Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2021 Jul 16;16(7):e0254383. doi: 10.1371/journal.pone.0254383

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis using a post-traumatic rat model

Ikufumi Takahashi 1,2,*, Keisuke Takeda 1, Taro Matsuzaki 3, Hiroshi Kuroki 2, Masahiro Hoso 3
Editor: Lin Han4
PMCID: PMC8284605  PMID: 34270585

Abstract

The purpose of this study was to clarify the histological effect of reducing the loading to knee on cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis (OA) using a post-traumatic rat model. Ten male rats were randomly allocated into two experimental groups: OA induction by surgical destabilization of medial meniscus (DMM, OA group) and hindlimb suspension after OA induction by DMM (OAHS group). The articular cartilage, osteophyte formation, and synovial membrane in the medial tibiofemoral joint were analyzed histologically and histomorphometrically at 2 and 4 weeks after surgery. The histological scores and changes in articular cartilage and osteophyte formation were significantly milder and slower in the OAHS group than in the OA group. At 2 and 4 weeks, there were no significant differences in cartilage thickness and matrix staining intensity between both the groups, but chondrocytes density was significantly lower in the OA group. Synovitis was milder in OAHS group than in OA group at 2 weeks. Reducing knee joint loading inhibited histological OA changes in articular cartilage, osteophyte formation, and synovial inflammation. This result supports the latest clinical guidelines for OA treatment. Further studies using biochemical and mechanical analyses are necessary to elucidate the mechanism underlying delayed OA progression caused by joint-load reduction.

Introduction

Mechanical stress, such as joint loading, is reported to influence osteoarthritis (OA) development [1, 2]. Moderate stress, such as physiological loading and exercise, are indispensable for metabolism of the articular cartilage and to help inhibit OA development [3, 4]. On the other hand, excessive mechanical stress has been reported to be the cause of the cartilage deterioration and OA onset [5, 6]. Hence, in the major international guidelines for OA treatment, such as the Osteoarthritis Research Society International (OARSI) issued in 2014 [7], the National Institute for Health and Care Excellence of the United Kingdom, National Health Service issued in 2014 [8] and those of the American Academy of Orthopedic Surgeons issued in 2013 [9], weight management and use of walking aids are recommended to avoid excessive loading on knee joints. In addition, the latest OARSI guidelines announced in 2019 strongly recommend weight management as the core treatment for all individuals regardless of comorbidity [10].

Therefore, clinical evidence has shown that reducing knee joint loading is effective and beneficial. However, little is known about the histological effects of reducing joint loading on articular cartilage. Focusing on this point, we previously analyzed the histological effect of reducing the loading on normal articular cartilage. We reported that reducing the loading on knee joints caused disuse atrophy of articular cartilage, including thinning of articular cartilage and decreased matrix staining [11, 12]. Moreover, we analyzed the histological effect of reduced loading on OA progression in a drug-induced rat model [13]. As a result, we revealed that OA progression was histologically suppressed by reducing the loading on knee joints in 2019 [13]. Although this finding is very useful, there were three major limitations. First, this OA model has no pathophysiological relationship with clinical OA because the model is drug induced and causes chondrocyte death directly and early [14, 15]. Second, only articular cartilage was analyzed. Recently, it has been reported that OA is a disease caused by the interaction of multiple joint components including articular cartilage, subchondral bone, and synovial membrane [15–18]. Third, only semiquantitative assessment by histological scores was performed to assess OA progression because it is difficult to evaluate the histomorphology due to the drug-induced model. We searched for previous studies that have clarified these three limitations but were unable to find any. Therefore, the histological and histomorphological effects of decreasing knee joint loading on articular cartilage, subchondral bone, osteophyte formation, and synovial membrane are not clear.

In view of the above, the purpose of this study is to overcome these three limitations and to clarify the effect of decreasing the load on the knee joint on OA progression of knee. Hence, we chose the surgical destabilization of medial meniscus (DMM) model as our rat OA model because surgically induced OA models have the advantage of similarity to post-traumatic OA [19], and the DMM model is the most widely used rodent OA model [20]. In addition, we analyzed histological changes in articular cartilage and multiple joint components, namely subchondral bone, osteophyte formation, and synovial membrane. Finally, we performed additional histomorphological analysis for cartilage thickness, intensity of matrix staining, chondrocyte density, osteophyte length, and synovial membrane thickness.

Materials and methods

Experimental animals and animal care

The study protocol was approved by the Animal Research Committee of the Graduate School of Medicine of Kanazawa University (Kanazawa, Japan; approval no. 204125) and conducted in accordance with the ARRIVE guidelines [21] and guidelines for the care and use of laboratory animals of Kanazawa University.

Twenty male Wistar rats (8 weeks old) were purchased from Japan SLC (Shizuoka, Japan) and housed under normal conditions for 5 weeks before the start of the experiments to acclimatize the animals to their new environment. One rat was housed per cage in a sanitary ventilated room under controlled temperature and humidity conditions and a 12-h light–dark cycle with ad libitum access to food and water. The experimental animals were monitored 2–3 times per week to control their health status, including general food and water intake, surgical wound condition, gait, and hindlimb suspension. The experimenter cleaned the cages once or twice every 2 weeks to keep the breeding environment clean.

The experimental protocol is shown in Fig 1. The rats were equally divided into two groups: post-traumatic OA (OA group) and hindlimb suspension after OA induction (OAHS group). OA was induced by surgical DMM. After surgical induction of OA, the rats were housed under normal conditions in the OA group and subjected to tail suspension during the experimental period in the OAHS group. The rats in the OAHS group were allowed to walk freely by using only their fore limbs. In both groups, we set the experimental period to 2 or 4 weeks after OA induction. Although it would have been ideal to evaluate OA until the end stage, long-term experimentation was difficult in the case of hindlimb suspension. Long-term suspension increases the suffering of the experimental animals and in some cases requires euthanasia. This also results in an increase in the number of experimental animals required. Consequently, this study focused only on early-stage OA.

Fig 1. The experimental design.

Fig 1

Open in a new tab

After starting the experiment, no further interventions, including range of motion exercise, were performed during the experimental period. No analgesics or anti-inflammatory drugs were administered to any of the rats during the peri-operative period. The non-use of these drugs was approved by the Animal Research Committee of our university as described above and was following previous studies [3, 12, 22, 23].

The rats in the OA group (n = 10) and OAHS group (n = 10) were kept under normal conditions for 5 weeks. All rats in both groups underwent DMM surgery on the left knee and sham surgery on the right knee. The rats in the OA group were kept under normal conditions for 2 or 4 weeks (n = 5, respectively). The rats in the OAHS group were subjected to tail suspension for 2 or 4 weeks (n = 5, respectively). After each experimental period, the rats were euthanized and assessed via histological analyses.

This study was concomitant to our previous study [12]. In order to reduce the number of animals used, based on the 3Rs principle of animal experiment (Reduction, Replacement and Refinement), a portion of the data used in this study was obtained from our previous study. Specifically, the data for the OA group at 2 and 4 weeks in the present study was the same as the data of the OA group at 2 and 4 weeks in the previous study. Since our previous study [12] is open-access and published under a CC-BY license, reuse of the data is permitted.

