and to assess the influence of KRG as an effective strategy for improving bone strength during growth. The reason for selecting jump exercise for comparison was that structural changes to bone caused by the physiological stimulation of jump exercise are considered representative of ideal change [19-21].

Materials and methods

Ethics statement and euthanasia

This study was approved by the Animal Ethics Committee of the Kawasaki University of Medical Welfare (Permit Number: 15–005). The experiment protocol and all animal care described in this investigation were performed in accordance with the Guidelines for Animal Experiments of the Kawasaki University of Medical Welfare. Each rat was euthanized using a combination of medetomidine hydrochloride (0.15 mg/kg, Dorbene® Vet; Kyoritsu Seiyaku Corporation, Tokyo, Japan), midazolam (2 mg/kg, Sandoz, Yamagata, Japan), and butorphanol (2.5 mg/kg, Vetorphale; Meiji Seika Pharma Co., Tokyo, Japan) administered via intraperitoneal injections at the end of the experiment, and all possible efforts were made to minimize the rat’s suffering.

Animal and experimental design

Thirty male Wistar rats were used in this study. Animals were obtained from Clea Japan (Osaka, Japan) at 10 weeks old. All rats were singly housed in standard cages (20 × 33 × 14 cm) under a constant temperature of 22 ± 1°C and a 12:12 light-dark cycle. Rats were given ad libitum access to standard laboratory animal chow (MF; Oriental Yeast, Chiba, Japan) containing 1.15% calcium and 0.88% phosphorus and water. Rats were habituated to the diet and new environment for 1 week. The health and behavior of animals were monitored at least twice daily. After 7 days of acclimation, rats were randomly assigned into three groups (n = 10 each) as follows: a sedentary control group (CON); a group administered KRG (KRG); and a jump exercise group (JUM). All rats were double-labeled with subcutaneous injections of 10 mg per kg of fluorescent calcein (Dojindo, Kumamoto, Japan) at 1 and 5 days before sacrifice. Soon after euthanasia, right calf muscles were collected from each rat and immediately weighed. Excised femora from each rat were cleaned of soft tissue, then femoral length was measured using digital calipers. Right femora were stored at -40°C until analysis using micro-computed tomography (micro-CT) and left femora were fixed in 70% ethanol for histomorphometric analysis.

Exercise protocol

We applied the jump exercise protocol we have used previously and the details have been described elsewhere [22, 24–26]. Briefly, rats in the JUM group were individually placed at the bottom of a wooden box, 40 cm × 40 cm and 40 cm high. The initial height of the box was 10 cm, and this was gradually increased to 40 cm during the first week. Since rat grasped the top of the board set at 40 cm with their forelimbs and then climbed onto it, the true jump height is estimated to be 30cm to 35cm. An electric stimulus was initially provided to force them to jump and to grasp the top of one of the sides of box with the forelimbs. The rat was then returned to the floor of the box for the next jump. The jump exercise program comprised 10 times per day, 5 days per week for 6 weeks. We improved the training environment as favorable as possible so that the rat can jump without much stress. For example, each jump exercise session was performed during the dark period at the same time each day. More importantly, since the rats have been conditioned to jump voluntarily soon after the start of exercise training, the electrical stimulus was required only in the initial few days. Even when the rat has interrupted the jump, light tap on the cage wall by a technician can restart the continuous jumping.

KRG preparation and administration

The KRG extract in concentrated form used in this study was purchased commercially from the Korea Ginseng Corporation (“Cheong Kwan Jang”, root extract of 6-year-old fresh KRG; Daejeon, Republic of Korea). The KRG extract dose was selected on the basis of previously published data [27]. The KRG extract was freshly prepared by dissolving in distilled water before administration. Each rat in the KRG group was orally administered KRG 5 days/week for 6 weeks at a dose of 200 mg/kg body weight/day of KRG solution dissolved in distilled water. KRG extract contained major ginsenoside-Rb1: 5.61 mg/g, -Rb2: 2.03 mg/g, -Rc: 2.20 mg/g, -Rd: 0.39 mg/g, -Re: 1.88 mg/g, -Rf: 0.89 mg/g, -Rg1: 3.06 mg/g, -Rg2s: 0.15 mg/g, -Rg3s: 0.17 mg/g, -Rg3r: 0.08 mg/g, and other minor ginsenosides. Rats in CON and JUM groups were orally administered volumes of distilled water equal to the volume of KRG solution. Oral administration was performed using an oral sonde to ensure the correct doses.

Micro-CT scanning

The bone microarchitecture and volumetric bone mineral density (vBMD, mg/cm3) of the right femur were analyzed by Ele Scan mini micro-CT system (Nittetsu Elex, Tokyo, Japan) as previously described [22, 24–26]. Measurement conditions for micro-CT were as follows: source energy, 30 kVp; electric current, 80 μA; filter, 0.1-mm copper plate; slice thickness, 18.11 μm; matrices, 512 × 512; pixel size, 18.11 μm. The sample area at the distal femoral metaphysis were scanned in an area 2.8–3.0 mm proximal to the distal end of the femur, including the border between the distal metaphysis and growth plate (Fig 1B). A total of 300 consecutive slices (5.4 mm) were taken for analysis of trabecular bone, with the region of interest was defined as the 130 slices (2.4 mm) above the most proximal portion of the growth plate (Fig 1C). After micro-CT scanning, the original image data were transferred to a workstation and analysed using 3D image analysis software (TRI/3D-BON; Ratoc System Engineering, Tokyo, Japan). Trabecular and cortical bone were separated (Fig 1D), and the following structural indices were calculated: trabecular bone volume fraction (BV/TV, %), trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, 1/mm), trabecular separation (Tb.Sp, μm), connectivity density (Conn.D, 1/mm3), trabecular bone pattern factor (TBPf, 1/mm), and structure model index (SMI) [28]. In addition, vBMD was determined using TRI/3D-Bon BMD software (Ratoc System Engineering, Tokyo, Japan). The densitometer’s BMD values were calibrated with a hydroxyapatite phantom (6 × 1 mm; 200, 300, 400, 500, 600, 700 and 800 mg/cm3; Kyoto Kagaku, Kyoto, Japan). The calibration phantom was scanned in the same conditions as actual bone.

Fig 1

Micro-CT analyses of trabecular bone microarchitecture in the distal femoral metaphysis.

