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Effects of short-term gentle treadmill walking on subchondral bone in a rat model of instability-induced osteoarthritis
Osteoarthritis and Cartilage 23 (2015) 1563e1574
Effects of short-term gentle treadmill walking on subchondral bone in a rat model of instability-induced osteoarthritis H. Iijima y, T. Aoyama z, A. Ito y, S. Yamaguchi y, M. Nagai y, J. Tajino y, X. Zhang y, H. Kuroki y * y Department of Motor Function Analysis, Human Health Sciences, Graduate School of Medicine, Kyoto University, Japan z Department of Development and Rehabilitation of Motor Function, Human Health Sciences, Graduate School of Medicine, Kyoto University, Japan
a r t i c l e i n f o
s u m m a r y
Article history: Received 9 October 2014 Accepted 15 April 2015
Objective: Subchondral bone cyst (SBC) growth, caused by osteoclast activity during early knee osteoarthritis (OA) pathogenesis, should be treated to prevent further progressions of OA. In the present study, we evaluated the effects of gentle treadmill walking on subchondral bone and cartilage changes in an experimental rat model of destabilized medial meniscus (DMM). Method: Twelve-week-old Wistar rats underwent DMM surgery in their right knee and sham surgery in their left knee and were assigned to either the sedentary group or walking group (n ¼ 42/group). Animals in the walking group were subjected to treadmill exercise 2 days after surgery, which included walking for 12 m/min, 30 min/day, 5 days/week for 1, 2, and 4 week(s). Subchondral bone and cartilage changes were evaluated by micro-CT analysis, histological analysis, and biomechanical analysis. Results: Treadmill walking had a tendency to suppress SBC growth, which was confirmed by micro-CT (P ¼ 0.06) and positive staining for tartrate-resistant acid phosphatase (TRAP) activity for the osteoclast number per bone surface (P ¼ 0.09) 4 weeks after surgery. These changes coincide with the prevention of cartilage degeneration as evaluated by the Osteoarthritis Research Society International (OARSI) score (P < 0.05) and biomechanically softening (P < 0.05). Furthermore, treadmill walking could suppressed increasing osteocyte deaths (P < 0.01), which was positively correlated with the OARSI score (r ¼ 0.77; P < 0.01). Conclusion: These results indicate biomechanical and biological links exist between cartilage and subchondral bone; preventive effects of treadmill walking on subchondral bone deterioration might be partly explained by the chondroprotective effects. © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Keywords: Osteoarthritis Bone mCT Exercise Osteoclasts Subchondral bone cyst
Introduction Knee osteoarthritis (OA) is considered a multifactorial wholejoint disease. Increased subchondral bone remodeling is an important factor that contributes to OA pathology through the crosstalk bone-cartilage unit1. The bone-cartilage unit forms a
* Address correspondence and reprint requests to: H. Kuroki, 53 Shogoin, Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Tel: 81-75-751-3963; Fax: 81-75751-3909. E-mail addresses: [email protected] (H. Iijima), aoyama. [email protected] (T. Aoyama), [email protected] (A. Ito), [email protected] (S. Yamaguchi), nagai.momoko.36v@st. kyoto-u.ac.jp (M. Nagai), [email protected] (J. Tajino), zhang. [email protected] (X. Zhang), [email protected] (H. Kuroki).
complex functional unit, which may play complementary roles in the load-bearing joint2, and the subchondral bone supports overlying cartilage biomechanically. In previous animal studies, it has been shown that subchondral bone cysts (SBCs), subchondral bone plate thinning, and cartilage degeneration were confirmed in early phase instability-induced OA, such as the destabilization of medial meniscus (DMM)3 through the dysregulation of osteoclast and osteoblast activity4. These animal studies recapitulated key features of human OA pathogenesis which coexist with meniscus degenerative changes5. Activated osteoclasts were confirmed in SBCs and further SBC growth was observed after drilling towards the articular cartilage6. According to an in vitro study, various cytokines and growth factors secreted by osteoclast and osteoblast of OA sclerotic bone promote loss of cartilage proteoglycans7. Therefore, preventing subchondral bone osteoporotic changes by suppressing increased subchondral
http://dx.doi.org/10.1016/j.joca.2015.04.015 1063-4584/© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
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bone remodeling in early OA may be a strategy to prevent the progression of OA8. Osteocytes of subchondral bone have been thought of as mechanosensing cells that influence osteoclast and osteoblast activity. Recently, osteocyte deaths were confirmed in OA subchondral bone, which may result in increased subchondral bone remodeling9. Cox et al. showed that increasing osteocyte deaths may cause SBC growth10. According to these previous studies, osteocyte deaths in subchondral bone lead to the dysregulation of osteoclast and osteoblast, which result in subchondral bone osteoporotic changes. Although the exact cause of increased subchondral bone remodeling is unknown, changes involved in overlying cartilage are proposed to be a principal cause. Mechanical loading is an important factor, which regulates the maintenance of both cartilage and subchondral bone of OA. Galois et al. showed that moderate mechanical loading has a potential preventive effect on cartilage degeneration in instability-induced OA11. Recently, Boudenot et al. and Siebelt et al. investigated the effects of treadmill running on both cartilage and subchondral bone changes caused by chemically induced OA; they showed that treadmill exercise influences subchondral or trabecular bone metabolism12,13. However, whether treadmill exercise has a preventive effects on subchondral bone changes, particularly SBC growth and osteocyte death, remains unclear and osteoclast and osteoblast activity should be examined further to clearly determine the effects of treadmill exercise on subchondral bone remodeling in OA. We hypothesized that in addition to its chondroprotective effects14, gentle treadmill walking has inhibitive effects on subchondral bone porosity. In this study, we evaluated the effects of gentle treadmill exercise, consisting of milder intensity exercise compared to previous studies12,13, on subchondral bone changes (by using histological techniques and micro-CT analysis) and cartilage degeneration in an experimental rat model of DMM. Methods Animals and surgical procedure This study was approved by the animal research committee of Kyoto University. Ninety male Wistar rats (12 weeks old; mean body weight ¼ 272.1 g) were purchased and placed in plastic cages with sawdust bedding, with three to four animals per cage. The room had a 12 h dark/light cycle and was at a constant temperature. Animals were allowed to move freely in the cages and had free access to food and water. As described previously, our preclinical model of DMM3 was performed under anesthesia using 0.85 mL/kg somnopentyl. All these surgery were conducted in the light phase. The surgery involved an incision of the medial capsule with a transection of the anterior medial meniscotibial ligament (MMTL) on the right knee. For internal controls, a sham operation was performed on the left knee joint using the same approach without MMTL transection. Exercise protocol Animals were randomly divided into either the sedentary group (n ¼ 42) or walking group (n ¼ 48). Animals in the walking group were subjected to treadmill exercise 2 days after surgery on a motor driven treadmill. A treadmill performance scale on a 1e5 Likert scale was used to assess trainability before the walking exercise in each animal15. Animals with a rating of 3 were included in the present study and those with a mean rating of 1 or 2 were excluded to avoid using differentially stressed animals16. Six out of the 48 rats in the walking group were excluded due to an insufficient score. In