Surgical induction of OA

The same highly experienced operators (IT and KT) performed all DMM surgeries [12]. DMM was induced by transecting the medial meniscotibial ligament (MMTL) in the left knee joint [22, 23]. In the OA and OAHS groups, for internal controls, a sham operation was performed on the right knee joint by using the same approach without MMTL transection. A previous study reported that there was no significant difference in OA severity between sham-operated and age-matched non-operated control joints [24].

Hindlimb suspension

In the OAHS group, the rats were subjected to tail suspension throughout the experiment [11–13, 25]. Hindlimb suspension was performed according to Andries Ferreira’s modified tail suspension method. Briefly, under inhalation anesthesia with isoflurane, the tail of each rat was disinfected. A sterile steel wire was then used to drill into the proximal coccyx in which the wire remained and shaped into a ring. The tail ring was then connected with another wire to a track hung above the cage, thereby enabling the animals to move freely on their forelimbs in the cage.

Histological preparation

As described previously [12], decalcified paraffin sections were prepared for histology. Both knees were excised frontally to evaluate the histological changes in the medial tibiofemoral joints [11, 13]. Three paraffin sections (3-μm thickness) spaced at 200-μm intervals spanning the center of the medial tibiofemoral joint were stained with hematoxylin–eosin and toluidine blue to evaluate the severity and extent of cartilage lesions [26, 27]. Finally, the sections were viewed under a light microscope and imaged by using a digital camera (BX-51 and DP-74; Olympus Corporation, Tokyo, Japan) to evaluate the histological changes in the articular cartilage.

Histological analyses for articular cartilage

To assess the histological changes of OA, we quantitatively evaluated the articular cartilage of the tibia in the medial tibiofemoral joint by using the OA cartilage histopathology assessment system [28]. The OARSI scoring system, consisting of six grades and four stages on a scale from 0 (normal) to 24 (severe cartilage lesion) was used for semiquantitative evaluation of cartilage lesion severity [28]. The maximum OARSI score was the highest score among 3 sections and provides a measure of the “severity” of the cartilage lesions [12, 26, 27]. The summed OARSI score was the total of the three section scores and provides a measure of the “extent” of the cartilage lesions [12, 26, 27].

All histological scores of these scoring systems were determined by two blinded and trained independent observers (MH, a pathologist and IT). In our previous study, interclass correlation coefficients for the intra- and inter-rater reliabilities of the OARSI score with 95% confidence intervals were excellent: 0.94 (0.92–0.95) and 0.91 (0.89–0.93), respectively [13].

Histological analyses for subchondral bone and synovial membrane

For evaluation of subchondral bone damage and synovial inflammation, we chose a single section with the highest OARSI score among the three sections. To quantitatively evaluate subchondral bone damage of the tibia in the medial tibiofemoral joint, we used the calcified cartilage and subchondral bone damage score [29]. This score was established by Gerwin et al. in 2010 and ranges from 0 to 5, with higher values indicating severe subchondral lesion (S1 Table. Subchondral bone damage score) [29]. In addition, to quantitatively evaluate synovial inflammation in the medial tibiofemoral joint, we used the scoring system for synovitis (SSS, S2 Table. Scoring system for synovitis) [30]. This system consists of three items of hyperplasia, infiltration, and stroma. Each of them is judged on a four-level scale, and the total score is calculated. Therefore, the score is 0–9, with a higher score indicating more severe synovial inflammation.

Histomorphometrical analyses for articular cartilage

As described previously [11, 12, 28, 31, 32], Adobe Photoshop CC imaging software (Adobe Systems, Inc., San Jose, CA, USA) was used to perform histomorphometrical analyses for articular cartilage to evaluate the following four parameters: cartilage thickness, intensity of matrix staining with toluidine blue, chondrocyte density, and osteophyte length. For evaluation of cartilage thickness, staining intensity, and chondrocyte density, we chose the single section with the highest OARSI score among 3 sections. For evaluation of osteophyte length, we chose the single section with the longest osteophyte among the three sections.

To evaluate cartilage thickness, digitized images of the sections stained with toluidine blue were used. The cartilage thickness was defined as the distance between the cartilage surface and the osteochondral junction. We used the measured area of the cartilage with a width of 1 mm in the center of the lesion at the tibia in the medial tibiofemoral joint as the average cartilage thickness. To evaluate matrix intensity, digitized images of cartilage stained with toluidine blue were converted to greyscale (white, 255; black, 0) to assess the relative intensity of toluidine blue staining. The average staining intensity was calculated at the same area in the same manner as performed for the measurement of articular cartilage thickness. To evaluate chondrocyte density, digitized images of the sections stained with hematoxylin–eosin were used. Chondrocyte density was determined as the number of chondrocytes per unit area of cartilage. This unit area was calculated by using the same method as used for the above cartilage thickness, and the width to be measured was set to 200 μm. Chondrocytes with visible nuclei within the area of interest were counted manually.

Histomorphometrical analyses for osteophyte and synovial membrane

For evaluation of osteophyte length, we chose a single section with the longest osteophyte among the three sections. To evaluate osteophyte formation of the tibia in the medial tibiofemoral joint, we used digitized images of sections stained with toluidine blue and measured the length from the deepest point of its base at the chondro-osseous junction to the surface of the overlying cartilage at its thickest point [29].

For evaluation of the inflammation of synovial membrane and measurement of osteophyte length, we chose a single section with the thickest synovial membrane among the three sections and measured the synovial thickness at the medial tibiofemoral joint.

Statistical analysis

All statistical analyses were performed by using JMP 14 software (SAS Institute, Cary, NC, USA). All data were statistically analyzed as parametric data. The sample size was five for each group. Descriptive statistics were calculated as the median with interquartile range for the OARSI score, subchondral bone score, and synovitis score and as the mean with standard deviation for body weight and histomorphometrical data. We considered P < .05 as indicative of statistical significance for all analyses; exact P values are shown in the figures. For all of the data, we performed analysis of variance followed by the post-hoc Tukey’s honest significant difference test.

A sample-size calculation was performed by using the sample size and power tool in G*power 3.1 (free; available at https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower.html) [33] and pilot experimental data of the main parameter and maximum and summed OARSI scores at 4 weeks, including the first five rats, in the OA and OAHS groups. For these scores, a minimum of 4 and 3 were required in the OA and OAHS groups, respectively, with a power of 0.95 and a significance level of P < .05. Therefore, we set the sample size to five rats per group.

Results

General condition

After a few minutes from the end of the inhalation-induced anesthesia, all rats attained consciousness and began to move around in their cages. No experimental animals died through accidents or other unexpected causes during any of the experiments. None of the rats developed infections in their knee joints or tail wounds. All animals completed the study safely, as per the experimental design, without any unexpected adverse events. Please refer to S3 Table (Body weight) for data on weight changes in the rats during the experiment.

Histological scores for articular cartilage and subchondral bone

In Figs 2 and 3, the maximum and summed OARSI score was significantly lower in OAHS group than in the OA group at 2 and 4 weeks after OA induction. In the subchondral bone damage scores, no significant differences were found between the two groups at 2 and 4 weeks. Please refer to the S4 Table (Histological scores) for detailed information on the histological scores.

Fig 2. Results of histological changes in the articular cartilage and subchondral bone.