A) Representative 3D images of the femoral trabecular bone architecture scanned by micro-CT. B) The distal femoral metaphysis was scanned in an area 2.8–3.0 mm proximal to the distal end of the femur, including the border between the metaphysis and growth plate (GP). C) The region of interest selected for analysis, including cortical and trabecular bone of the distal femoral metaphysis (130 slices). D) Cortical and trabecular bone were subsequently separated and 3D structural indices were calculated. E) After analyzing the trabecular bone architecture, samples were subjected to FEA for prediction of fracture loads. The nodes located at the proximal surface of the femur was fully fixed in all directions, and compression test was simulated at a load of 400N applied to the distal surface of the femur.

Finite element analysis (FEA) for micro-CT images

Micro-FEA were performed using TRI/3D-FEM64 software (RATOC System Engineering, Tokyo, Japan). Reconstructed 3D images of the distal femur obtained by micro-CT (130 slices with a voxel size of 18.11 μm) were exploited for FEA. Compression loading condition was simulated by fully constraining the nodes located at the proximal surface of the femur and a distributed load was applied on the distal surface along the perpendicular direction (Fig 1E). The magnitude of the applied force was chosen to be 400 N. The trabecular bone tissue was modeled as an isotropic, linear elastic material. Young’s modulus of the trabecular bone was calculated based on Carter’s equation and mineralization value from micro-CT images [29] according to the following equation: E = 16.311 (-5) × [trabecular mineralization density (mg/cm3)]3. Where E is the Young’s modulus (measured in MPa). Poisson’s ratio was set to the regular value of 0.3. The element on this surface was restrained to one voxel. Fracture load was defined as the load when 2.8% of the elements reached a shear stress of 68 Mpa or more. Concurrently, the total reaction force on the bottom surface (= fixed area) was analyzed as a fracture load.

Bone histomorphometry

After sacrifice, the left femur was isolated, fixed in 70% pure ethanol, and embedded in methyl-methacrylate (Wako Pure Chemical Industries, Osaka, Japan) without bone decalcification. Frontal plane sections (5 μm thick) of the distal metaphysis were cut using a RM2255 rotary microtome (Leica, Wetzlar, Germany) and stained with Villanueva osteochrome bone stain (basic fuchsin, fast green, orange G, and azure II; Merck, Darmstadt, Germany) for bone histomorphometry as previously reported [24]. Static and dynamic histomorphometric analyses were measured at the Ito Bone Histomorphometry Institute (Niigata, Japan). The following parameters were measured using a semiautomatic graphic system (Histometry RT CAMERA; System Supply Co., Nagano, Japan): BV/TV (%); Tb.Th (μm); Tb.N (N/mm); Tb.Sp (μm); osteoclast surface per bone surface (Oc.S/BS, %); eroded surface per BS (ES/BS, %); number of osteoclasts per BS (N.Oc/BS, N/mm); number osteoblast surface per BS (Ob.S/BS, %); mineralizing surface per BS (MS/BS, %); number of osteoblasts per BS (N.Ob/BS, N/mm); bone formation rate per BS (BFR/BS, μm3/μm2/day), and mineral apposition rate (MAR, μm/day) [30].

Data analysis

Data analysis was performed using IBM SPSS Statistics version 26.0 software package (SPSS, Chicago, IL). The experimental results are expressed in the form of the mean and standard deviation (SD). Data were tested for normal distribution using the Shapiro-Wilk test and homogeneity of variance using Levene’s test. If data failed these tests, the significance of differences across groups was evaluated using the Kruskal-Wallis nonparametric test. For the variables that exhibited normal distribution, one-way ANOVA and Tukey’s post hoc tests were used for the statistical analysis of significance. Values of p < 0.05 were considered statistically significant.

Results

Physical parameters of rats

Body weight before and after the experiment, calf muscle weight and femoral length of rats from each group are shown in Table 1. During the course of the experiment, no differences in body weight were noted among the CON group, JUM group, and KRG group. Calf muscle weight and femoral length did not significantly differ among the CON, JUM, and KRG groups at the end of the experiment.

Table 1

Body weight, hindlimb muscle weight, and femoral length in experimental rats.

	CON	KRG	JUM
Body weight before experiment (g)	309.10 ± 17.15	304.60 ± 08.18	301.30 ± 09.82
Body weight after experiment (g)	423.80 ± 31.16	419.20 ± 20.78	422.00 ± 24.19
Calf muscle weight (g)	2.03 ± 0.16	2.03 ± 0.12	2.05 ± 0.11
Femoral length (mm)	36.31 ± 0.57	36.41 ± 0.37	35.73 ± 0.70

Values are shown as mean ± SD. Male Wistar rats that were 11 weeks old at the beginning of the study were distributed into 3 groups: a group administered 200 mg/kg body weight of Korean red ginseng daily (KRG); a group that jumped 10 times/day from a height of 40 cm (JUM); and control group without exercise or KRG administration (CON).

Microstructural properties, vBMD and FEA-predicted fracture load

Results for 3D microstructural parameters and vBMD in the distal metaphysis of the femur are shown in Fig 2. In terms of femoral trabecular bone parameters, BV/TV, Tb.Th, and Tb.N were significantly higher (72%, p<0.001; 51%, p<0.001; and 14%, p<0.001, respectively) and Tb.Sp, SMI, TBPf, and DA were significantly lower (-23%, p<0.001; -66%, p<0.001; -75%, p<0.001; and -6%, p<0.001, respectively) in the JUM group compared with the CON group. In the KRG group, BV/TV, Tb.N, and Conn.D were significantly higher than in the CON group (17%, p<0.05; 13%, p<0.001; and 17%, p<0.05, respectively). In contrast, BV/TV and Tb.Th were significantly higher (48%, p<0.001; and 46%, p<0.001, respectively), whereas DA was significantly lower in the JUM group than in the KRG group (-5%, p<0.01). A significant increase in vBMD was seen in the JUM group compared to both CON and KRG groups (118%, p<0.001 and 55%, p<0.001, respectively). Finally, FEA-predicted fracture loads were significantly higher in the JUM group than in the CON and KRG groups (196%, p<0.01 and 108%, p<0.05, respectively) (Fig 3).

Fig 2

Microstructural parameters in the distal femoral metaphysis as measured by micro-CT.