total, 42 rats were included into each group (i.e., sedentary and walking groups) for the present study. All these exercise training were conducted in the light phase. Animals in the walking group were then subjected to treadmill exercise for 1, 2, and 4 week(s) (n ¼ 14 per time point) for 5 days/ week, 30 min/day. The initial velocity of 6 m/min at 2 days was increased to 12 m/min at or after 3 days. Animals in the sedentary group were allowed to move freely in standardized cages without any treadmill exercise for 1, 2, and 4 week(s) (n ¼ 14 per time point). Seven rats in the sedentary and walking groups at each time point were used for biomechanical analysis and the remaining seven rats at each time point were used for micro-CT and histological analysis. Biomechanical analysis After the rats at each time point were sacrificed, a microindentation test at the center of the medial tibia plateau was performed, to determine the biomechanical properties of cartilage in each group, according to a previously validated method17,18. A preload of 0.01 N was applied and allowed to equilibrate for 100 s, followed by loading at a strain rate of 0.005 mm/s up to 0.1 N, which was maintained for 300 s17. A stressestrain curve and creep (mm) were obtained from the micro-indentation test17,18. Micro-CT analysis of subchondral bone changes Prior to histological sectioning, all rat knee joints were scanned using a micro-CT system (SMX-100CT, Shimadzu, Kyoto, Japan) with the following parameters: 600 views over 360 increment, 20 exposures averaged per view, voltage of 43 kV, current of 40 mA, voxel size of 21 mm, and a scan time of approximately 7 min per knee. The reconstructed data sets were examined using threedimensional data analysis software (Amira5.4, Visage, Berlin, Germany). To measure subchondral bone plate thickness (Sb thickness), we selected subchondral bone of the weight-bearing region in the medial tibia, which was defined as a mediolateral width of 0.5 mm and a ventrodorsal length of 1 mm in the frontal plane19. In addition, the maximum SBC diameter4 and the average diameter of 3 SBCs were compared between the DMM knee of the sedentary and walking groups at each time point. To further analyze the proximal tibia, 2 separate cylindrical regions of interest (ROI), with a diameter of 1 mm were placed at the trabecular bone of the epiphysis in the medial tibia. The first cylinder, which had a height of 0.6 mm, was placed on epiphysis distal to the growth plate in the trabecular bone. These ROIs were determined based on anatomical landmarks with reference to previous report such as that by McErlain et al.20 Details about the ROIs are presented in Supplementary Fig. 4. Then, the following parameters were calculated for the trabecular bone of the epiphysis: trabecular bone volume fraction (Trab BV/TV), trabecular bone thickness (Tb.Th), and trabecular spacing (Tb.Sp)21. To examine whether Trab BV/TV could be changed locally as indicated by Botter et al.21, the other cylinder, which had a height of 0.2 mm, was placed in the trabecular bone which was located epiphysis proximal to the medial subchondral bone plate of the weight-bearing region. By using the same algorithm as that used for the first cylindrical ROI, the subchondral trabecular bone volume fraction (Sb BV/TV) was also calculated21. Histological and immunohistochemical analysis As described previously3, decalcified paraffin sections were prepared from the medial tibial plateau in the frontal plane. A
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minimum of three tissue sections, each 6 mm thick, were stained with toluidine blue. The Osteoarthritis Research Society International (OARSI) scoring system22 was used to grade and stage OA development. The most severe score in all three sections was determined by a single trained observer (HI). OA development was also quantified using the osteophyte score according to a previously validated method23. Six mm thick histological tissue sections stained with H&E in the weight-bearing region of the medial tibia were digitized with a light microscope at x100 magnification (Eclipse 80i, Nikon, Tokyo, Japan). These tissue sections were serial sections of toluidine bluestained sections in the frontal plane of the medial tibia. To determine depth-specific changes of subchondral bone, the sequentially bonded 4 standardized rectangular fields (100 mm deep and 500 mm long) were superimposed in the photo image using the Image-J software from the surface of subchondral bone towards the deep subchondral bone. The number of empty osteocyte lacunae per square millimeter (E. Lac/mm2), the number of healthy osteocyte lacunae per square millimeter (Healthy Ot/mm2), the total number of osteocyte lacunae per square millimeter (Total Ot/mm2) including the number of empty and healthy osteocyte lacunae, and the osteocyte lacunar occupancy (%) which was the healthy osteocyte lacunae number divided by the total osteocyte lacunae
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number, were calculated in each rectangular field from three tissue sections per knee joint. Furthermore, the healthy osteocyte lacunae surface (mm2) and empty osteocyte lacunae surface (mm2) were determined with the Image-J freehand tool by drawing their contour line in each ROI24. Osteoclasts and osteoblasts were visualized using histochemical staining for tartrate-resistant acid phosphatase (TRAP) activity and alkaline phosphatase (ALP) activity, respectively, using the TRAP/ ALP stain kit (Wako, Osaka, Japan). TRAP-positive osteoclasts per bone surface, which were adherent to calcified cartilage (subchondral bone region) within a region containing a mediolateral width of 0.5 mm in the middle region of the medial tibia25, were counted. Furthermore, TRAP-positive osteoclasts per bone surface in osteophyte (osteophyte region) were also counted25. Immunostaining of type II collagen (Diluted 1:200; Fine Chemical Co., Toyama, Japan) was also performed according to the method as previously described18. In situ cell death detection (apoptosis) of articular cartilage and subchondral bone plate Articular cartilage and osteocyte apoptosis were assessed by TUNEL assay using an in situ apoptosis detection kit (Takara, Shiga,
Fig. 1. Effects of treadmill walking on the biomechanical properties of cartilage in the medial tibia. (A) Stress-displacement curve at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; **P < 0.01 sham vs DMM group; xP < 0.05, xxP < 0.01 sham vs DMM þ walking group). (B) Creep response of cartilage in the medial tibia (n ¼ 7; *P < 0.05, **P < 0.01).
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Japan) according to the manufacturer's instructions. The tissue sections were also incubated with propidium iodide (PI) to label the nuclei. Fluorescence micrographs were obtained using fluorescence microscopy (Fluoview FV10i, Olympus, Tokyo, Japan). Chondrocyte and osteocyte apoptosis were determined as the percentage of TUNEL-positive chondrocytes and osteocytes in the PI-labeled chondrocytes and osteocytes within a region with a mediolateral width of 0.5 mm and depths of 0.25 mm (chondrocyte) and 0.1 mm (osteocyte) from the cartilage and subchondral bone plate surface respectively, in the middle region of the medial tibia, by using the Image-J software. Statistical analysis Statistical analyses were performed with the JMP 11 software program (SAS Institute, Cary, NC USA) or R (the R Foundation for Statistical Computing, Vienna, Austria). To evaluate effects of treadmill walking on the DMM knee, we analyzed and compared biomechanical properties, subchondral bone changes, and cartilage degeneration among the following three groups: (1) DMM
operated knee of sedentary group (DMM group), (2) DMM operated knee of walking group (DMM þ walking group), and (3) sham operated knee of sedentary group (sham group). Graphical results were displayed as mean with uncertainly expressed by 95% confidence intervals for continuous data, and as medians and interquartile ranges for categorical data. All the continuous data were assessed for normality by using the ShapiroeWilk test and for homoscedasticity by using the Bartlett or a F-test. To evaluate the differences among the three groups, an analysis of variance with a subsequent post hoc Tukey-Kramer or Games-Howell test was used for parametric continuous data. Meanwhile, the KruskaleWallis test with subsequent post hoc Steel-Dwass test was used for nonparametric continuous data or categorical data. The unpaired ttest was used for pair-wise differences of the SBC diameter between the DMM and DMM þ walking groups. Welch correction was applied for variables with unequal variance. A Spearman's rank test was used to calculate the correlations between OARSI scores and E. Lac/mm2 in the superficial zone (0e100 mm) and the total zone (i.e., sum of the superficial to deep zones as the total zone; 0e400 mm) from the averaged OARSI scores and E. Lac/mm2 from three tissue
Fig. 2. The chondroprotective effects of treadmill walking. (A) Gross appearance of tibia in the sham, DMM, and DMM þ walking groups at 4 weeks after surgery. The surface of cartilage was smooth in the sham group and in the disrupted middle region of the medial tibia particularly in the DMM group (dot-line area). (B) Histological findings of toluidine blue section (a, c, e) and immunohistochemical staining of type II collagen (b, d, f) 4 weeks after surgery. AC, articular cartilage; SB, subchondral bone. Magnification: 100. Scale bars ¼ 100 mm. (C) Osteoarthritis development evaluated by OARSI scores. Boxplots display median values and interquartile ranges (n ¼ 7; *P < 0.05, **P < 0.01). (D) Osteophyte scores as evaluated by histological sections Boxplots display median values and interquartile ranges (n ¼ 7; *P < 0.05, **P < 0.01).
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Fig. 3. Osteocyte deaths in the subchondral bone, which was partly prevented by treadmill walking. (A) The number of empty osteocyte lacunae per square millimeter (E. Lac/mm2) according to depth at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; *P < 0.05, **P < 0.01). (B) The number of healthy osteocyte lacunae per square millimeter (Healthy Ot/mm2) according to depth at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; *P < 0.05). (C) The total number of osteocyte lacunae per square millimeter (Total Ot/mm2) according to depth at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; *P < 0.05, **P < 0.01). (D) The osteocyte lacunar occupancy (%) according to depth at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; *P < 0.05, **P < 0.01).
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sections per knee joint. Throughout the text, “n” refers to the number of animals. In all cases, P < 0.05 was considered statistically significant.