Fig 2

Open in a new tab

The maximum OARSI score (A) and summed OARSI score (B) were significantly lower in the OAHS group than those in the OA group at 2 and 4 weeks. For the subchondral bone score (C), no significant difference was found between the two groups at 2 and 4 weeks. Representative histological changes in the articular cartilage are shown in (D) (2 weeks) and (E) (4 weeks). At 2 weeks, in the OA and OAHS groups, the articular surface was intact, but the staining intensity with toluidine blue was partially reduced in the OA group. At 4 weeks, fibrillation and fissures were detected and the area of reduced staining with toluidine blue was expanded in the OA group. In the OAHS group, a slight decrease in staining intensity on the surface of the articular cartilage was found, but the surface remained almost intact. Scale bar = 500 μm.

Fig 3. Histological findings of the tibial articular cartilage at a high magnification.

Fig 3

Open in a new tab

(A) and (B) represent histological images at 2 and 4 weeks, respectively. In the OA group, fibrillation was detected at 2 weeks, and a moderate decrease in toluidine blue staining intensity was found from the surface to the middle layer. At 4 weeks, fibrillation and fissures were detected, and the staining intensity was further decreased. In the OAHS group, the articular surface was intact at both 2 and 4 weeks, but the overall staining intensity was slightly reduced. Scale bar = 100 μm.

Histomorphometrical analyses for articular cartilage and osteophyte

No significant differences in cartilage thickness in the operated limb were found between the OA and OAHS groups at 2 and 4 weeks after surgery. However, the cartilage thickness in the operated limb was significantly less than that in the sham limb in both groups at 2 and 4 weeks after surgery (Fig 4 and S5 Table. Histomorphometrical results).

Fig 4. Results of histomorphological changes in the articular cartilage.

Fig 4

Open in a new tab

(A-C) present cartilage thickness, staining intensity, and cell density, respectively. No significant differences in cartilage thickness and matrix intensity of the operated limb were found between the two groups at 2 and 4 weeks. Cell density of the operated limb in the OA group was significantly less than that in the OAHS group at 2 and 4 weeks.

No significant differences in matrix staining intensity by toluidine blue of the operated limb were found between the two groups at 2 and 4 weeks (Fig 4 and S5 Table. Histomorphometrical results).

Chondrocyte density of the operated limb was significantly lower in the OA group than that in the OAHS group at 2 and 4 weeks. And operated limb density was significantly less than sham limb density in the OA group at 2 and 4 weeks after surgery, but no significant differences were found between the operated and sham limbs in the OAHS group at 2 and 4 weeks (Fig 4 and S5 Table. Histomorphometrical results).

In the osteophyte length of the operated limb, no significant difference was found between the OA and OAHS groups at 2 weeks after surgery, but the length was longer in the OA group than in the OAHS group at 4 weeks. There was greater osteophyte formation in the operated limb than in the sham limb in both the groups at 2 and 4 weeks (Fig 5 and S5 Table. Histomorphometrical results).

Fig 5. Results of histological and histomorphological changes in osteophyte formation.

Fig 5

Open in a new tab

As the graph (A) and histological images (B) show, no significant difference in the operated limb was found between both groups at 2 weeks, but osteophyte length was longer in the OA group than that in the OAHS group at 4 weeks. Scale bar = 500 μm.

Histological score and histomorphometrical analyses for synovial membrane

In SSS, no significant differences in the operated limb were found between both groups at 2 and 4 weeks after surgery (Fig 6 and Supplementary Result). In synovial thickness, at 2 weeks after surgery, the thickness of operated limb was significantly thinner in OAHS group than in OA group; however, at 4 weeks, no significant difference in the operated limb was found between the OA and OAHS groups (Fig 6, S4 Table. Histological scores, and S5 Table. Histomorphometrical results).

Fig 6. Results of histological and histomorphological changes in synovial inflammation.

Fig 6

Open in a new tab

As the graph (A and B) and histological images (C) show, for SSS, no significant difference in the operated limb was found between the OA and OAHS groups throughout the experimental period. Regarding synovial thickness, at 2 weeks after surgery, the operated limb was significantly thinner in the OAHS group than that in the OA group. In the histological images, no significant difference in the operated limb was found between the OA and OAHS groups at 4 weeks. Slight hyperplasia of the synovial surface cells was observed in the OA group at 2 weeks. In both groups, slight inflammatory cell infiltration and slight stroma activation were observed at 2 and 4 weeks. Scale bar = 50 μm.

Discussion

The purpose of this study was to determine the histopathological effect of reduced knee joint loading on OA progression using surgical model that is pathologically relevant to human secondary OA, and hindlimb suspension model. As a result, it was demonstrated that the histological OA changes in cartilage, osteophyte formation, and synovial inflammation developed more slowly when knee joint loading was reduced. Furthermore, reducing joint loading maintained matrix staining and chondrocyte density by inhibiting OA progression, thereby keeping the articular cartilage condition closer to normal histologically and histomorphologically. Therefore, these histological findings provide strong basic evidence to support the recommendation of weight management for OA in the above-mentioned guidelines [7–10].

In the present study, multiple joint components were evaluated, and the results showed no obvious significant difference in histological changes in the subchondral bone. Also, no significant difference in thickness and matrix staining of the articular cartilage was observed. This may be due to the fact that the evaluation period was only in the early postoperative period of 2–4 weeks. Ideally, the long-term course should be evaluated until the end of OA, such as 8–16 weeks, but the tail suspension for more than 8 weeks often leads to tail injury. For long-term experiments in tail suspension, tail injury may cause distress to the experimental animal and dropout by euthanasia from the experimental design may be necessary. In such cases, more experimental animals would be needed, but such cases are inappropriate from the standpoint of experimental ethics. Therefore, our focus was to observe the early postoperative period of 2–4 weeks.

As shown by these histological and histomorphological results described above, OA progression was significantly suppressed by reducing the load on the knee joints. However, in hindlimb suspension models such as the one used in this study, disuse atrophy of the articular cartilage is a result of the unloading environment [4, 11, 12]. In the results of the present study, compared to the degree of OA progression, the OAHS group showed a decrease in cartilage thickness almost equal to that of the OA group. Therefore, we believe that it is highly likely that disuse atrophy of articular cartilage associated with non-loading has occurred. In addition, disuse atrophy of articular cartilage is one of the factors that accelerates OA progression, as we reported in our previous study [12]. Therefore, clinically, to suppress OA progression and not cause disuse atrophy of articular cartilage, intermittent loading or partial loading may be effective during long-term unloading. Further studies are necessary to clarify the appropriate amount and time of the loading.

The present study consists of the results of our previous study and additional experiments specified herein [12]. To avoid significant differences in experimental techniques between the two experiments, we paid careful attention to three aspects: surgery, staining, and evaluation. OA models were created by the same surgeon using the same surgical technique as in the previous study [22, 23]. Additionally, the surgeon’s proficiency in the surgical technique was confirmed by preliminary experimentation. Furthermore, there was no marked difference in operative time between experiments. Staining was performed in the same environment and conditions, including staining solution used, staining time, room temperature, and laboratory equipments. Before conducting additional experiments, we preliminarily verified that there was no marked difference in staining intensity by the same technique. Histological evaluation was performed by two well-trained double-blinded experimenters. The same experimenter performed the histological evaluation in the two experiments and the two had high intra- and inter-rater reliability [13]. Based on the above, we consider our two experiments to have been conducted with high reliability and reproducibility.