Trabecular microarchitecture parameters of the distal femoral metaphysis are shown as: BV/TV, trabecular bone volume fraction (A); Tb.Th, trabecular thickness (B); Tb.N, trabecular number (C); Tb.Sp, trabecular separation (D); Conn.D, connectivity density (E); TBPf, trabecular bone pattern factor (F); SMI, structure model index (G); vBMD, volumetric bone mineral density (H). Male Wistar rats that were 11 weeks old at the beginning of the study were distributed into 3 groups: a group administered 200 mg/kg body weight of Korean red ginseng daily (KRG); a group that jumped 10 times/day from a height of 40 cm (JUM); and control group without exercise or KRG administration (CON). All data represent mean ± SD. Significant difference vs. CON group: *p<0.05; **p<0.01; ***p<0.001. Significant difference vs. KRG group: ††p<0.01; †††p<0.001.

Fig 3

Fracture load as predicted by FEA.

CON, sedentary control group; KRG, KRG administration group; JUM, jump exercise group. All data represent mean ± SD. Significant difference vs. CON group: ***p<0.001. Significant difference vs. KRG group: †††p<0.001.

Figs 4 and 5 show representative 3D trabecular microarchitecture and histological images in the distal femoral metaphysis for a rat from each group. Both jump-exercised and KRG-administered rats demonstrated that diffuse increases in trabecular bone and differential effects of jump exercise as compared to KRG on trabecular microarchitecture can be confirmed visually from these images.

Fig 4

Representative three-dimensional micro-CT images of trabecular bone of the distal femoral metaphyseal region from each group were reconstructed.

KRG, group administered 200 mg/kg body weight of Korean red ginseng daily; JUM, group that jumped 10 times/day from a height of 40 cm; CON, control group without exercise or KRG administration.

Fig 5

Histological images of the distal femoral metaphyseal region from each group.

A) Representative sections of distal femurs under natural light. Red arrows show bone trabeculae. B) Representative sections of distal femurs under fluorescent light. The labeling surface with calcein is indicated by white arrows. Sections were stained by Villanueva staining. GP, growth plate; MS, marrow space; Scale bars (yellow) = 500 μm. KRG, group administered 200 mg/kg body weight of Korean red ginseng daily; JUM, group that jumped 10 times/day from a height of 40 cm; CON, control group without exercise or KRG administration.

Histomorphometry

Results for static and dynamic histomorphometric at the distal femoral metaphysis of rats from each group are shown in Table 2. Regarding static histomorphometry, BV/TV and Tb.N in both JUM (85%, p<0.001; and 57%, p<0.001, respectively) and KRG groups (57%, p<0.01; and 37%, p<0.01, respectively) were significantly higher than in the CON group, whereas Tb.Sp was significantly lower in the JUM (-44%, p<0.001) and KRG groups (-32%, p<0.001) than in the CON group. Conversely, Tb.Th was highest in the JUM group and differed significantly from that in the CON group. With respect to bone resorption, Oc.S/BS, ES/BS, and N.Oc/BS in both JUM (-30%, p<0.05; -33%, p<0.01; and -31%, p<0.05, respectively) and KRG groups (-36%, p<0.01; -20%, p<0.05; and -42%, p<0.001, respectively) were significantly lower than in the CON group. In contrast, values for N.Ob/BS reflecting bone formation were higher in the JUM group than in the CON and KRG groups (p = 0.363 and p = 0.688, respectively).

Table 2

Static and dynamic bone histomorphometric parameters of the distal femoral metaphysis.

	CON	KRG	JUM
BV/TV (%)	12.1 ± 3.2	18.7 ± 3.9**	22.4 ± 4.4***
Tb.Th (μm)	60.7 ± 9.4	70.2 ± 5.9	72.3 ± 7.2**
Tb.N (N/mm)	2.0 ± 0.3	2.7 ± 0.5**	3.1 ± 0.4***
Tb.Sp (μm)	456.2 ± 79.4	308.3 ± 58.1***	256.1 ± 46.6***
Ob.S/BS (%)	21.8 ± 7.4	23.3 ± 5.2	22.6 ± 2.7
MS/BS (%)	39.1 ± 4.5	36.2 ± 4.1	38.6 ± 1.5
N.Ob/BS (N/mm)	15.0 ± 5.1	18.8 ± 5.3	19.3 ± 5.7
Oc.S/BS (%)	13.4 ± 2.8	8.5 ± 3.6**	9.4 ± 2.5*
ES/BS (%)	32.8 ± 5.4	26.1 ± 7.6*	22.1 ± 4.5**
N.Oc/BS (N/mm)	4.6 ± 1.0	2.7 ± 1.1***	3.2 ± 0.7**
MAR (μm/day)	2.8 ± 0.2	2.7 ± 0.2	2.8 ± 0.1
BFR/BS (μm3/μm2/day)	1.09 ± 0.14	0.97 ± 0.14	1.07 ± 0.06

Values are shown as means ± SD. BV/TV, trabecular bone volume fraction; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; Ob.S/BS, osteoblast surface per BS; MS/BS, mineralizing surface per BS; N.Ob/BS, number of osteoblasts per BS; Oc.S/BS, osteoclast surface per BS; ES/BS, eroded surface per BS; N.Oc/BS, number of osteoclasts per BS; MAR, mineral apposition rate; BFR/BS, bone formation rate per BS. Male Wistar rats that were 11 weeks old at the beginning of the study were distributed into 3 groups: a group administered 200 mg/kg body weight of Korean red ginseng daily (KRG); a group that jumped 10 times/day from a height of 40 cm (JUM); and control group without exercise or KRG administration (CON). Significant difference vs. CON group:

*p < 0.05,

**p < 0.01,

***p < 0.001.

Discussion

Our primary focus in this study was to compare the effects of KRG administration on trabecular microarchitecture with changes caused by jump exercise. We found that jump exercise and KRG administration exerted different effects on trabecular microarchitecture in the distal femoral metaphysis of growing male rats. Namely, jump exercise improved both trabecular thickness and number, whereas KRG predominantly improved trabecular number.