(P < 0.05); however, there were no significant differences at 1 (P ¼ 0.99) and 2 weeks (P ¼ 0.66) after surgery. Osteoarthritic changes in articular cartilage and osteophyte growth
Results Changes in biomechanical properties One week after surgery, there were no significant differences in the stressestrain curve among the three groups [Fig. 1(A)], but both the DMM and DMM þ walking groups had a significantly increased displacement after 2 and 4 weeks compared to the sham group (P < 0.05). Four weeks after surgery, the DMM þ walking group had a lower displacement compared to the DMM group; however, there were no significant differences between the DMM and DMM þ walking groups at any compressive stress value. The creep behaviors among groups were also evaluated at each time point [Fig. 1(B)]. From 2 weeks after surgery, the DMM group showed a significantly higher creep compared to the sham group (P < 0.01). The DMM þ walking group had a significantly lower creep compared to the DMM group 4 weeks after surgery
During gross observation 4 weeks after surgery [Fig. 2(A)], the surface of the cartilage was smooth in the sham group and had a disrupted middle region of the medial tibia with evidence of fibrillation particularly in the DMM group. The surface of the cartilage in DMM þ walking group seemed to be less macroscopic changes than those in DMM group. The histology of cartilage in the DMM group was characterized by vertical fissure reaching the deep zone, decreased chondrocytes, and strong proteoglycan depletion shown by the toluidine blue and type II collagen staining [Fig. 2(B)]. Cartilage in the DMM þ walking group had less vertical fissure, which was located only in the superficial zone, compared to the DMM group. The OARSI score [Fig. 2(C)] in the DMM þ walking group was significantly lower than that in the DMM group 4 weeks after surgery (P < 0.05). There were no significant differences in the osteophyte scores between the DMM and DMM þ walking groups at any of the time points [Fig. 2(D)].
Fig. 4. In situ cell deaths detected by TUNEL staining. (A) Fluorescence micrograph of the medial tibia 4 weeks after surgery. Although few apoptotic chondrocytes were confirmed in the sham group (aeb), there were many apoptotic chondrocytes confirmed particularly around the degenerated cartilage region in both the DMM (ced) and DMM þ walking groups (eef). AC, articular cartilage; SB, subchondral bone; BM, bone marrow. The white box represents a magnification of the osteochondral region shown on the right. Scale bars ¼ 100 mm. (B) Quantitative analysis of TUNEL staining of chondrocytes; number of PI-labeled chondrocytes (a), TUNEL-positive chondrocytes (b), and the percentage of TUNELpositive chondrocytes (c) (n ¼ 5; *P < 0.05, **P < 0.01).
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Histological analysis of subchondral bone plate In OA conditions, a lumber of empty osteocyte lacunae were confirmed especially in the superficial zone (0e100 mm from the osteochondral region) of subchondral bone plate, which were located underneath degenerated cartilage. Treadmill walking suppressed the increase of E. Lac/mm2 in the superficial zone from 2 to 4 weeks [Fig. 3(A)], which became significantly lower level compared to the DMM group 4 weeks after surgery (P < 0.05). Healthy Ot/mm2 and Total Ot/mm2 were higher in the DMM and DMM þ walking groups compared to the sham group [Fig. 3(B), (C)]; no significant differences were confirmed between the DMM and DMM þ walking groups at any of the time points. As with E. Lac/mm2, lacunar occupancy was decreased over time in the superficial zone in the DMM group [Fig. 3(D)], to a significantly lower level compared to the DMM þ walking group 4 weeks after surgery (P < 0.01). Additionally, healthy osteocyte lacunae surface in the DMM group was significantly larger compared to the sham and the DMM þ walking groups particularly in the superficial zone of the subchondral bone 4 weeks after surgery (Supplementary Fig. 1). There were no significant differences in empty osteocyte lacunae surface among three groups at any of the time points. There were significant correlations between the OARSI score and E. Lac/mm2 in the superficial zone (r ¼ 0.77, P < 0.01) as shown in Supplementary Fig. 2. This correlation became unclear when E. Lac/mm2 in the total zone (0e400 mm) was considered (r ¼ 0.58, P < 0.01). In situ cell deaths detected by TUNEL staining To examine the cell death mechanisms, particularly that of osteocytes on the subchondral bone surface as mentioned earlier [Fig. 3], we performed TUNEL staining [Fig. 4(A)]. However, there were few TUNEL-positive osteocytes in the subchondral bone of any group (Supplementary Fig. 3). Equally, many TUNEL-positive chondrocytes were confirmed in the
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cartilage of all groups [Fig. 4(A), (B)]. The number of PI-labeled chondrocytes was significantly lower in the DMM group compared to the sham group at 4 weeks after surgery (P < 0.01). The number of TUNEL-positive chondrocytes in the DMM group had a tendency to increase compared to the sham group at 4 weeks after surgery (P ¼ 0.06). The percentage of TUNELpositive chondrocytes in the DMM þ walking group was significantly lower than that in the DMM group at 4 weeks after surgery (P < 0.05). Micro-CT analysis of subchondral bone changes According to the micro-CT observation, SBCs were present in the medial tibia of all animals in the DMM and DMM þ walking groups underneath the region of degenerated cartilage throughout the time-course of the study [Fig. 5(A)]. There were no significant changes in Sb BV/TV and Sb thickness in the weight-bearing region of the three groups at 1 and 2 week(s) after surgery [Fig. 5(B)]. Four weeks after surgery, the Sb BV/TV in the DMM þ walking group was significantly higher than the DMM group (P < 0.05). Prominent subchondral bone thickening was confirmed in the DMM þ walking group at 4 weeks after surgery which was significantly higher compared to the sham group (P < 0.01), but not significantly different compared to the DMM group (P ¼ 0.06). The DMM surgery did not affect the parameters of subchondral trabecular bone which are epiphysis distal to the growth plate (Supplementary Fig. 4). The formation of subchondral bone perforations created by the SBCs in the medial tibia is shown in Fig. 6(A). There were no significant differences in the maximum and average SBC diameters between the DMM and DMM þ walking groups at 1 and 2 weeks after surgery [Fig. 6(B)]. The maximum SBC diameter was not different between the DMM and DMM þ walking group 4 weeks after surgery (P ¼ 0.53); however the DMM þ walking group tended to have a short average SBC diameter compared to the DMM group (P ¼ 0.06).
Fig. 5. Effect of treadmill walking on subchondral bone plate evaluated by micro-CT. (A) Micro-CT observations of the sham, DMM, and DMM þ walking groups 4 weeks after surgery. Subchondral bone cysts were confirmed in the middle region of the medial tibia in the DMM and DMM þ walking groups (white arrow head). (B) The subchondral trabecular bone volume fraction (Sb BV/TV) (a) and subchondral bone plate thickness (Sb thickness) (b) of the medial tibia at each time point (n ¼ 7; *P < 0.05, **P < 0.01).
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Fig. 6. Formation of SBCs. (A) Three-dimensional image of the subchondral bone of the tibial plateau 4 weeks after surgery. Several subchondral perforations were confirmed, particularly in the severe sample (black arrow head). Osteophyte mineralization was confirmed in all samples of DMM and DMM þ walking groups (white arrow head). (B) The maximum (a) and average (b) diameters of SBCs in the medial tibia after surgery (n ¼ 7).
Changes in TRAP and ALP staining
Discussion
Increased TRAP-positive osteoclasts were confirmed in SBCs of the DMM group after drilling towards the articular cartilage underneath the degenerated cartilage; however, this was not markedly observed in the DMM þ walking group [Fig. 7(A)]. The number of osteoclasts [Fig. 7(B)] in the subchondral bone region was significantly increased in the DMM group compared to the sham group from 2 weeks after surgery (P < 0.05), which was not pronounced in the DMM þ walking group at 2 (P ¼ 0.16) and 4 (P ¼ 0.09) weeks after surgery. In the osteophyte region, the DMM group also showed increased osteoclasts number over time; the DMM þ walking group had a significantly lower osteoclasts number compared to the DMM group 4 weeks after surgery (P < 0.05). ALP-positive osteoblasts were particularly observed in SBCs of the DMM group [Fig. 8]. ALP-positive chondrocytes in the articular cartilage were diminished underneath the degenerated cartilage particularly in the DMM group [Fig. 8(C) and (D)].