This study had four limitations. First, only histological and histomorphometrical analyses were performed. To clarify the effect of load reduction on OA progression, further biochemical studies for catabolic markers, such as MMP-13 and ADAMTS5, or anabolic markers, such as aggrecan and type II collagen, would be necessary, and further studies using mechanical tests would also be needed. Second, the sample size was relatively small. We used G*power to calculate the required sample size, but due to the use of multiple comparisons in the statistical analysis and the large individual differences in scores, a larger sample size may be desired. Third, the sex of the experimental animals we used was male only; since OA generally occurs more frequently in females [34], it may have been necessary to equalize the sex of the experimental animals in this study. Fourth, there is no quantitative assessment of the mechanical stress occurring in the knee joint. Tail suspension is an experimental method that allows the rats to reduce bodyweight load on the knee joint. However, the rats can flex and extend their knee joints freely (actively or passively), and with this movement, mechanical stresses such as shear and compressive forces are generated in the articular cartilage. The metabolism of articular cartilage is influenced by these mechanical stresses [35, 36]. In addition, the number of steps and other activities of the experimental animals in their cages were not evaluated.

Conclusions

Reducing knee joint loading inhibited histological changes in articular cartilage, osteophyte formation, and synovial inflammation in early-stage OA. In addition, reduced loading kept the articular cartilage condition closer to normal histologically and histomorphologically. Therefore, our findings support the latest clinical guidelines for OA treatment. Further studies using biochemical and mechanical analyses are required to clarify the structure underlying delayed OA progression caused by joint-load reduction.

Supporting information

S1 Table. Subchondral bone damage score.

(DOCX)

Click here for additional data file. (23.2KB, docx)
S2 Table. Scoring system for synovitis.

(DOCX)

Click here for additional data file. (22.8KB, docx)
S3 Table. Body weight.

(DOCX)

Click here for additional data file. (29.7KB, docx)
S4 Table. Histological scores.

(DOCX)

Click here for additional data file. (30.3KB, docx)
S5 Table. Histomorphometrical results.

(DOCX)

Click here for additional data file. (33.7KB, docx)

Acknowledgments

The authors thank the members of the Department of Human Pathology at the Kanazawa University Graduate School of Medicine for providing advice about the histopathological techniques.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by a JSPS KAKENHI grant-in-aid for Young Scientists B (number: 17K13051) and for Early Career Scientists (number: 20K19444).

References

  • 1.Tsezou A. Osteoarthritis year in review 2014: genetics and genomics. Osteoarthr Cartil. 2014;22: 2017–2024. doi: 10.1016/j.joca.2014.07.024 [DOI] [PubMed] [Google Scholar]
  • 2.Moyer RF, Ratneswaran A, Beier F, Birmingham TB. Osteoarthritis year in review 2014: mechanics—basic and clinical studies in osteoarthritis. Osteoarthr Cartil. 2014;22: 1989–2002. doi: 10.1016/j.joca.2014.06.034 [DOI] [PubMed] [Google Scholar]
  • 3.Iijima H, Aoyama T, Ito A, Yamaguchi S, Nagai M, Tajino J, et al. Effects of short-term gentle treadmill walking on subchondral bone in a rat model of instability-induced osteoarthritis. Osteoarthr Cartil. 2015;23: 1563–1574. doi: 10.1016/j.joca.2015.04.015 [DOI] [PubMed] [Google Scholar]
  • 4.Vincent TL, Wann AKT. Mechanoadaptation: articular cartilage through thick and thin. J Physiol. 2019;597: 1271–1281. doi: 10.1113/JP275451 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bader DL, Salter DM, Chowdhury TT. Biomechanical influence of cartilage homeostasis in health and disease. Arthritis 2011;2011: 979032. doi: 10.1155/2011/979032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Clements KM, Bee ZC, Crossingham GV, Adams MA, Sharif M. How severe must repetitive loading be to kill chondrocytes in articular cartilage? Osteoarthr Cartil. 2001;9: 499–507. doi: 10.1053/joca.2000.0417 [DOI] [PubMed] [Google Scholar]
  • 7.McAlindon TE, Bannuru RR, Sullivan MC, Gee TL, Bourne R, Wells GA. OARSI guidelines for the non-surgical management of knee osteoarthritis. Osteoarthr Cartil. 2014;22: 363–388. doi: 10.1016/j.joca.2014.01.003 [DOI] [PubMed] [Google Scholar]
  • 8.Osteoarthritis: care and management NICE guidelines [ICG177] [Internet]. London: The National Institute for Health and Care Excellence (NICE) provides national guidance and advice to improve health and social care [updated 2014 February; cited 2018 April 5]. Available from: http://www.nice.org.uk/guidance/cg177.
  • 9.Treatment of Osteoarthritis of the Knee - 2nd Edition (PDF) [Internet]. Illinois: The American Academy of Orthopaedic Surgeons [updated 2013 May 18; cited 2018 March 28]. Available from: http://www.aaos.org/cc_files/aaosorg/research/guidelines/treatmentofosteoarthritisofthekneeguideline.pdf.
  • 10.Bannuru RR, Osani MC, Vaysbrot EE, Arden NK, Bennell K, Bierma-Zeinstra SM, et al. OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis. Osteoarthr Cartil. 2019;27: 1578–1589. doi: 10.1016/j.joca.2019.06.011 [DOI] [PubMed] [Google Scholar]
  • 11.Takahashi I, Matsuzaki T, Kuroki H, Hoso M. Disuse histological changes of an unloading environment on joint components in rat knee joints. Osteoarthr Cartil. Open 2019;1: 100008. doi: 10.1016/j.ocarto.2019.100008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Takahashi I, Matsuzaki T, Kuroki H, Hoso M. Disuse atrophy of articular cartilage induced by unloading condition accelerates histological progression of osteoarthritis in a post-traumatic rat model. Cartilage, in press. [DOI] [PMC free article] [PubMed]
  • 13.Takahashi I, Matsuzaki T, Kuroki H, Hoso M. Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis. Osteoarthr Cartil. 2019;27: 1084–1093. doi: 10.1016/j.joca.2019.03.001 [DOI] [PubMed] [Google Scholar]
  • 14.Ferreira-Gomes J, Adaes S, Sousa RM, Mendonca M, Castro-Lopes JM. Dose-dependent expression of neuronal injury markers during experimental osteoarthritis induced by monoiodoacetate in the rat. Mol Pain. 2012;8: 50. doi: 10.1186/1744-8069-8-50 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Lampropoulou-Adamidou K, Lelovas P, Karadimas EV, Liakou C, Triantafillopoulos IK, Dontas I, et al. Useful animal models for the research of osteoarthritis. Eur J Orthop Surg Traumatol: Orthopedie Traumatologie 2014;24: 263–271. doi: 10.1007/s00590-013-1205-2 [DOI] [PubMed] [Google Scholar]
  • 16.Yuan XL, Meng HY, Wang YC, Peng J, Guo QY, Wang AY, et al. Bone-cartilage interface crosstalk in osteoarthritis: potential pathways and future therapeutic strategies. Osteoarthr Cartil. 2014;22: 1077–1089. doi: 10.1016/j.joca.2014.05.023 [DOI] [PubMed] [Google Scholar]
  • 17.Kühn K, D’Lima DD, Hashimoto S, Lotz M. Cell death in cartilage. Osteoarthr Cartil. 2004;12: 1–16. [DOI] [PubMed] [Google Scholar]
  • 18.Sandell LJ, Aigner T. Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res. 2001;3: 107–113. doi: 10.1186/ar148 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Teeple E, Jay GD, Elsaid KA, Fleming BC. Animal models of osteoarthritis: challenges of model selection and analysis. AAPS J 2013;15: 438–446. doi: 10.1208/s12248-013-9454-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.van der Kraan PM. Factors that influence outcome in experimental osteoarthritis. Osteoarthr Cartil. 2017;25: 369–375. doi: 10.1016/j.joca.2016.09.005 [DOI] [PubMed] [Google Scholar]
  • 21.du Sert NP, Ahluwalia A, Alam S, Avey MT, Baker M., Browne WJ. Reporting animal research: explanation and elaboration for the arrive guidelines 2.0. PLoS Biology, 2020;18: 3000411. doi: 10.1371/journal.pbio.3000411 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Glasson SS, Blanchet TJ, Morris EA. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthr Cartil. 2007;15: 1061–1069. doi: 10.1016/j.joca.2007.03.006 [DOI] [PubMed] [Google Scholar]
  • 23.Iijima H, Aoyama T, Ito A, Tajino J, Nagai M, Zhang X, et al. Destabilization of the medial meniscus leads to subchondral bone defects and site-specific cartilage degeneration in an experimental rat model. Osteoarthr Cartil. 2014;22: 1036–1043. doi: 10.1016/j.joca.2014.05.009 [DOI] [PubMed] [Google Scholar]
  • 24.Loeser RF, Olex AL, McNulty MA, Carlson CS, Callahan MF, Ferguson CM, et al. Microarray analysis reveals age-related differences in gene expression during the development of osteoarthritis in mice. Arthritis Rheum 2012;64: 705–717. doi: 10.1002/art.33388 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Ferreira JA, Crissey JM, Brown M. An alternant method to the traditional NASA hindlimb unloading model in mice. J Vis Exp. 2011;49. doi: 10.3791/2467 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Iijima H, Aoyama T, Ito A, Tajino J, Yamaguchi S, Nagai M, et al. Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model. Osteoarthr Cartil. 2017;25: 964–975. doi: 10.1016/j.joca.2016.12.008 [DOI] [PubMed] [Google Scholar]
  • 27.Iijima H, Aoyama T, Ito A, Tajino J, Yamaguchi S, Nagai M, et al. Exercise intervention increases expression of bone morphogenetic proteins and prevents the progression of cartilage-subchondral bone lesions in a post-traumatic rat knee model. Osteoarthr Cartil. 2016;24: 1092–1102. doi: 10.1016/j.joca.2016.01.006 [DOI] [PubMed] [Google Scholar]
  • 28.Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthr Cartil. 2006;14: 13–29. doi: 10.1016/j.joca.2005.07.014 [DOI] [PubMed] [Google Scholar]
  • 29.Gerwin N, Bendele AM, Glasson S, Carlson CS. The OARSI histopathology initiative—recommendations for histological assessments of osteoarthritis in the rat. Osteoarthr Cartil. 2010;18: S24–S34. doi: 10.1016/j.joca.2010.05.030 [DOI] [PubMed] [Google Scholar]
  • 30.Krenn V, Morawietz L, Häupl T, Neidel J, Petersen I, König A. Grading of chronic synovitis–a histopathological grading system for molecular and diagnostic pathology. Pathol Res Pract. 2002;198: 317–325. doi: 10.1078/0344-0338-5710261 [DOI] [PubMed] [Google Scholar]
  • 31.Nomura M, Sakitani N, Iwasawa H, Kohara Y, Takano S, Wakimoto Y, et al. Thinning of articular cartilage after joint unloading or immobilization. An experimental investigation of the pathogenesis in mice. Osteoarthr Cartil. 2017;25: 727–736. doi: 10.1016/j.joca.2016.11.013 [DOI] [PubMed] [Google Scholar]
  • 32.Iijima H, Aoyama T, Ito A, Tajino J, Nagai M, Zhang X, et al. Immature articular cartilage and subchondral bone covered by menisci are potentially susceptive to mechanical load. BMC Musculoskelet Disord. 2014;15: 101. doi: 10.1186/1471-2474-15-101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Faul F, Erdfelder E, Lang AG, Buchner A. G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav ResMethods 2007;39: 175–191. doi: 10.3758/bf03193146 [DOI] [PubMed] [Google Scholar]
  • 34.Salmon JH, Rat AC, Achit H, Ngueyon-Sime W, Gard C, Guillemin F, et al. Health resource use and costs of symptomatic knee and/or hip osteoarthritis. Osteoarthr Cartil. 2019;27: 1011–1017. doi: 10.1016/j.joca.2019.03.005 [DOI] [PubMed] [Google Scholar]
  • 35.Flachsmann R, Broom ND, Hardy AE. Deformation and rupture of the articular surface under dynamic and static compression. J Orthop Res. 2001;19: 1131e9. doi: 10.1016/S0736-0266(01)00049-3 [DOI] [PubMed] [Google Scholar]
  • 36.Nugent-Derfus GE, Takara T, O’Neilly JK, Cahill SB, Gortz S, Pong T, et al. Continuous passive motion applied to whole joints stimulates chondrocyte biosynthesis of PRG41. Osteoarthr Cartil. 2007;15: 566e74. [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Lin Han