The effects of exercise on bone mass and strength depend on loading magnitude and strain rate applied to the bone during the activity. A higher strain rate is associated with a greater adaptive bone response [31]. Several studies with rats have found that jumping yields greater increases in bone mass and strength because of the greater mechanical stress and higher strain rate imposed [19-21]. Previous histomorphometric analyses have shown that the increase in trabecular bone mass that occurs with resistance exercise is primarily due to increased trabecular thickness, rather than noticeable changes in numbers of trabeculae [32, 33]. In previous experiments with 3D micro-CT of the distal femoral metaphysis, we observed that the gain in trabecular bone induced by jump exercise during the remobilization period was also predominantly attributable to increases in trabecular thickness rather than number [25]. The present study showed similar results, with a 72% increase in trabecular bone mass, a 51% increase in trabecular thickness, and a 14% increase in trabecular number. In the bone histomorphometric analysis of the left femur distal metaphysis, the results of static histomorphometry are very similar to data from 3D morphometric parameters of current study.

In contrast to jump exercise, however, administration of KRG extract increased trabecular number (13%) when compared with the sedentary control group, but trabecular thickness was unaffected. These results imply that the trabecular bone gain induced by KRG administration is mainly due to increases in trabecular number as opposed to trabecular thickness. Several previous studies support this finding. For example, Kim et al. showed that Panax ginseng prevented age-related decreases in trabecular number without significant effects on trabecular thickness in a rat model [34]. Lee et al. also showed that oral administration of KRG extract to irradiated mice for 12 weeks prevented loss of trabecular bone mass of the proximal tibia, primarily by significant alterations in trabecular number [23]. Furthermore, we have previously demonstrated that both running and jump exercises exerted different effects on trabecular microarchitecture in the distal metaphysis in rats while both of them similarly enhanced trabecular bone mass [22, 26]. Taken together, these results suggest that KRG and jump exercises may produce trabecular bones with different structural and mechanical characteristics.

Bone mineral density is a major determinant of bone strength, but it does not capture all aspects that define the various mechanical behavior of trabecular bone. In addition to bone mass, microarchitecture also play an important role in determining the mechanical properties of trabecular bone [35]. In this study, several non-metric parameters characterizing bone architecture were also evaluated in addition to trabecular thickness, number and separation. Among these non-metric parameters Conn.D was significantly higher in the KRG group compared with CON, whereas TBPf and SMI showed significant changes in favor of increased bone strength in the JUM group. These results suggest that KRG and jump exercise may exert different effects on the structural and mechanical characteristics of trabecular bone. Although the fracture load predicted by FEA was higher in both KRG and JUM groups compared with the CON group, the difference was significant only in the JUM group. Since the magnitude of increase in bone mass was smaller in the KRG group than in the JUM group, the statistical power of the present investigation may have been insufficient to detect differences in fracture load between KRG and CON.

Several studies have demonstrated that ginsenosides inhibit osteoclastogenesis and reduce bone resorption [15-18], although this is not uniformly the case [36, 37]. The histomorphometric data in the present study showed similar results. Osteoclastic parameters such as Oc.S/BS, ES/BS, and N.Oc/BS were inhibited more by KRG, without changes in osteoblastic parameters. A previous study found that the restoration of trabecular bone architecture induced by treatment with an osteoclastic inhibitor (tiludronate) during remobilization after suspension-induced osteopenia in rats was predominantly attributable to increased trabecular number [38], and the present results are similar to this. From this perspective, the data obtained in the present study imply that the effect of KRG extract in increasing trabecular number was predominantly mediated by inhibition of bone resorption rather than stimulation of bone formation. On the other hand, the mechanical loading induced by physical exercise is well known to increase bone formation and reduce bone resorption, leading to the maintenance of a healthy skeleton [39]. In addition, jump exercise that principally promotes osteogenesis of osteoblasts have been reported to increase trabecular thickness of the distal femoral metaphysis in rats, but not trabecular number, resulting in an elevated trabecular bone volume [26]. The upshot of the present study was that jump exercise can significantly increase trabecular bone thickness by inhibiting bone resorption and increasing bone formation. Further synergistic effects are expected when KRG is used in combination with jump exercise, because KRG extract and jump exercises may change the trabecular bone architecture via different mechanisms of action.

Some limitations to the animal model used and the length of KRG administration applied during this study must be considered when interpreting the results. First, although there are similarities in bone metabolism between rats and humans, the inherent differences in bone structure, remodeling, and skeletal loading patterns should be considered before extrapolating our results to humans. Second, evaluation of the KRG effect on improvements in trabecular microarchitecture was performed at only a single dose selection (dose of 200mg/kg) and one time point (6 weeks after treatment). A longer administration duration and higher KRG dose could have lead to a greater increase in trabecular bone mass in KRG group. Further studies are warranted to confirm whether a dose higher than 200mg/kg and long-term use would provide better improvements to trabecular bone microarchitecture. However, maximizing this response was not the main purpose of the present study. Nonetheless, the dose and duration of KRG extract administration selected for the present study was sufficient to achieve increases in trabecular bone volume in the distal femoral metaphysis of growing male rats. Third, in this study, in order to clarify the relationship between trabecular bone structure and bone strength, FEA was performed using a model in which only the trabecular bone region was extracted. Therefore, the mechanical condition in the FEM model may be slightly different from the actual anatomical condition. Finally, this study did not include the combination of KRG and jump exercise. The present study showed that KRG has a weak but significant positive effect on bone mass and further suggest that the effect on trabecular microarchitecture is different from that by jump exercise. Thus the combination of KRG and jump exercise may synergistically lead to increase in trabecular bone mass and strength.

In conclusion, we demonstrated that the effects on trabecular bone mass differed between jump exercise and KRG, in that jump exercise increased trabecular bone volume by thickening trabeculae, whereas administration of KRG extract added to trabecular bone volume by increasing trabecular number without accompanying increases in trabecular thickness. Whether combining KRG and jump exercise could increase trabecular bone mass and strength in an additive or even synergistic manner warrants investigation, as the mechanisms of bone response differed. Further studies are required to clarify this relationship. Taken together, our data may provide basic insights into the efficacy of KRG extract on trabecular bone architecture, supporting its development as a safe therapeutic agent and functional food with beneficial effects on bone.

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PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods, please provide the product number for the KRG extract you obtained from Korea Ginseng Corporation, and include details of any quality assessments provided.

3. Please provide details about any efforts to minimise distress during the jumping task.

4. To comply with PLOS ONE submissions requirements, please provide the method of euthanasia in the Methods section of your manuscript.