According to the best of our knowledge, this was the first study conducted to show that 4 weeks of gentle treadmill walking tended to suppress the growth of SBCs and TRAP-positive osteoclasts in subchondral bone surface. These preventive effects of treadmill walking coincide with the chondroprotective effects, as indicated by OARSI scores, TUNEL assay results, and changes in biomechanical properties. Furthermore, treadmill walking could suppress increasing osteocyte deaths induced by DMM surgery, which is positively correlated with OARSI scores. SBC growth, produced by osteoclasts, plays an important role in early stages of OA pathogenesis, which should be treated to prevent the further progression of OA26. Cox et al. showed SBC growth was proposed to be accelerated by the death of osteocytes that surround SBCs10. Also, other studies showed that existing osteoclast-created SBCs correspond with the presence of necrotic bone4, and that osteocyte debris could elevate osteoclastogenesis27. In the present study, we described that gentle treadmill walking could partly
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Fig. 7. Histological findings of TRAP staining. (A) Histological section of TRAP-positive osteoclasts (stained red-violet) in the medial tibia 4 weeks after surgery. There were few osteoclasts in the osteochondral region in the sham group (aeb); however, a high number of osteoclasts were confirmed in subchondral bone cysts located underneath degenerated cartilage, particularly in the DMM (ced) group compared to the DMM þ walking (eef) groups (black arrow head). Osteoclasts were also confirmed in the osteophyte region of the DMM group (geh). AC, articular cartilage; SB, subchondral bone; BM, bone marrow; OP, osteophyte. The black box represents a magnification of the figure on the right. Scale bars ¼ 100 mm. (B) Numbers of TRAP-positive osteoclasts per bone surface (N. Oc/BS) in subchondral bone region (a) and osteophyte region (b) (n ¼ 7; *P < 0.05, **P < 0.01 sham vs DMM group; xP < 0.05, xxP < 0.01 sham vs DMM þ walking group; yP < 0.01 DMM vs DMM þ walking group).
prevent subchondral bone osteocyte deaths in the superficial zone, which could contribute to the prevention of the SBC growth generated by osteoclasts in the DMM knee. Currently, there are few reports to directly explain the cause of these preventive effects of exercise on subchondral bone deterioration in vivo; however one possibility could be the chondroprotective effect of treadmill exercise through mechanical and biological interactions at the bone-cartilage interface. As shown in several studies, degenerated and softened cartilage might transmit excessive mechanical loading to the breached underlying subchondral bone surface resulting in osteocyte deaths, which further leads to SBC growth10. In addition to mechanical interactions, biological processes also affect the pathogenesis of structural changes through bone-cartilage molecular crosstalk. In a recent study, the RANKL was released by chondrocytes of degenerated cartilage28. RANKL acts in a paracrine manner on the subchondral bone plate through calcified cartilage, which results in osteoclast stimulation. Furthermore, TNF-a and IL-1, which are released by degenerated cartilage, could induce osteocyte deaths29. According to the mechanisms of the chondroprotective effect of treadmill walking, such as elevation of anti-apoptotic capacity11 and anti-oxidative stress capacity30, that have been discussed in previous in vivo studies, these might involve suppression of osteocyte deaths and osteoclast-induced SBC growth. However, we could not consider separately the effects of treadmill walking from its direct effects on the regulation of
subchondral bone homeostasis12,31. Further studies are needed to evaluate exercise effects along with various exercise intensities which could affect the effectiveness of exercise on cartilage and subchondral bone11,32,33. Osteocytes are known to regulate mineralization surrounding their lacunae. Lane et al. showed that elastic modulus of subchondral bone surrounding large osteocyte lacunae were reduced in glucocorticoid-treated mice, thereby increasing bone fragility34. These large osteocyte lacunae may be able to dissipate loads and may crack around the surface35. In the present study, we described the large osteocyte lacunae were confirmed in the DMM group particularly around micro-cracks but not so pronounced in the DMM þ walking group (Supplementary Fig. 1). Actually little is known about the mechanism of changes in osteocyte lacunae surface in musculo-skeletal pathologies, specifically regarding subchondral bone and exercise, our results indicate that increased osteocyte lacunae surface of subchondral bone in the DMM group may be associated with low mineral material strength that could cause OA development, and that gentle treadmill walking may prevent bone fragility via improvement of microenvironment surrounding osteocyte lacunae. Surprisingly, osteocyte apoptosis was not detected in the TUNEL staining even in the DMM and DMM þ walking groups which is consistent with a recent study reported by Boudenot et al.12 Several studies showed that necrotic bone was confirmed adjacent to osteoclastic bone resorption and degenerated cartilage4,36. These
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Fig. 8. Histological section of ALP staining in the medial tibia 4 weeks after surgery. ALP-stained cell (brown) was localized in hypertrophic chondrocytes or the subchondral bone marrow in the sham group (aeb). ALP-stained chondrocytes on the top of the subchondral cyst were diminished (arrow head) in DMM group (ced). Strongly ALP-stained osteoblasts were confirmed in subchondral bone cysts of the DMM group (d, black arrow) compared to the DMM þ walking group (eef). ALP-positive cells were increased in the osteophyte region in the DMM group (geh). AC, articular cartilage; SB, subchondral bone; BM, bone marrow; OP, osteophyte. The black box represents a magnification of the figure shown on the right. Scale bars ¼ 100 mm.
studies indicate that the cell death mechanisms might include factors other than apoptosis, such as osteonecrosis, and might not be affected by short-term exercise. To explain these cell death mechanisms in early OA warrant further studies. There are several limitations in the present study. Firstly, there was a lack of statistical power to determine the effects of treadmill walking; therefore, no significant differences, particularly regarding SBC growth and osteoclasts activity in subchondral bone region, were confirmed between the DMM and DMM þ walking groups. Secondly, the present study involved short-term intervention periods in the early stage of OA which is a transient period. Hence, we could not affirm the effectiveness of gentle treadmill exercise in late stage of OA. Therefore, the effects of long-term treadmill walking on cartilage and subchondral bone should be confirmed. Thirdly, we used an experimental rat model; thus, our findings may not translate directly to humans. Finally, there are
controversies regarding the initial structural changes in the DMM knee and whether these alterations first occur in the subchondral bone or the articular cartilage. In conclusion, short-term gentle treadmill walking suppressed increasing osteocyte deaths and had a tendency to prevent SBC growth generated by osteoclasts in the DMM knee. These changes coincide with the prevention of cartilage degeneration, including biomechanical softening. Interestingly, there was a positive correlation between cartilage degeneration and osteocyte deaths, indicating that biomechanical and biological links exist between cartilage and subchondral bone, and the preventive effects of treadmill walking on subchondral bone deterioration might be partly explained by the chondroprotective effect. Further researches are required for investigating the effects of gentle treadmill walking on subchondral bone changes in an instabilityinduced OA.
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Author contributions All authors have made substantial contributions to (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data; (2) drafting the article or revising it critically for important intellectual content; and (3) final approval of the version to be submitted. The specific contributions of the authors are as follows: (1) Conception and design of the study: HI, TA, AI, and SY. (2) Analysis and interpretation of the data: HI, TA, AI, MN, XZ, SY, and HK. (3) Drafting of the article: HI, TA, JT, and HK. (4) Critical revision of the article for important intellectual content: HI, TA, AI, MN, and HK. (5) Final approval of the article: HI, TA, and HK. (6) Statistical expertise: HI, TA, and JT. (7) Obtaining of funding: HK. (8) Collection and assembly of data: HI.
Role of the funding source This study was supported in part by a JSPS KAKENHI Grant-in-Aid for Scientific Research (A) (number 20240057) and a JSPS KAKENHI Grant-in-Aid for Challenging Exploratory Research (number 25560258). Conflict of interest The authors have no competing interests. Acknowledgements This study was supported in part by a JSPS KAKENHI Grant-inAid for Scientific Research (A) (no. 25242055) and a JSPS KAKENHI Grant-in-Aid for Challenging Exploratory Research (no. 25560258). Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.joca.2015.04.015. References 1. 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. Osteoarthritis Cartilage 2014;22:1077e89. 2. Pan J, Zhou X, Li W, Novotny JE, Doty SB, Wang L. In situ measurement of transport between subchondral bone and articular cartilage. J Orthop Res 2009;27:1347e52. 3. 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. Osteoarthritis Cartilage 2014;22: 1036e43. 4. McErlain DD, Ulici V, Darling M, Gati JS, Pitelka V, Beier F, et al. An in vivo investigation of the initiation and progression of subchondral cysts in a rodent model of secondary osteoarthritis. Arthritis Res Ther 2012;14:R26. 5. Bloecker K, Guermazi A, Wirth W, Benichou O, Kwoh CK, Hunter DJ, et al. Tibial coverage, meniscus position, size and damage in knees discordant for joint space narrowing e data from the Osteoarthritis Initiative. Osteoarthritis Cartilage 2013;21:419e27.