15 Apr 2021

PONE-D-21-06339

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early stage using a post-traumatic rat model

PLOS ONE

Dear Dr. Takahashi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 30 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Lin Han

Academic Editor

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 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

2. Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, some of which you are an author.

https://journals.sagepub.com/doi/10.1177/1947603520982350

https://www.oarsijournal.com/article/S1063-4584(19)30896-9/fulltext

We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.

Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.

3. As part of your revisions, please update your Methods/Results to address the following items pertaining to animal research, animal health and welfare.  (1) The frequency of animal monitoring, including the specific criteria you used to monitor animal health as well as a description of humane endpoints. (2) Specific animal welfare considerations, including efforts to alleviate suffering (supportive care - gel-packs, cage modifications/environmental enrichment, administration of analgesics, anesthetics, etc.), (3) The rate of mortality during the study (if applicable) and if animals died unexpectedly or prior to the experimental endpoint, an explanation for the cause of death. (4) Details of unanticipated adverse events such as illness or injury as a result of the experimental procedures; (5) Lastly, please complete and submit the ARRIVE Guidelines 2.0 checklist (Essential 10 version): https://arriveguidelines.org/resources/author-checklists.

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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: In their manuscript “Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early stage using a post-traumatic rat model” Takahashi et al. report the effect of reducing the loading on knee joints during early osteoarthritis (OA) progression by using rat OA model. They find that the histological changes in articular cartilage and osteophyte formation were milder and slower with decreasing load to the knee. With more loading, the chondrocyte density is significantly lower. Synovitis was severe with loading increase. In summary, with reducing knee joint loading, the histological OA changes in cartilage, osteophyte formation as well as synovial inflammation got inhibited in early OA progression.

The authors present a well-written manuscript. They have investigated the knee joint component changes in histomorphological level such as knee cartilage, osteophyte and synovium. The manuscript is of interest to the readership of PlosOne and should be published after the following minor points have been addressed and corrected by authors:

1. Title: Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early stage using a post-traumatic rat model. Missing a subject for early stage, consider adding “early stage of osteoarthritis” or consider change title to “Reduction of knee joint load alleviates the early stage of osteoarthritis progression by suppressing cartilage degeneration, osteophyte formation, and synovitis using a post traumatic rat model”.