5. As part of your revision, please complete and submit a copy of the Full ARRIVE 2.0 Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://arriveguidelines.org/sites/arrive/files/Author%20Checklist%20-%20Full.pdf (PDF). Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article.

6.Please state specifically whether the animal ethics committee specifically approved the study.

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

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Reviewer #1: No

Reviewer #2: No

**********

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Reviewer's comments on paper “Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercise” submitted to Plos One (PONE-D-21-20663).

Although some interesting results seems to be reported in this work, Authors didn't draft the manuscript well and I cannot recommend the publication of the article in its present form.

The (L24) abstract and introduction (L49-59) suggest that the aim of the study was to compare the effect of KRG with Jump-exercise in rat osteoporosis model. There is no explanation how 11 wk-old (male rats can serve as a osteoporosis model animal - no osteoporosis agent was introduced to animals in this study (ovariectomy, orchidectomy, hypophysectomy, parathyroidectomy, immobilization, or dietary manipulation). In rats, first osteoporotic changes in long bone metaphysis occurs in older animals, peak bone mass in trabeculae of femur metaphysis is reached at the age of ca. 6 months. On the other hand, the last sentences of the introduction indicate that the true aim of the study was to assess the influence of KRG as an effective strategy for improving bone strength during growth (L91) and compare this effect with well-known method of stimulation of bone formation in young adults – exercises. Therefore, I suggest the correct the manuscript by removing most of the osteoporosis-related fragments and make it cleaner that this study is focused on the young individuals during development.

There is only one dose examined, which selectin was based on a single reference data in which a different osteoporosis model (OVX) was under study. In this manner, the idea comparison of KRG (dietary supplement) with jump-exercised group is not sufficiently explained.

Limitations of the study should be extended as there are three main limitation of this study: the mentioned issue with the single-dose selection (and a single jump regime in lesser extent), the lack of the KRG+JUM group and the single time-point analysis.

Distal femoral histomorphometry was performed both using uCT and histological analysis. Were these results somehow consistent? It should be discussed.

Minor comments (selected):

L31 the information about KRG form is missing – extract? powder?

L53 the direct reference to WHO should be given (but I recommend remove all fragments related to primary osteoporosis)

L53 “bone destruction” – rephrase

L85 add information that this correspond to “rat model studies” as all references [25-27] are rat-related

L117 was it pure EtOH or phosphate-buffered EtOH?

L142 copper

L157 what was the mineral density of hydroxyapatite phantom

L173-174 I think that this information should be placed earlier, when animal management is described

L178 Correct to Villanueva osteochrome bone stain. Also was it ready-to-use kit ? If yes, please give the information of manufacturer.

L179-186 How all of these indices were calculated? what software was used ?

Statistics: As you present the results of statistical analysis as a single comparison of KGR group vs CON or vs JMP, why didn't you perform simple t-test or contrast analysis of KGR vs appropriate group instead of Tukey’s test where all three groups are examined ?

L203 What was the idea of presenting the weight of the calf muscle ? Was the increase of muscle weight expected (any references?)? Also, why tight muscle, which is more appropriate for any femur analysis than calf, was not examined? Finally, if some changes in muscle structure were expected, muscle histology also should be performed.

L301 hPTH? What is the connection of human parathyroid hormone with current study ?

Fig 2 - No A, B ... H on figure 2.

Fig 5 - more interesting than showing trabeculae would be showing calcenin labeling lines

which are barely visible in presented figures

Reviewer #2: Review of PONE-D-21-20663

Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercise

General Comments

This study examined the effects of KRG on the trabecular bone of the distal femur in young growing male Wistar rats. Trabecular architecture and dynamic histomorphometry were employed as primary measures, complementing with computational models of the extracted trabecular architecture. Only the distal femur trabecular compartment was measured, with no axial skeleton examined and no diaphyseal cortex examined. As a comparator, the jump exercise model was examined in a matched cohort of rats, demonstrating a larger positive effect on this compartment. Both models suggested a shift to increased osteoblast numbers and decreased osteoclast numbers. The typical combined therapeutic (KRG + exercise) was not examined in this study, but has likely been pursued by this group (I encourage inclusion of such data in this paper if available).

Many details regarding the computational models are not included and need to be clarified in the manuscript. Some additional computational series should be run to answer the questions posed in the specific comments below.

Jump exercise had a measured effect of reducing femur length by 0.6-0.7 mm, which is a substantial amount. As an initial bound on this examination, t-tests performed without statistical penalty for JUM vs KRG and JUM vs CON demonstrate p values of 0.014 and 0.057, substantially more indicative of real difference than afforded by osteoblast numbers that are noted in presentation of results with p values > 0.5 due to variation. What occurs with jump exercise to stunt longitudinal growth? Is there damage to the physis?

The importance of ginseng processing is detailed, but the processing and verification of constituents and potency of the commercial product used in this study were not measured or reported. A test of residual KRG product should be performed prior to publication of this study.

The study would be improved with the inclusion of measurements for the axial skeleton (e.g. lumbar vertebrae) and cortical bone (e.g., femoral mid-diaphysis cortex) as is customary for bone morphometry measurements in studies of this nature. These data should be included for publication.

Specific Comments

Line 33: State the actual estimated jump height, and not the box height of 40 cm, as that seems misleading. See comment below regarding line 122.

Lines 75-77: These studies thus implicate direct action on osteoclasts. Did any of these in vivo studies perform histological measurements?

Lines 93-94: Cite references that provide data demonstrating this conclusion of ideal.

Line 109: Should “anesthetized” be “euthanized”?

Line 117: Why are no histological data reported in the abstract methods and results? They are very important.

Line 122: As described with a 40 cm tall box, how high must the rats jump from standing stretched position in order to grasp the box wall? It seems that the jump height would be 10-20 cm. Please clarify in the manuscript.

Lines 168-169: The method and algorithm implemented for assigning elastic modulus based on micro-CT derived tissue mineral density (using the Carter & Hayes relationship) must be provided. Was there any volumetric averaging of discrete values or was every voxel assigned a unique value? (unique assignment to each voxel does not seem justified)? Are your results different if one homogeneous value of elastic modulus is assigned to all regions of all models? Typically, trabecular structure dominates the variance in modulus assigned via such mineralization rules. Most importantly, what failure criterion was implemented for strength?