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6. Botter SM, van Osch GJ, Waarsing JH, van der Linden JC, Verhaar JA, Pols HA, et al. Cartilage damage pattern in relation to subchondral plate thickness in a collagenase-induced model of osteoarthritis. Osteoarthritis Cartilage 2008;16:506e14. 7. Westacott CI, Webb GR, Warnock MG, Sims JV, Elson CJ. Alteration of cartilage metabolism by cells from osteoarthritic bone. Arthritis Rheum 1997;40:1282e91. 8. Shimizu S, Asou Y, Itoh S, Chung UI, Kawaguchi H, Shinomiya K, et al. Prevention of cartilage destruction with intraarticular osteoclastogenesis inhibitory factor/osteoprotegerin in a murine model of osteoarthritis. Arthritis Rheum 2007;56:3358e65. 9. Jaiprakash A, Prasadam I, Feng JQ, Liu Y, Crawford R, Xiao Y. Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones: a possible pathological role in subchondral bone sclerosis. Int J Biol Sci 2012;8:406e17. 10. Cox LG, Lagemaat MW, van Donkelaar CC, van Rietbergen B, Reilingh ML, Blankevoort L, et al. The role of pressurized fluid in subchondral bone cyst growth. Bone 2011;49:762e8. 11. Galois L, Etienne S, Grossin L, Watrin-Pinzano A, CournilHenrionnet C, Loeuille D, et al. Dose-response relationship for exercise on severity of experimental osteoarthritis in rats: a pilot study. Osteoarthritis Cartilage 2004;12:779e86. 12. Boudenot A, Presle N, Uzbekov R, Toumi H, Pallu S, Lespessailles E. Effect of interval-training exercise on subchondral bone in a chemically-induced osteoarthritis model. Osteoarthritis Cartilage 2014;22:1176e85. 13. Siebelt M, Groen HC, Koelewijn SJ, de Blois E, Sandker M, Waarsing JH, et al. Increased physical activity severely induces osteoarthritic changes in knee joints with papain induced sulphate-glycosaminoglycan depleted cartilage. Arthritis Res Ther 2014;16:R32. 14. Chang TK, Huang CH, Huang CH, Chen HC, Cheng CK. The influence of long-term treadmill exercise on bone mass and articular cartilage in ovariectomized rats. BMC Musculoskelet Disord 2010;11:185. 15. Dishman RK, Armstrong RB, Delp MD, Graham RE, Dunn AL. Open-field behavior is not related to treadmill performance in exercising rats. Physiol Behav 1988;43:541e6. 16. Franciozi CE, Tarini VA, Reginato RD, Goncalves PR, Medeiros VP, Ferretti M, et al. Gradual strenuous running regimen predisposes to osteoarthritis due to cartilage cell death and altered levels of glycosaminoglycans. Osteoarthritis Cartilage 2013;21:965e72. 17. Cao L, Youn I, Guilak F, Setton LA. Compressive properties of mouse articular cartilage determined in a novel microindentation test method and biphasic finite element model. J Biomech Eng 2006;128:766e71. 18. 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. 19. Botter SM, van Osch GJ, Clockaerts S, Waarsing JH, Weinans H, van Leeuwen JP. Osteoarthritis induction leads to early and temporal subchondral plate porosity in the tibial plateau of mice: an in vivo microfocal computed tomography study. Arthritis Rheum 2011;63:2690e9. 20. McErlain DD, Appleton CT, Litchfield RB, Pitelka V, Henry JL, Bernier SM, et al. Study of subchondral bone adaptations in a rodent surgical model of OA using in vivo micro-computed tomography. Osteoarthritis Cartilage 2008;16:458e69. 21. Botter SM, Glasson SS, Hopkins B, Clockaerts S, Weinans H, van Leeuwen JP, et al. ADAMTS5-/- mice have less subchondral bone changes after induction of osteoarthritis through surgical instability: implications for a link between cartilage and
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subchondral bone changes. Osteoarthritis Cartilage 2009;17: 636e45. Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, et al. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage 2006;14: 13e29. Gerwin N, Bendele AM, Glasson S, Carlson CS. The OARSI histopathology initiative e recommendations for histological assessments of osteoarthritis in the rat. Osteoarthritis Cartilage 2010;18(Suppl 3):S24e34. Maurel DB, Jaffre C, Rochefort GY, Aveline PC, Boisseau N, Uzbekov R, et al. Low bone accrual is associated with osteocyte apoptosis in alcohol-induced osteopenia. Bone 2011;49: 543e52. Hayami T, Pickarski M, Wesolowski GA, McLane J, Bone A, Destefano J, et al. The role of subchondral bone remodeling in osteoarthritis: reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model. Arthritis Rheum 2004;50:1193e206. Siebelt M, Waarsing JH, Groen HC, Muller C, Koelewijn SJ, de Blois E, et al. Inhibited osteoclastic bone resorption through alendronate treatment in rats reduces severe osteoarthritis progression. Bone 2014;66:163e70. Kogianni G, Mann V, Noble BS. Apoptotic bodies convey activity capable of initiating osteoclastogenesis and localized bone destruction. J Bone Miner Res 2008;23:915e27. Martinez-Calatrava MJ, Prieto-Potin I, Roman-Blas JA, Tardio L, Largo R, Herrero-Beaumont G. RANKL synthesized by articular
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Effects of short-term gentle treadmill walking on subchondral bone in a rat model of instability-induced osteoarthritis H. Iijima y, T. Aoyama z, A. Ito y, S. Yamaguchi y, M. Nagai y, J. Tajino y, X. Zhang y, H. Kuroki y * y Department of Motor Function Analysis, Human Health Sciences, Graduate School of Medicine, Kyoto University, Japan z Department of Development and Rehabilitation of Motor Function, Human Health Sciences, Graduate School of Medicine, Kyoto University, Japan
a r t i c l e i n f o
s u m m a r y
Article history: Received 9 October 2014 Accepted 15 April 2015
Objective: Subchondral bone cyst (SBC) growth, caused by osteoclast activity during early knee osteoarthritis (OA) pathogenesis, should be treated to prevent further progressions of OA. In the present study, we evaluated the effects of gentle treadmill walking on subchondral bone and cartilage changes in an experimental rat model of destabilized medial meniscus (DMM). Method: Twelve-week-old Wistar rats underwent DMM surgery in their right knee and sham surgery in their left knee and were assigned to either the sedentary group or walking group (n ¼ 42/group). Animals in the walking group were subjected to treadmill exercise 2 days after surgery, which included walking for 12 m/min, 30 min/day, 5 days/week for 1, 2, and 4 week(s). Subchondral bone and cartilage changes were evaluated by micro-CT analysis, histological analysis, and biomechanical analysis. Results: Treadmill walking had a tendency to suppress SBC growth, which was confirmed by micro-CT (P ¼ 0.06) and positive staining for tartrate-resistant acid phosphatase (TRAP) activity for the osteoclast number per bone surface (P ¼ 0.09) 4 weeks after surgery. These changes coincide with the prevention of cartilage degeneration as evaluated by the Osteoarthritis Research Society International (OARSI) score (P < 0.05) and biomechanically softening (P < 0.05). Furthermore, treadmill walking could suppressed increasing osteocyte deaths (P < 0.01), which was positively correlated with the OARSI score (r ¼ 0.77; P < 0.01). Conclusion: These results indicate biomechanical and biological links exist between cartilage and subchondral bone; preventive effects of treadmill walking on subchondral bone deterioration might be partly explained by the chondroprotective effects. © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Keywords: Osteoarthritis Bone mCT Exercise Osteoclasts Subchondral bone cyst
Introduction Knee osteoarthritis (OA) is considered a multifactorial wholejoint disease. Increased subchondral bone remodeling is an important factor that contributes to OA pathology through the crosstalk bone-cartilage unit1. The bone-cartilage unit forms a
* Address correspondence and reprint requests to: H. Kuroki, 53 Shogoin, Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Tel: 81-75-751-3963; Fax: 81-75751-3909. E-mail addresses: [email protected] (H. Iijima), aoyama. [email protected] (T. Aoyama), [email protected] (A. Ito), [email protected] (S. Yamaguchi), nagai.momoko.36v@st. kyoto-u.ac.jp (M. Nagai), [email protected] (J. Tajino), zhang. [email protected] (X. Zhang), [email protected] (H. Kuroki).