2. Line 28: The study aim was… consider change to “The aim of this study was” or “The study’s aim was”.

3. Line 109: 2 weeks and 4 weeks. You have mentioned the reason why to study 2 weeks and 4 weeks later in your discussion, it would be better to specify it here as well.

4. Line 146: Three paraffin sections spanning across the entire knee joint? Please explain or add a reference of why three paraffin sections were chosen.

5. Line 208: All data were analyzed as parametric data? Please identify the data are normally distributed or not.

6. Line 287: “Histological effect of reduced knee joint loading to on osteophyte”, please delete “to”.

7. Line 329: “euthanasia by dropout from the experimental design”. It should be “dropout by euthanasia from the experimental design”.

8. For the body weight, there is a significant difference between OAHS group and OA group at 2 weeks. Can you give some speculations of why the body weight drops at 2 weeks in OAHS group?

9. Figure 4B, y-axis label incorrect. The graph data are not consistent with S5 Table Histomorphometrical results-Matrix intensity data.

10. In the figure, the operated group showing as DMM. In manuscript and supplementary document, the operated group showing as operated. Please keep consistent by using either DMM or operated.

11. Ref: some references are without doi, formats are not consistent.

Reviewer #2: The paper investigated on the effect of reduced load on a DMM-induced OA progression. The topic is of great interest and the results show promising evidence that the reduction of knee joint load supressed the OA progression. However I have 2 major concerns.

1. As the author mentioned, the OA group at 2 and 4 weeks data are from the previous study. However I did not see any explanation about how the surgical procedure was controlled to be consistent between the previous study and this study (I assume the surgeries are done at different times and/or by different personnel?). Only staining method control was described. DMM surgeries performed by different personnels can result in different outcomes due to operational variations so it is important the author have this addressed.

2. Also mentioned in the discussion section, the study used only histological and histomorphometrical analysis to support the conclusion. This does not seem scientifically justifiable. There are several studies on the mechanical properties of articular cartilage and it does not always go consistent with histological signs. For example in Doyran et al 2017 Osteoarthritis Cartilage, it was demonstrated that after the DMM surgery, mechanical properties start to decrease just a few days after the surgery while histological signs doesn't show up until 2-4 weeks after the surgery. The reduced histological sign suggests that the DMM-induced OA is delayed by reduced load but at least one more evidence is needed to demonstrate the results, for example immunohistochemistry staining, biochemical assay, or mechanical test.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Jul 16;16(7):e0254383. doi: 10.1371/journal.pone.0254383.r002

Author response to Decision Letter 0

11 May 2021

Responses to Journal Editor’s Additional Comments

Comment 1

As part of your revisions, PLOS ONE asked to provide additional methodological details pertaining to animal research, health and well-being. In response to this request, you have kindly revised your manuscript to include the following details (lines 101 - 103):

"After starting the experiment, no further interventions, including range of motion exercise, were performed during the experimental period. No analgesics or anti-inflammatory drugs were administered to any of the rats."

Can you please clarify - did you provide animals with peri- or post-operative analgesics? If you did not, please provide a scientific justification and indicate whether your animal research oversight committee/IACUC specifically approved this failure to provide pain relief.

Author Action:

In accordance with the editor’s comment, we have revised the following sentences.

Original sentence:

No analgesics or anti-inflammatory drugs were administered to any of the rats.

Revised sentences (page 6, lines 102–105):

No analgesics or anti-inflammatory drugs were administered to any of the rats during the peri-operative period. The non-use of these drugs was approved by the Animal Research Committee of our university as described above and was following previous studies [3,12,22,23].

Comment 2

Additionally, you write the following (lines 141-142):"Since our previous study [12] is open-access and copyrighted under a CC-BY license, reuse of the data is permitted." Please replace the word, "copyrighted" with "published".

(From https://creativecommons.org/about/program-areas/open-access/: "Open access literature is defined as 'digital, online, free of charge, and free of most copyright and licensing restrictions. The recommendations of the Budapest Open Access Declaration—including the use of liberal licensing (such as CC BY)— is widely recognized in the community as a means to make a work truly open access.")

Author Action:

In accordance with the editor’s comment, we have replaced the word "copyrighted" with "published" in the corresponding sentence.

 

Responses to Academic Editor’s Comments

Comment 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

Author Action:

We have revised the manuscript, file naming, and author information in accordance with the journal’s style requirements.

Comment 2

Thank you for submitting the above manuscript to PLOS ONE. During our internal evaluation of the manuscript, we found significant text overlap between your submission and the following previously published works, some of which you are an author.

https://journals.sagepub.com/doi/10.1177/1947603520982350

https://www.oarsijournal.com/article/S1063-4584(19)30896-9/fulltext

We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications.

Please revise the manuscript to rephrase the duplicated text, cite your sources, and provide details as to how the current manuscript advances on previous work. Please note that further consideration is dependent on the submission of a manuscript that addresses these concerns about the overlap in text with published work.

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.

Author Action:

We completely agree with the editor’s comment. As the editor pointed out, we identified sections that were identical or similar to our previous study. We have revised all the relevant sections, aside from the Methods section. However, if by any chance, identical or similar phrasing remains, please let us know; we will do our best to correct it immediately.

Comment 3

As part of your revisions, please update your Methods/Results to address the following items pertaining to animal research, animal health and welfare. (1) The frequency of animal monitoring, including the specific criteria you used to monitor animal health as well as a description of humane endpoints. (2) Specific animal welfare considerations, including efforts to alleviate suffering (supportive care - gel-packs, cage modifications/environmental enrichment, administration of analgesics, anesthetics, etc.), (3) The rate of mortality during the study (if applicable) and if animals died unexpectedly or prior to the experimental endpoint, an explanation for the cause of death. (4) Details of unanticipated adverse events such as illness or injury as a result of the experimental procedures; (5) Lastly, please complete and submit the ARRIVE Guidelines 2.0 checklist (Essential 10 version): https://arriveguidelines.org/resources/author-checklists.

Author Action:

In accordance with the reviewer’s comment, we have added the following sentences. We have updated and resubmitted the ARRIVE Guidelines 2.0 checklist (Essential 10 version) to be based on the revisions.

Added information (page 5–6, lines 86–89):

The experimental animals were monitored 2–3 times per week to control their health status, including general food and water intake, surgical wound condition, gait, and hindlimb suspension. The experimenter cleaned the cages once or twice every 2 weeks to keep the breeding environment clean.

Added information (page 13, lines 222–225):

No experimental animals died through accidents or other unexpected causes during any of the experiments. None of the rats developed infections in their knee joints or tail wounds. All animals completed the study safely, as per the experimental design, without any unexpected adverse events.

 

Responses to Reviewer #1 Comments

Comment 1:

Title: Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early stage using a post-traumatic rat model. Missing a subject for early stage, consider adding “early stage of osteoarthritis” or consider change title to “Reduction of knee joint load alleviates the early stage of osteoarthritis progression by suppressing cartilage degeneration, osteophyte formation, and synovitis using a post traumatic rat model”.

Author Action:

In accordance with the reviewer’s comment, we have revised the title.

Original title:

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early stage using a post-traumatic rat model

Revised title:

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis using a post-traumatic rat model

Comment 2:

Line 28: The study aim was… consider change to “The aim of this study was” or “The study’s aim was”.

Author Action:

In response to the reviewer’s comment, we have revised the sentence.