Lines 129-130: Measurements should have been performed to assess the true composition of ginsenosides, etc. within this commercial sample of KRG. Very frequently the true composition of such products is not as stated on the label.

Line 166: The boundary condition of uniform applied force rather than uniform displacement (Fig 1E) does not seem realistic, given the heterogeneous assignment of modulus values. That is, the lack of geometrical constraint due to extraction of trabecular bone, along with the spatial difference in elastic modulus, would seemingly allow each boundary location of applied force to deform differentially and unrealistically relative to the anatomical condition.

Line 166-168: What is the basis for selecting 2.8% as a threshold for defining failure. How do the results change if that value is perturbed upward and downward? Most importantly, what material failure criterion was applied in the definition of failure? This is a critical part of this method.

Lines 216-217: The stated differences in vBMD of 2% and 4% for jump exercise relative to control and KRG do not match the bar charts in Figure 2, which show large percentage differences similar to BV/TV (as they should). The data in figure 2 are almost certainly values of vBMD (i.e., apparent density) and report essentially the same comparative data as BV/TV. Presumably this statement regarding 2-4% pertains to the tissue-level density, which typically varies at most by a few percent. However, the manuscript makes no reference to tissue mineral density measurement throughout.

Lines 237-238: It does appear that osteoblast numbers were 25-30% higher in the experimental groups, but that measurement variation swamped the statistical comparison. It might be more insightful to the reader to provide the information in that manner.

Table 2: The histological data indicate that KRG lowered BFR/BS relative to control and jump exercise. Is this secondary to depletion of osteoclasts?

**********

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Reviewer #1: No

Reviewer #2: No

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24 Feb 2022

February 25, 2022

Dear. Ewa Tomaszewska

Academic Editor

PLOS ONE

Manuscript ID PONE-D-21-20663

My colleagues and I wish to thank you and the reviewers for analysis of our manuscript entitled “Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercise” (PONE-D-21-20663).

We have followed the suggestions of the editor and the reviewers and have made the following revisions:

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We have confirmed that our manuscript meets PLOS ONE’s style requirements.

2. In your Methods, please provide the product number for the KRG extract you obtained from Korea Ginseng Corporation, and include details of any quality assessments provided.

We have added accordingly (page 8, lines 154-157).

3. Please provide details about any efforts to minimise distress during the jumping task.

We have added accordingly (page 8, lines 139-145).

4. To comply with PLOS ONE submissions requirements, please provide the method of euthanasia in the Methods section of your manuscript.

We have added accordingly (page 6, lines 106-111).

5. As part of your revision, please complete and submit a copy of the Full ARRIVE 2.0 Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://arriveguidelines.org/sites/arrive/files/Author%20Checklist%20-%20Full.pdf (PDF). Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article.

We have included the ARRIVE Guidelines Checklist accordingly.

6.Please state specifically whether the animal ethics committee specifically approved the study.

We have added accordingly (page 6, lines 103-106).

Responses to the Reviewers

Reviewer #1

Comments to the Author

The (L24) abstract and introduction (L49-59) suggest that the aim of the study was to compare the effect of KRG with Jump-exercise in rat osteoporosis model. There is no explanation how 11 wk-old (male rats can serve as a osteoporosis model animal - no osteoporosis agent was introduced to animals in this study (ovariectomy, orchidectomy, hypophysectomy, parathyroidectomy, immobilization, or dietary manipulation). In rats, first osteoporotic changes in long bone metaphysis occurs in older animals, peak bone mass in trabeculae of femur metaphysis is reached at the age of ca. 6 months. On the other hand, the last sentences of the introduction indicate that the true aim of the study was to assess the influence of KRG as an effective strategy for improving bone strength during growth (L91) and compare this effect with well-known method of stimulation of bone formation in young adults – exercises. Therefore, I suggest the correct the manuscript by removing most of the osteoporosis-related fragments and make it cleaner that this study is focused on the young individuals during development.

In accordance with the reviewer’s comment, the manuscript has been revised (page 2, line 24 and page 4, lines 51-58).

There is only one dose examined, which selectin was based on a single reference data in which a different osteoporosis model (OVX) was under study. In this manner, the idea comparison of KRG (dietary supplement) with jump-exercised group is not sufficiently explained.

According to our knowledge, the effect of KRG extract on trabecular bone microarchitecture has not been verified in growing rat. Thus, we conducted explorative experiment at different dose of KRG extract (100mg/kg, 200mg/kg and 300mg/kg) using the smallest sample size for each condition before starting the present study. Bone mass in both dose of 200mg/kg and 300mg/kg showed a greater increase than in the dose of 100mg/kg, but no difference was observed between the dose of 200mg/kg and 300mg/kg. A similar effect has also been described by Avsar et al., (Cell Mol Biol. 2013;59(Suppl):OL1835-1841) who reported that BMD in 200mg/kg administered group showed a greater increase than 100ng/kg administered group. Thus, using data from explorative experiment in our laboratory and other studies, we determined the dose of KRG administration required for the experiment.

Comments on the idea comparison of KRG with jump exercise have been added to the manuscript (page 5, lines 88-92).

Limitations of the study should be extended as there are three main limitation of this study: the mentioned issue with the single-dose selection (and a single jump regime in lesser extent), the lack of the KRG+JUM group and the single time-point analysis.

In accordance with the reviewer’s suggestion, comments on limitations of the study have been added to the manuscript (pages 17-18, lines 344-350 and lines 357-361).

In the study by Umemura et al (J Bone Miner Res. 1997;12:1480-1485), they showed that although a slight tendency toward increase according to the number of jumps per day was observed, there were few differences in bone morphological and mechanical parameters among the 5-, 10-, 20-, and 40-jump groups. In this study, therefore, we did not consider the dose-effect in jump exercise.

Distal femoral histomorphometry was performed both using uCT and histological analysis. Were these results somehow consistent? It should be discussed.

We have added accordingly (page 15, lines 288-290).

Minor comments (selected):

L31 the information about KRG form is missing – extract? powder?

We have added accordingly (page 2, line 31).

L53 the direct reference to WHO should be given (but I recommend remove all fragments related to primary osteoporosis)

We have modified accordingly (page 4, lines 51-58).