complex functional unit, which may play complementary roles in the load-bearing joint2, and the subchondral bone supports overlying cartilage biomechanically. In previous animal studies, it has been shown that subchondral bone cysts (SBCs), subchondral bone plate thinning, and cartilage degeneration were confirmed in early phase instability-induced OA, such as the destabilization of medial meniscus (DMM)3 through the dysregulation of osteoclast and osteoblast activity4. These animal studies recapitulated key features of human OA pathogenesis which coexist with meniscus degenerative changes5. Activated osteoclasts were confirmed in SBCs and further SBC growth was observed after drilling towards the articular cartilage6. According to an in vitro study, various cytokines and growth factors secreted by osteoclast and osteoblast of OA sclerotic bone promote loss of cartilage proteoglycans7. Therefore, preventing subchondral bone osteoporotic changes by suppressing increased subchondral
http://dx.doi.org/10.1016/j.joca.2015.04.015 1063-4584/© 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
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bone remodeling in early OA may be a strategy to prevent the progression of OA8. Osteocytes of subchondral bone have been thought of as mechanosensing cells that influence osteoclast and osteoblast activity. Recently, osteocyte deaths were confirmed in OA subchondral bone, which may result in increased subchondral bone remodeling9. Cox et al. showed that increasing osteocyte deaths may cause SBC growth10. According to these previous studies, osteocyte deaths in subchondral bone lead to the dysregulation of osteoclast and osteoblast, which result in subchondral bone osteoporotic changes. Although the exact cause of increased subchondral bone remodeling is unknown, changes involved in overlying cartilage are proposed to be a principal cause. Mechanical loading is an important factor, which regulates the maintenance of both cartilage and subchondral bone of OA. Galois et al. showed that moderate mechanical loading has a potential preventive effect on cartilage degeneration in instability-induced OA11. Recently, Boudenot et al. and Siebelt et al. investigated the effects of treadmill running on both cartilage and subchondral bone changes caused by chemically induced OA; they showed that treadmill exercise influences subchondral or trabecular bone metabolism12,13. However, whether treadmill exercise has a preventive effects on subchondral bone changes, particularly SBC growth and osteocyte death, remains unclear and osteoclast and osteoblast activity should be examined further to clearly determine the effects of treadmill exercise on subchondral bone remodeling in OA. We hypothesized that in addition to its chondroprotective effects14, gentle treadmill walking has inhibitive effects on subchondral bone porosity. In this study, we evaluated the effects of gentle treadmill exercise, consisting of milder intensity exercise compared to previous studies12,13, on subchondral bone changes (by using histological techniques and micro-CT analysis) and cartilage degeneration in an experimental rat model of DMM. Methods Animals and surgical procedure This study was approved by the animal research committee of Kyoto University. Ninety male Wistar rats (12 weeks old; mean body weight ¼ 272.1 g) were purchased and placed in plastic cages with sawdust bedding, with three to four animals per cage. The room had a 12 h dark/light cycle and was at a constant temperature. Animals were allowed to move freely in the cages and had free access to food and water. As described previously, our preclinical model of DMM3 was performed under anesthesia using 0.85 mL/kg somnopentyl. All these surgery were conducted in the light phase. The surgery involved an incision of the medial capsule with a transection of the anterior medial meniscotibial ligament (MMTL) on the right knee. For internal controls, a sham operation was performed on the left knee joint using the same approach without MMTL transection. Exercise protocol Animals were randomly divided into either the sedentary group (n ¼ 42) or walking group (n ¼ 48). Animals in the walking group were subjected to treadmill exercise 2 days after surgery on a motor driven treadmill. A treadmill performance scale on a 1e5 Likert scale was used to assess trainability before the walking exercise in each animal15. Animals with a rating of 3 were included in the present study and those with a mean rating of 1 or 2 were excluded to avoid using differentially stressed animals16. Six out of the 48 rats in the walking group were excluded due to an insufficient score. In
total, 42 rats were included into each group (i.e., sedentary and walking groups) for the present study. All these exercise training were conducted in the light phase. Animals in the walking group were then subjected to treadmill exercise for 1, 2, and 4 week(s) (n ¼ 14 per time point) for 5 days/ week, 30 min/day. The initial velocity of 6 m/min at 2 days was increased to 12 m/min at or after 3 days. Animals in the sedentary group were allowed to move freely in standardized cages without any treadmill exercise for 1, 2, and 4 week(s) (n ¼ 14 per time point). Seven rats in the sedentary and walking groups at each time point were used for biomechanical analysis and the remaining seven rats at each time point were used for micro-CT and histological analysis. Biomechanical analysis After the rats at each time point were sacrificed, a microindentation test at the center of the medial tibia plateau was performed, to determine the biomechanical properties of cartilage in each group, according to a previously validated method17,18. A preload of 0.01 N was applied and allowed to equilibrate for 100 s, followed by loading at a strain rate of 0.005 mm/s up to 0.1 N, which was maintained for 300 s17. A stressestrain curve and creep (mm) were obtained from the micro-indentation test17,18. Micro-CT analysis of subchondral bone changes Prior to histological sectioning, all rat knee joints were scanned using a micro-CT system (SMX-100CT, Shimadzu, Kyoto, Japan) with the following parameters: 600 views over 360 increment, 20 exposures averaged per view, voltage of 43 kV, current of 40 mA, voxel size of 21 mm, and a scan time of approximately 7 min per knee. The reconstructed data sets were examined using threedimensional data analysis software (Amira5.4, Visage, Berlin, Germany). To measure subchondral bone plate thickness (Sb thickness), we selected subchondral bone of the weight-bearing region in the medial tibia, which was defined as a mediolateral width of 0.5 mm and a ventrodorsal length of 1 mm in the frontal plane19. In addition, the maximum SBC diameter4 and the average diameter of 3 SBCs were compared between the DMM knee of the sedentary and walking groups at each time point. To further analyze the proximal tibia, 2 separate cylindrical regions of interest (ROI), with a diameter of 1 mm were placed at the trabecular bone of the epiphysis in the medial tibia. The first cylinder, which had a height of 0.6 mm, was placed on epiphysis distal to the growth plate in the trabecular bone. These ROIs were determined based on anatomical landmarks with reference to previous report such as that by McErlain et al.20 Details about the ROIs are presented in Supplementary Fig. 4. Then, the following parameters were calculated for the trabecular bone of the epiphysis: trabecular bone volume fraction (Trab BV/TV), trabecular bone thickness (Tb.Th), and trabecular spacing (Tb.Sp)21. To examine whether Trab BV/TV could be changed locally as indicated by Botter et al.21, the other cylinder, which had a height of 0.2 mm, was placed in the trabecular bone which was located epiphysis proximal to the medial subchondral bone plate of the weight-bearing region. By using the same algorithm as that used for the first cylindrical ROI, the subchondral trabecular bone volume fraction (Sb BV/TV) was also calculated21. Histological and immunohistochemical analysis As described previously3, decalcified paraffin sections were prepared from the medial tibial plateau in the frontal plane. A
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minimum of three tissue sections, each 6 mm thick, were stained with toluidine blue. The Osteoarthritis Research Society International (OARSI) scoring system22 was used to grade and stage OA development. The most severe score in all three sections was determined by a single trained observer (HI). OA development was also quantified using the osteophyte score according to a previously validated method23. Six mm thick histological tissue sections stained with H&E in the weight-bearing region of the medial tibia were digitized with a light microscope at x100 magnification (Eclipse 80i, Nikon, Tokyo, Japan). These tissue sections were serial sections of toluidine bluestained sections in the frontal plane of the medial tibia. To determine depth-specific changes of subchondral bone, the sequentially bonded 4 standardized rectangular fields (100 mm deep and 500 mm long) were superimposed in the photo image using the Image-J software from the surface of subchondral bone towards the deep subchondral bone. The number of empty osteocyte lacunae per square millimeter (E. Lac/mm2), the number of healthy osteocyte lacunae per square millimeter (Healthy Ot/mm2), the total number of osteocyte lacunae per square millimeter (Total Ot/mm2) including the number of empty and healthy osteocyte lacunae, and the osteocyte lacunar occupancy (%) which was the healthy osteocyte lacunae number divided by the total osteocyte lacunae
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number, were calculated in each rectangular field from three tissue sections per knee joint. Furthermore, the healthy osteocyte lacunae surface (mm2) and empty osteocyte lacunae surface (mm2) were determined with the Image-J freehand tool by drawing their contour line in each ROI24. Osteoclasts and osteoblasts were visualized using histochemical staining for tartrate-resistant acid phosphatase (TRAP) activity and alkaline phosphatase (ALP) activity, respectively, using the TRAP/ ALP stain kit (Wako, Osaka, Japan). TRAP-positive osteoclasts per bone surface, which were adherent to calcified cartilage (subchondral bone region) within a region containing a mediolateral width of 0.5 mm in the middle region of the medial tibia25, were counted. Furthermore, TRAP-positive osteoclasts per bone surface in osteophyte (osteophyte region) were also counted25. Immunostaining of type II collagen (Diluted 1:200; Fine Chemical Co., Toyama, Japan) was also performed according to the method as previously described18. In situ cell death detection (apoptosis) of articular cartilage and subchondral bone plate Articular cartilage and osteocyte apoptosis were assessed by TUNEL assay using an in situ apoptosis detection kit (Takara, Shiga,
Fig. 1. Effects of treadmill walking on the biomechanical properties of cartilage in the medial tibia. (A) Stress-displacement curve at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; **P < 0.01 sham vs DMM group; xP < 0.05, xxP < 0.01 sham vs DMM þ walking group). (B) Creep response of cartilage in the medial tibia (n ¼ 7; *P < 0.05, **P < 0.01).