Original sentence:

The study aim was to clarify the histological effect of reducing the loading to knee on cartilage degeneration, osteophyte formation, and synovitis as osteoarthritis (OA) progresses in the early stage using a post-traumatic rat model.

Revised sentence (page 2, lines 17–19):

The purpose of this study was to clarify the histological effect of reducing the loading to knee on cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis (OA) using a post-traumatic rat model.

Comment 3:

Line 109: 2 weeks and 4 weeks. You have mentioned the reason why to study 2 weeks and 4 weeks later in your discussion, it would be better to specify it here as well.

Author Action:

In accordance with the reviewer’s comment, we have added the following sentences.

Added sentences (page 6, lines 96–100):

Although it would have been ideal to evaluate OA until the end stage, long-term experimentation was difficult in the case of hindlimb suspension. Long-term suspension increases the suffering of the experimental animals and in some cases requires euthanasia. This also results in an increase in the number of experimental animals required. Consequently, this study focused only on early-stage OA.

Comment 4:

Line 146: Three paraffin sections spanning across the entire knee joint? Please explain or add a reference of why three paraffin sections were chosen.

Author Action:

We evaluated the articular cartilage of the center of the tibia in the medial tibiofemoral joint. We chose three paraffin sections to assess the extent of the osteoarthritis lesions, as shown in lines 166–169 of the manuscript. This method has been reported in previous studies by Iijima et al1, 2. These two references have been added as citations. In the present study, we used the same method as in the previous studies to evaluate histological changes. Specifically, three paraffin sections were created every 200 μm, and the total OARSI score was calculated by summing the OARSI scores of the three sections to evaluate the extent of the lesion. Also, the total OARSI score of the three paraffin sections was used as the total OARSI score to evaluate the severity of osteoarthritis.

Following the reviewer’s comment, we have revised the below-mentioned sentences. Additionally, the following two references have been added as citations.

Original sentence:

As described previously [12], decalcified paraffin sections were prepared for histology. Both knees were excised frontally to evaluate the histological changes in the medial tibiofemoral joints [11,13]. Three paraffin sections (3-µm thickness) spaced at 200-µm intervals spanning the entire knee joint were stained with hematoxylin–eosin and toluidine blue to evaluate the severity of cartilage lesions.

Revised sentence (page 8, lines 137–141):

As described previously [12], decalcified paraffin sections were prepared for histology. Both knees were excised frontally to evaluate the histological changes in the medial tibiofemoral joints [11,13]. Three paraffin sections (3-µm thickness) spaced at 200-µm intervals spanning the center of the medial tibiofemoral joint were stained with hematoxylin–eosin and toluidine blue to evaluate the severity and extent of cartilage lesions [26,27].

1. Iijima H, Aoyama T, Ito A, Tajino J, Yamaguchi S, Nagai M, et al. Exercise intervention increases expression of bone morphogenetic proteins and prevents the progression of cartilage-subchondral bone lesions in a post-traumatic rat knee model. Osteoarthr Cartil. 2016;24: 1092-1102. doi:10.1016/j.joca.2016.01.006.

2. Iijima H, Aoyama T, Ito A, Tajino J, Yamaguchi S, Nagai M, et al. Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model. Osteoarthr Cartil. 2017;25: 964-975. doi:10.1016/j.joca.2016.12.008.

Comment 5:

Line 208: All data were analyzed as parametric data? Please identify the data are normally distributed or not.

Author Action:

Thank you for pointing this out. We had received the critique previously, in the peer review of our submission1. In that case, the reviewer, presumed to be a biostatistician, had instructed us as follows: “With sample size of 5 per group, it is not possible to assess the distribution of the data from the data itself and use of tests, such as Shapiro–Wilk or Levene is misleading. The authors should consider what is known about the distribution of weight, OARSI and Mankin scores and choose a statistical model thereafter. I would recommend using parametric methods.”

In addition, at that time, because it was difficult for us to accurately resolve this reviewer’s comment, we consulted with a Professor and the Chief of the Department of Biostatistics at the Innovative Clinical Research Center, Kanazawa University Hospital, who suggested using parametric methods, similar to the advice of the peer reviewer.

Therefore, we did not test the normality and homogeneity of variance between each group because of the small sample size and used Tukey’s Honest Significant Difference test for all groups.

Thus, in the present study, we used a parametric method because we performed the study with a small sample size. In addition, we used G*power to ensure statistical sample size validity.

1. Takahashi I, Matsuzaki T, Kuroki H, Hoso M. Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis. Osteoarthr Cartil. 2019;27: 1084-1093. doi:10.1016/j.joca.2019.03.001.

Comment 6:

Line 287: “Histological effect of reduced knee joint loading to on osteophyte”, please delete “to”.

Author Action:

According to the reviewer’s comment, we have revised the following sentence.

Original sentence:

Figure 5. Histological effect of reduced knee joint loading to on osteophyte formation of the medial tibia.

Revised sentence (page 16, lines 282):

Fig 5. Results of histological and histomorphological changes in osteophyte formation.

Comment 7:

Line 329: “euthanasia by dropout from the experimental design”. It should be “dropout by euthanasia from the experimental design”.

Author Action:

According to the reviewer’s comment, we have revised the following sentence.

Original sentence:

For long-term experiment in tail suspension, tail injury may cause distress to the experimental animal and euthanasia by dropout from the experimental design may be necessary.

Revised sentence (page 19, lines 323–325):

For long-term experiments in tail suspension, tail injury may cause distress to the experimental animal and dropout by euthanasia from the experimental design may be necessary.

Comment 8:

For the body weight, there is a significant difference between OAHS group and OA group at 2 weeks. Can you give some speculations of why the body weight drops at 2 weeks in OAHS group?

Author Action:

There are several possible causes for significant weight loss in the OAHS group at 2 weeks. We believe that one factor is stress associated with environmental changes, leading to an associated decrease in food intake and water consumption. Therefore, we believe that the significant weight loss observed at 2 weeks after hindlimb suspension disappeared at 4 weeks after hindlimb suspension due to adaptation to the rearing environment. However, we did not measure the food and water consumption of the animals or the serum albumin content and total protein level, so this remains speculative. To the best of our knowledge, there are some previous studies on hindlimb suspension using rats. In previous studies that described changes in body weight, there is currently no consensus on the cause of these weight changes. Some studies have reported that body weight of hindlimb-suspension rats was not significantly different than that of the control rats1-3. Conversely, other studies have reported that body weight of the hindlimb-suspension rats was lower than that of the control rats4,5.

1. Matsuzaki T, Yosida S, Ikeda A, Hoso M. Changes in joint components after knee immobilization associated with hindlimb unweighting in rats. Journal of Wellness and Health Care 2018;42:33-40.

2. Kaneguchi A, Ozawa J, Kawamata S, Kurose T, Yamaoka K. Intermittent whole-body vibration attenuates a reduction in the number of the capillaries in unloaded rat skeletal muscle. BMC Musculoskeltal Disorders 2014;15:315.

3. Ferreira JA, Crissey JM, Brown M. An alternant method to the traditional NASA hindlimb unloading model in mice. Journal of Visualized Experiments 2011;(49):e2467.

4. O'Connor KM. Unweighting accelerates tidemark advancement in articular cartilage at the knee joint of rats. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 1997;12:580-9.

5. Takahashi I, Matsuzaki T, Kuroki H, Hoso M. Joint unloading inhibits articular cartilage degeneration in knee joints of a monosodium iodoacetate-induced rat model of osteoarthritis. Osteoarthritis and Cartilage 2019;27:1084-93.