L53 “bone destruction” – rephrase

We have modified accordingly (page 4, lines 51-58).

L85 add information that this correspond to “rat model studies” as all references [25-27] are rat-related

The information regarding "rat model studies" has been added to the manuscript (page 5, lines 84-85).

L117 was it pure EtOH or phosphate-buffered EtOH?

It was pure EtOH. We have added accordingly (page 11, line 201).

L142 copper

We have corrected a typing error "coper" to "copper" (page 9, line 166).

L157 what was the mineral density of hydroxyapatite phantom

The information regarding the mineral density of hydroxyapatite phantom has been added to the manuscript (page 10, line 180).

L173-174 I think that this information should be placed earlier, when animal management is described

The manuscript has been revised as suggested (page 7, lines 122-124).

L178 Correct to Villanueva osteochrome bone stain. Also was it ready-to-use kit ? If yes, please give the information of manufacturer.

The information of manufacturer has been added to the manuscript (page 11, lines 204-206).

L179-186 How all of these indices were calculated? what software was used ?

The information about histomorphometric image analysis system has been added to the manuscript (page 11, lines 208-209).

Statistics: As you present the results of statistical analysis as a single comparison of KGR group vs CON or vs JMP, why didn't you perform simple t-test or contrast analysis of KGR vs appropriate group instead of Tukey’s test where all three groups are examined ?

In this study, instead of simple pairwise comparison with CON, we adopted a more robust method of ANOVA and post-hoc analysis among three equivalent groups.

L203 What was the idea of presenting the weight of the calf muscle ? Was the increase of muscle weight expected (any references?)? Also, why tight muscle, which is more appropriate for any femur analysis than calf, was not examined? Finally, if some changes in muscle structure were expected, muscle histology also should be performed.

Change in muscle mass may play an important role in the regulation of bone mass. However, there was no significant difference in muscle weight between the groups. This phenomenon is not unusual in studies using rats. The comparability of muscle weight between the study groups strengthens the conclusion that the bone changes observed in our study are derived primarily from the exercise per se.

The anatomy of the thigh muscles is markedly different in rat and human, with the biceps femoris being a far larger muscle in rat, with an extensive attachment along the tibia. Therefore, it is very unlikely that these large muscles will increase muscle weight through 10 time jump exercise protocol. Furthermore, since the removal procedure of large muscles such as biceps femoris, vastus medialis, and rectus femoris of rat is more complicated than the simple extraction of calf muscles, it could be an error factor in the comparison of muscle weight.

L301 hPTH? What is the connection of human parathyroid hormone with current study?

Since the direct relationship with the results of this study is small, we have deleted the word of hPTH (page 17, line 332).

Fig 2 - No A, B ... H on figure 2.

We have added accordingly (Figure 2).

Fig 5 - more interesting than showing trabeculae would be showing calcenin labeling lines

which are barely visible in presented figures

We much appreciate the reviewer’s suggestion. We have modified accordingly (page 26, lines 524-526; Figure 5).

Reviewer: 2

Comments to the Author

General Comments

This study examined the effects of KRG on the trabecular bone of the distal femur in young growing male Wistar rats. Trabecular architecture and dynamic histomorphometry were employed as primary measures, complementing with computational models of the extracted trabecular architecture. Only the distal femur trabecular compartment was measured, with no axial skeleton examined and no diaphyseal cortex examined. As a comparator, the jump exercise model was examined in a matched cohort of rats, demonstrating a larger positive effect on this compartment. Both models suggested a shift to increased osteoblast numbers and decreased osteoclast numbers. The typical combined therapeutic (KRG + exercise) was not examined in this study, but has likely been pursued by this group (I encourage inclusion of such data in this paper if available).

In this study, we elucidated the differential effects of jump and KRG on trabecular bone architecture in rats. Thus the combination of KRG and jump exercise may synergistically lead to increase in trabecular bone mass and strength. We are currently conducting additional experiments to find out about that.

Comments on the lack of the combination of KRG and jump exercise have been added to the manuscript (pages 17-18, lines 353-357).

Many details regarding the computational models are not included and need to be clarified in the manuscript. Some additional computational series should be run to answer the questions posed in the specific comments below.

Jump exercise had a measured effect of reducing femur length by 0.6-0.7 mm, which is a substantial amount. As an initial bound on this examination, t-tests performed without statistical penalty for JUM vs KRG and JUM vs CON demonstrate p values of 0.014 and 0.057, substantially more indicative of real difference than afforded by osteoblast numbers that are noted in presentation of results with p values > 0.5 due to variation. What occurs with jump exercise to stunt longitudinal growth? Is there damage to the physis?

According to our previous studies (Bone. 2003;33:485-493, J Appl Physiol. 2008;104:1594-1600, 2012;112:766-772, 2015;119:990-997, PLoS One. 2014;9:e107953, Phys Act Nutr. 2020;24:1-8), the decrease in femoral length was not always by exercise stress. Various factors can be considered as the cause such as intake of food and water, body weight, breeding conditions, and measurement variation. Even if some impaired longitudinal bone growth occurs by these factors, the influence of these factors is even among the experimental groups. Although we cannot completely exclude the possibility of the influence of jump exercise on the longitudinal bone growth, we believe the effects would be negligible, if any.

The importance of ginseng processing is detailed, but the processing and verification of constituents and potency of the commercial product used in this study were not measured or reported. A test of residual KRG product should be performed prior to publication of this study.

The composition of ginsenosides and a broad range of efficacy of the KRG extract used in this study have been approved by the Korean Food and Drug Administration (KFDA), and we contacted the Korea Ginseng Corporation directly and confirmed the true composition of ginsenosides.

We have added accordingly (page 8, lines 154-157).

The study would be improved with the inclusion of measurements for the axial skeleton (e.g. lumbar vertebrae) and cortical bone (e.g., femoral mid-diaphysis cortex) as is customary for bone morphometry measurements in studies of this nature. These data should be included for publication.

It has been reported that bone mass and strength in the lumbar vertebrae of rat are not significantly affected by exercise (Iwamoto et al., Bone. 1999;24:163-169, J Bone Miner Metab. 2004;22:26-31, Notomi et al., J Appl Physio. 2002;93:1152-1158). Also, in the previous report, exercise showed a positive effect on bone mass in long bones at weight-bearing sites such as tibia and femur compared with vertebral bone. Furthermore, the adaptive response of bone to exercise differs between regions of the same bone because the stress distribution by exercise differ among skeletal sites. In our laboratory’s previous studies in rats (data not presented), both jump and running exercises had less impact on cortical bone structural changes. For these reasons, axial skeleton and cortical bone were not included in the present study. The analysis of these sites would be included in future studies.