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Japan) according to the manufacturer's instructions. The tissue sections were also incubated with propidium iodide (PI) to label the nuclei. Fluorescence micrographs were obtained using fluorescence microscopy (Fluoview FV10i, Olympus, Tokyo, Japan). Chondrocyte and osteocyte apoptosis were determined as the percentage of TUNEL-positive chondrocytes and osteocytes in the PI-labeled chondrocytes and osteocytes within a region with a mediolateral width of 0.5 mm and depths of 0.25 mm (chondrocyte) and 0.1 mm (osteocyte) from the cartilage and subchondral bone plate surface respectively, in the middle region of the medial tibia, by using the Image-J software. Statistical analysis Statistical analyses were performed with the JMP 11 software program (SAS Institute, Cary, NC USA) or R (the R Foundation for Statistical Computing, Vienna, Austria). To evaluate effects of treadmill walking on the DMM knee, we analyzed and compared biomechanical properties, subchondral bone changes, and cartilage degeneration among the following three groups: (1) DMM
operated knee of sedentary group (DMM group), (2) DMM operated knee of walking group (DMM þ walking group), and (3) sham operated knee of sedentary group (sham group). Graphical results were displayed as mean with uncertainly expressed by 95% confidence intervals for continuous data, and as medians and interquartile ranges for categorical data. All the continuous data were assessed for normality by using the ShapiroeWilk test and for homoscedasticity by using the Bartlett or a F-test. To evaluate the differences among the three groups, an analysis of variance with a subsequent post hoc Tukey-Kramer or Games-Howell test was used for parametric continuous data. Meanwhile, the KruskaleWallis test with subsequent post hoc Steel-Dwass test was used for nonparametric continuous data or categorical data. The unpaired ttest was used for pair-wise differences of the SBC diameter between the DMM and DMM þ walking groups. Welch correction was applied for variables with unequal variance. A Spearman's rank test was used to calculate the correlations between OARSI scores and E. Lac/mm2 in the superficial zone (0e100 mm) and the total zone (i.e., sum of the superficial to deep zones as the total zone; 0e400 mm) from the averaged OARSI scores and E. Lac/mm2 from three tissue
Fig. 2. The chondroprotective effects of treadmill walking. (A) Gross appearance of tibia in the sham, DMM, and DMM þ walking groups at 4 weeks after surgery. The surface of cartilage was smooth in the sham group and in the disrupted middle region of the medial tibia particularly in the DMM group (dot-line area). (B) Histological findings of toluidine blue section (a, c, e) and immunohistochemical staining of type II collagen (b, d, f) 4 weeks after surgery. AC, articular cartilage; SB, subchondral bone. Magnification: 100. Scale bars ¼ 100 mm. (C) Osteoarthritis development evaluated by OARSI scores. Boxplots display median values and interquartile ranges (n ¼ 7; *P < 0.05, **P < 0.01). (D) Osteophyte scores as evaluated by histological sections Boxplots display median values and interquartile ranges (n ¼ 7; *P < 0.05, **P < 0.01).
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Fig. 3. Osteocyte deaths in the subchondral bone, which was partly prevented by treadmill walking. (A) The number of empty osteocyte lacunae per square millimeter (E. Lac/mm2) according to depth at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; *P < 0.05, **P < 0.01). (B) The number of healthy osteocyte lacunae per square millimeter (Healthy Ot/mm2) according to depth at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; *P < 0.05). (C) The total number of osteocyte lacunae per square millimeter (Total Ot/mm2) according to depth at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; *P < 0.05, **P < 0.01). (D) The osteocyte lacunar occupancy (%) according to depth at 1 (a), 2 (b), and 4 (c) week(s) after surgery (n ¼ 7; *P < 0.05, **P < 0.01).
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sections per knee joint. Throughout the text, “n” refers to the number of animals. In all cases, P < 0.05 was considered statistically significant.
(P < 0.05); however, there were no significant differences at 1 (P ¼ 0.99) and 2 weeks (P ¼ 0.66) after surgery. Osteoarthritic changes in articular cartilage and osteophyte growth
Results Changes in biomechanical properties One week after surgery, there were no significant differences in the stressestrain curve among the three groups [Fig. 1(A)], but both the DMM and DMM þ walking groups had a significantly increased displacement after 2 and 4 weeks compared to the sham group (P < 0.05). Four weeks after surgery, the DMM þ walking group had a lower displacement compared to the DMM group; however, there were no significant differences between the DMM and DMM þ walking groups at any compressive stress value. The creep behaviors among groups were also evaluated at each time point [Fig. 1(B)]. From 2 weeks after surgery, the DMM group showed a significantly higher creep compared to the sham group (P < 0.01). The DMM þ walking group had a significantly lower creep compared to the DMM group 4 weeks after surgery
During gross observation 4 weeks after surgery [Fig. 2(A)], the surface of the cartilage was smooth in the sham group and had a disrupted middle region of the medial tibia with evidence of fibrillation particularly in the DMM group. The surface of the cartilage in DMM þ walking group seemed to be less macroscopic changes than those in DMM group. The histology of cartilage in the DMM group was characterized by vertical fissure reaching the deep zone, decreased chondrocytes, and strong proteoglycan depletion shown by the toluidine blue and type II collagen staining [Fig. 2(B)]. Cartilage in the DMM þ walking group had less vertical fissure, which was located only in the superficial zone, compared to the DMM group. The OARSI score [Fig. 2(C)] in the DMM þ walking group was significantly lower than that in the DMM group 4 weeks after surgery (P < 0.05). There were no significant differences in the osteophyte scores between the DMM and DMM þ walking groups at any of the time points [Fig. 2(D)].
Fig. 4. In situ cell deaths detected by TUNEL staining. (A) Fluorescence micrograph of the medial tibia 4 weeks after surgery. Although few apoptotic chondrocytes were confirmed in the sham group (aeb), there were many apoptotic chondrocytes confirmed particularly around the degenerated cartilage region in both the DMM (ced) and DMM þ walking groups (eef). AC, articular cartilage; SB, subchondral bone; BM, bone marrow. The white box represents a magnification of the osteochondral region shown on the right. Scale bars ¼ 100 mm. (B) Quantitative analysis of TUNEL staining of chondrocytes; number of PI-labeled chondrocytes (a), TUNEL-positive chondrocytes (b), and the percentage of TUNELpositive chondrocytes (c) (n ¼ 5; *P < 0.05, **P < 0.01).