Comment 9:

Figure 4B, y-axis label incorrect. The graph data are not consistent with S5 Table Histomorphometrical results-Matrix intensity data.

Author Action:

We thank the reviewer for pointing this out, but we would like to defend the current presentation of the graph. We intentionally reversed the y-axis from 0-255. The reason for this is that darker staining is normal for toluidine blue staining, while lighter staining is abnormal, such as in osteoarthritis. However, in the data, darker staining intensity is calculated as the lower value and lighter staining intensity as the higher value. In other words, if the graph is represented without inverting the y-axis, specimens stained darker, closer to normal, will show lower values, which may be mistakenly understood as worsening. Therefore, we inverted the y-axis to enhance visual understanding of the graph.

Comment 10:

In the figure, the operated group showing as DMM. In manuscript and supplementary document, the operated group showing as operated. Please keep consistent by using either DMM or operated.

Author Action:

In response to the reviewer’s comment, we have standardized our terminology to “operated.”

Comment 11:

Ref: some references are without doi, formats are not consistent.

Author Action:

We have revised the reference format according to the journal’s style requirements.

 

Responses to Reviewer #2 Comments

Comment 1:

As the author mentioned, the OA group at 2 and 4 weeks data are from the previous study. However I did not see any explanation about how the surgical procedure was controlled to be consistent between the previous study and this study (I assume the surgeries are done at different times and/or by different personnel?). Only staining method control was described. DMM surgeries performed by different personnels can result in different outcomes due to operational variations so it is important the author have this addressed.

Author Action:

We completely agree with the reviewer’s comment. As pointed out by the reviewer, we created the OA and OAHS groups in two phases. Therefore, we have made efforts to standardize the surgery and staining techniques. Specifically, throughout the two surgeries, we used the same experimental technique. Further, the same two surgeons conducted the experiments using identical protocols and techniques to create OA rats by DMM. In addition, we confirmed their proficiency in the surgical technique via preliminary experiments. Furthermore, when the surgical time was measured, no obvious difference was observed between the two experiments. As for the experimental technique, we followed that of a previous study1. Hence, we believe that we have ensured high reproducibility and accuracy in the two surgical procedures.

1. Glasson SS, Blanchet TJ, Morris EA. The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthr Cartil. 2007;15: 1061–1069. doi:10.1016/j.joca.2007.03.006.

Accordingly, we have revised and added a description on this in the Discussion as follows:

Original sentences

In the present study, we conducted additional experiments after the primary one [12]. To avoid any obvious differences in staining between the two experiments, we made four technical checks. First, we used the same staining conditions and environmental factors (laboratory, dyeing solution, room temperature, and used goods). Second, before conducting the experiments, we confirmed the absence of apparent differences in the staining intensity. Third, in the histological evaluation, the evaluator was blinded to all experimental conditions, including group assignment and the experimental period. Fourth, the intra- and inter-rater reliabilities were very high [13]. Therefore, it was possible to perform highly scientific experiments with assurance of objectivity and reproducibility.

Revised sentences (pages 20, lines 340-353):

The present study consists of the results of our previous study and additional experiments specified herein [12]. To avoid significant differences in experimental techniques between the two experiments, we paid careful attention to three aspects: surgery, staining, and evaluation. OA models were created by the same surgeon using the same surgical technique as in the previous study [22,23]. Additionally, the surgeon’s proficiency in the surgical technique was confirmed by preliminary experimentation. Furthermore, there was no marked difference in operative time between experiments. Staining was performed in the same environment and conditions, including staining solution used, staining time, room temperature, and laboratory equipment. Before conducting additional experiments, we preliminarily verified that there was no marked difference in staining intensity by the same technique. Histological evaluation was performed by two well-trained double-blinded experimenters. The same experimenter performed the histological evaluation in the two experiments and the two had high intra- and inter-rater reliability [13]. Based on the above, we consider our two experiments to have been conducted with high reliability and reproducibility.

Comment 2:

Also mentioned in the discussion section, the study used only histological and histomorphometrical analysis to support the conclusion. This does not seem scientifically justifiable. There are several studies on the mechanical properties of articular cartilage and it does not always go consistent with histological signs. For example in Doyran et al 2017 Osteoarthritis Cartilage, it was demonstrated that after the DMM surgery, mechanical properties start to decrease just a few days after the surgery while histological signs doesn't show up until 2-4 weeks after the surgery. The reduced histological sign suggests that the DMM-induced OA is delayed by reduced load but at least one more evidence is needed to demonstrate the results, for example immunohistochemistry staining, biochemical assay, or mechanical test.

Author Action:

We completely understand the concern mentioned in the reviewer’s comment. Ideally, we also believe that multiple analytical methods should be performed. However, in terms of study period, funding, and experimental equipment, we were unable to conduct any additional experiments using these analytical methods. Fortunately, we were able to obtain novel results through histological and histomorphometrical analyses alone. Therefore, we have added a description of study limitations to the Discussion and revised the concluding statement of the Discussion as follows:

Revised description

Original sentences

First, only histological and histomorphometrical analysis method was performed. In order to clarify the mechanism of the effect of load reduction on OA progression, further studies for catabolic markers, such as MMP-13 and ADAMTS5, or anabolic markers, such as aggrecan and type II collagen, would be necessary using immunohistochemical staining, situ hybridization, western blotting, and polymerase chain reaction.

Revised sentences (page 21, lines 354–358):

First, only histological and histomorphometrical analyses were performed. To clarify the effect of load reduction on OA progression, further biochemical studies for catabolic markers, such as MMP-13 and ADAMTS5, or anabolic markers, such as aggrecan and type II collagen, would be necessary, and further studies using mechanical tests would also be needed.

Revised description

Original sentences

Further studies using biochemical analyses to examine protein and gene expression are required to elucidate the mechanism underlying delayed OA progression caused by joint-load reduction.

Revised sentences (pages 22, lines 377–378):

Further studies using biochemical and mechanical analyses are required to clarify the structure underlying delayed OA progression caused by joint-load reduction.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (43KB, docx)

Decision Letter 1

Lin Han

25 Jun 2021

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis using a post-traumatic rat model

PONE-D-21-06339R1

Dear Dr. Takahashi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Lin Han

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

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

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. 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: (No Response)

Reviewer #2: All my previous comments have been properly addressed. Thank you for the great opportunity to review this paper and great work!

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Acceptance letter

Lin Han

7 Jul 2021

PONE-D-21-06339R1

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis using a post-traumatic rat model

Dear Dr. Takahashi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Lin Han

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. Subchondral bone damage score.

    (DOCX)

    Click here for additional data file. (23.2KB, docx)
    S2 Table. Scoring system for synovitis.

    (DOCX)

    Click here for additional data file. (22.8KB, docx)
    S3 Table. Body weight.

    (DOCX)

    Click here for additional data file. (29.7KB, docx)
    S4 Table. Histological scores.

    (DOCX)

    Click here for additional data file. (30.3KB, docx)
    S5 Table. Histomorphometrical results.

    (DOCX)

    Click here for additional data file. (33.7KB, docx)
    Attachment

    Submitted filename: Response to Reviewers.docx

    Click here for additional data file. (43KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

    Articles from PLoS ONE are provided here courtesy of PLOS

    RESOURCES