Specific Comments

Line 33: State the actual estimated jump height, and not the box height of 40 cm, as that seems misleading. See comment below regarding line 122.

The actual estimated jump height has been included in the manuscript (pages 7-8, lines 133-136).

Lines 75-77: These studies thus implicate direct action on osteoclasts. Did any of these in vivo studies perform histological measurements?

These previous studies have not been histologically examined and the relationship between KRG and bone histomorphometric parameters is unknown. We have modified the manuscript (page 5, lines 74-76).

Lines 93-94: Cite references that provide data demonstrating this conclusion of ideal.

We have added accordingly (page 6, line 99).

Line 109: Should “anesthetized” be “euthanized”?

We have corrected accordingly (page 6, lines 106-111).

Line 117: Why are no histological data reported in the abstract methods and results? They are very important.

We have added accordingly (page 2, lines 35-36 and page 3, lines 42-43).

Line 122: As described with a 40 cm tall box, how high must the rats jump from standing stretched position in order to grasp the box wall? It seems that the jump height would be 10-20 cm. Please clarify in the manuscript.

The actual estimated jump height has been included in the manuscript (pages 7-8, lines 133-136).

Lines 168-169: The method and algorithm implemented for assigning elastic modulus based on micro-CT derived tissue mineral density (using the Carter & Hayes relationship) must be provided. Was there any volumetric averaging of discrete values or was every voxel assigned a unique value? (unique assignment to each voxel does not seem justified)? Are your results different if one homogeneous value of elastic modulus is assigned to all regions of all models? Typically, trabecular structure dominates the variance in modulus assigned via such mineralization rules. Most importantly, what failure criterion was implemented for strength?

We have added accordingly (page 10, lines 191-198).

Lines 129-130: Measurements should have been performed to assess the true composition of ginsenosides, etc. within this commercial sample of KRG. Very frequently the true composition of such products is not as stated on the label.

The composition of ginsenosides and a broad range of efficacy of the KRG extract used in this study have been approved by the Korean Food and Drug Administration (KFDA), and we contacted the Korea Ginseng Corporation directly and confirmed the true composition of ginsenosides.

We have added accordingly (page 8, lines 154-157).

Line 166: The boundary condition of uniform applied force rather than uniform displacement (Fig 1E) does not seem realistic, given the heterogeneous assignment of modulus values. That is, the lack of geometrical constraint due to extraction of trabecular bone, along with the spatial difference in elastic modulus, would seemingly allow each boundary location of applied force to deform differentially and unrealistically relative to the anatomical condition.

In accordance with the reviewer’s suggestion, comments on limitations of the study have been added to the manuscript (pages 17-18, lines 353-357).

Line 166-168: What is the basis for selecting 2.8% as a threshold for defining failure. How do the results change if that value is perturbed upward and downward? Most importantly, what material failure criterion was applied in the definition of failure? This is a critical part of this method.

In this study, we chose 2.8% as the threshold for defining destruction based on literature that failure load was defined as the load when 2% of the elements exceeded a strain of 0·007 using HR-pQCT images (Samelson et al., Lancet Diabetes Endocrinol. 2019;7:34-43). If it is determined as a fracture when one element is destroyed, an element that is destroyed by the model error may come out. To avoid this error, we had a few percent margin to meet the occurrence of a sure local fractures.

We have modified the manuscript accordingly (page 10, lines 196-198).

Lines 216-217: The stated differences in vBMD of 2% and 4% for jump exercise relative to control and KRG do not match the bar charts in Figure 2, which show large percentage differences similar to BV/TV (as they should). The data in figure 2 are almost certainly values of vBMD (i.e., apparent density) and report essentially the same comparative data as BV/TV. Presumably this statement regarding 2-4% pertains to the tissue-level density, which typically varies at most by a few percent. However, the manuscript makes no reference to tissue mineral density measurement throughout.

We much appreciate the reviewer’s careful review. We made a typo and corrected "2% and 4%" as "118% and 55%" (page 13, line 246).

Lines 237-238: It does appear that osteoblast numbers were 25-30% higher in the experimental groups, but that measurement variation swamped the statistical comparison. It might be more insightful to the reader to provide the information in that manner.

We agree with your comment. Using data from previous studies in our laboratory and other studies, we determined the minimum number of animals required for the experiment. However, the statistical power seems to be insufficient for some evaluation items, such as the results of static and dynamic histomorphometric. Perhaps a larger sample size may confirm these findings.

Table 2: The histological data indicate that KRG lowered BFR/BS relative to control and jump exercise. Is this secondary to depletion of osteoclasts?

We agree with your comment. Since both bone resorption (Oc.S/BS(%), ES/BS(%), and N.Oc/BS) and BFR/BS were lower, it is considered that the bone metabolism has shifted to low turnover rate. Namely, we speculate the low BFR/BS is secondary to the decrease of osteoclastic bone resorption by KRG.

We believe the thoughtful comments of the reviewers have substantially improved the manuscript. We hope that the manuscript will now be acceptable for publication in PLOS ONE.

We thank you very much for your kind consideration.

Cordially yours,

Yong-In Ju, Ph.D.

Kawasaki University of Medical Welfare

288 Matsushima, Kurashiki

Okayama 701-0193, Japan

Tel: +81-86-462-1111, Fax: +81-86-464-1109

E-mail: jyi.k051@mw.kawasaki-m.ac.jp

Submitted filename: Resposes to the Reviewer_(PloS One).docx

Click here for additional data file.

11 Apr 2022

Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercise

PONE-D-21-20663R1

Dear Dr. Yong-In Ju ,

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Reviewer #1: (No Response)

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The manuscript has been substantially improved, so my recommendation is to accept the manuscript in its present form.

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Reviewer #1: No

25 Apr 2022

PONE-D-21-20663R1

Effects of Korean red ginseng on three-dimensional trabecular bone microarchitecture and strength in growing rats: comparison with changes due to jump exercise

Dear Dr. Ju:

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