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Histological analysis of subchondral bone plate In OA conditions, a lumber of empty osteocyte lacunae were confirmed especially in the superficial zone (0e100 mm from the osteochondral region) of subchondral bone plate, which were located underneath degenerated cartilage. Treadmill walking suppressed the increase of E. Lac/mm2 in the superficial zone from 2 to 4 weeks [Fig. 3(A)], which became significantly lower level compared to the DMM group 4 weeks after surgery (P < 0.05). Healthy Ot/mm2 and Total Ot/mm2 were higher in the DMM and DMM þ walking groups compared to the sham group [Fig. 3(B), (C)]; no significant differences were confirmed between the DMM and DMM þ walking groups at any of the time points. As with E. Lac/mm2, lacunar occupancy was decreased over time in the superficial zone in the DMM group [Fig. 3(D)], to a significantly lower level compared to the DMM þ walking group 4 weeks after surgery (P < 0.01). Additionally, healthy osteocyte lacunae surface in the DMM group was significantly larger compared to the sham and the DMM þ walking groups particularly in the superficial zone of the subchondral bone 4 weeks after surgery (Supplementary Fig. 1). There were no significant differences in empty osteocyte lacunae surface among three groups at any of the time points. There were significant correlations between the OARSI score and E. Lac/mm2 in the superficial zone (r ¼ 0.77, P < 0.01) as shown in Supplementary Fig. 2. This correlation became unclear when E. Lac/mm2 in the total zone (0e400 mm) was considered (r ¼ 0.58, P < 0.01). In situ cell deaths detected by TUNEL staining To examine the cell death mechanisms, particularly that of osteocytes on the subchondral bone surface as mentioned earlier [Fig. 3], we performed TUNEL staining [Fig. 4(A)]. However, there were few TUNEL-positive osteocytes in the subchondral bone of any group (Supplementary Fig. 3). Equally, many TUNEL-positive chondrocytes were confirmed in the
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cartilage of all groups [Fig. 4(A), (B)]. The number of PI-labeled chondrocytes was significantly lower in the DMM group compared to the sham group at 4 weeks after surgery (P < 0.01). The number of TUNEL-positive chondrocytes in the DMM group had a tendency to increase compared to the sham group at 4 weeks after surgery (P ¼ 0.06). The percentage of TUNELpositive chondrocytes in the DMM þ walking group was significantly lower than that in the DMM group at 4 weeks after surgery (P < 0.05). Micro-CT analysis of subchondral bone changes According to the micro-CT observation, SBCs were present in the medial tibia of all animals in the DMM and DMM þ walking groups underneath the region of degenerated cartilage throughout the time-course of the study [Fig. 5(A)]. There were no significant changes in Sb BV/TV and Sb thickness in the weight-bearing region of the three groups at 1 and 2 week(s) after surgery [Fig. 5(B)]. Four weeks after surgery, the Sb BV/TV in the DMM þ walking group was significantly higher than the DMM group (P < 0.05). Prominent subchondral bone thickening was confirmed in the DMM þ walking group at 4 weeks after surgery which was significantly higher compared to the sham group (P < 0.01), but not significantly different compared to the DMM group (P ¼ 0.06). The DMM surgery did not affect the parameters of subchondral trabecular bone which are epiphysis distal to the growth plate (Supplementary Fig. 4). The formation of subchondral bone perforations created by the SBCs in the medial tibia is shown in Fig. 6(A). There were no significant differences in the maximum and average SBC diameters between the DMM and DMM þ walking groups at 1 and 2 weeks after surgery [Fig. 6(B)]. The maximum SBC diameter was not different between the DMM and DMM þ walking group 4 weeks after surgery (P ¼ 0.53); however the DMM þ walking group tended to have a short average SBC diameter compared to the DMM group (P ¼ 0.06).
Fig. 5. Effect of treadmill walking on subchondral bone plate evaluated by micro-CT. (A) Micro-CT observations of the sham, DMM, and DMM þ walking groups 4 weeks after surgery. Subchondral bone cysts were confirmed in the middle region of the medial tibia in the DMM and DMM þ walking groups (white arrow head). (B) The subchondral trabecular bone volume fraction (Sb BV/TV) (a) and subchondral bone plate thickness (Sb thickness) (b) of the medial tibia at each time point (n ¼ 7; *P < 0.05, **P < 0.01).
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Fig. 6. Formation of SBCs. (A) Three-dimensional image of the subchondral bone of the tibial plateau 4 weeks after surgery. Several subchondral perforations were confirmed, particularly in the severe sample (black arrow head). Osteophyte mineralization was confirmed in all samples of DMM and DMM þ walking groups (white arrow head). (B) The maximum (a) and average (b) diameters of SBCs in the medial tibia after surgery (n ¼ 7).
Changes in TRAP and ALP staining
Discussion
Increased TRAP-positive osteoclasts were confirmed in SBCs of the DMM group after drilling towards the articular cartilage underneath the degenerated cartilage; however, this was not markedly observed in the DMM þ walking group [Fig. 7(A)]. The number of osteoclasts [Fig. 7(B)] in the subchondral bone region was significantly increased in the DMM group compared to the sham group from 2 weeks after surgery (P < 0.05), which was not pronounced in the DMM þ walking group at 2 (P ¼ 0.16) and 4 (P ¼ 0.09) weeks after surgery. In the osteophyte region, the DMM group also showed increased osteoclasts number over time; the DMM þ walking group had a significantly lower osteoclasts number compared to the DMM group 4 weeks after surgery (P < 0.05). ALP-positive osteoblasts were particularly observed in SBCs of the DMM group [Fig. 8]. ALP-positive chondrocytes in the articular cartilage were diminished underneath the degenerated cartilage particularly in the DMM group [Fig. 8(C) and (D)].
According to the best of our knowledge, this was the first study conducted to show that 4 weeks of gentle treadmill walking tended to suppress the growth of SBCs and TRAP-positive osteoclasts in subchondral bone surface. These preventive effects of treadmill walking coincide with the chondroprotective effects, as indicated by OARSI scores, TUNEL assay results, and changes in biomechanical properties. Furthermore, treadmill walking could suppress increasing osteocyte deaths induced by DMM surgery, which is positively correlated with OARSI scores. SBC growth, produced by osteoclasts, plays an important role in early stages of OA pathogenesis, which should be treated to prevent the further progression of OA26. Cox et al. showed SBC growth was proposed to be accelerated by the death of osteocytes that surround SBCs10. Also, other studies showed that existing osteoclast-created SBCs correspond with the presence of necrotic bone4, and that osteocyte debris could elevate osteoclastogenesis27. In the present study, we described that gentle treadmill walking could partly
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Fig. 7. Histological findings of TRAP staining. (A) Histological section of TRAP-positive osteoclasts (stained red-violet) in the medial tibia 4 weeks after surgery. There were few osteoclasts in the osteochondral region in the sham group (aeb); however, a high number of osteoclasts were confirmed in subchondral bone cysts located underneath degenerated cartilage, particularly in the DMM (ced) group compared to the DMM þ walking (eef) groups (black arrow head). Osteoclasts were also confirmed in the osteophyte region of the DMM group (geh). AC, articular cartilage; SB, subchondral bone; BM, bone marrow; OP, osteophyte. The black box represents a magnification of the figure on the right. Scale bars ¼ 100 mm. (B) Numbers of TRAP-positive osteoclasts per bone surface (N. Oc/BS) in subchondral bone region (a) and osteophyte region (b) (n ¼ 7; *P < 0.05, **P < 0.01 sham vs DMM group; xP < 0.05, xxP < 0.01 sham vs DMM þ walking group; yP < 0.01 DMM vs DMM þ walking group).
prevent subchondral bone osteocyte deaths in the superficial zone, which could contribute to the prevention of the SBC growth generated by osteoclasts in the DMM knee. Currently, there are few reports to directly explain the cause of these preventive effects of exercise on subchondral bone deterioration in vivo; however one possibility could be the chondroprotective effect of treadmill exercise through mechanical and biological interactions at the bone-cartilage interface. As shown in several studies, degenerated and softened cartilage might transmit excessive mechanical loading to the breached underlying subchondral bone surface resulting in osteocyte deaths, which further leads to SBC growth10. In addition to mechanical interactions, biological processes also affect the pathogenesis of structural changes through bone-cartilage molecular crosstalk. In a recent study, the RANKL was released by chondrocytes of degenerated cartilage28. RANKL acts in a paracrine manner on the subchondral bone plate through calcified cartilage, which results in osteoclast stimulation. Furthermore, TNF-a and IL-1, which are released by degenerated cartilage, could induce osteocyte deaths29. According to the mechanisms of the chondroprotective effect of treadmill walking, such as elevation of anti-apoptotic capacity11 and anti-oxidative stress capacity30, that have been discussed in previous in vivo studies, these might involve suppression of osteocyte deaths and osteoclast-induced SBC growth. However, we could not consider separately the effects of treadmill walking from its direct effects on the regulation of
subchondral bone homeostasis12,31. Further studies are needed to evaluate exercise effects along with various exercise intensities which could affect the effectiveness of exercise on cartilage and subchondral bone11,32,33. Osteocytes are known to regulate mineralization surrounding their lacunae. Lane et al. showed that elastic modulus of subchondral bone surrounding large osteocyte lacunae were reduced in glucocorticoid-treated mice, thereby increasing bone fragility34. These large osteocyte lacunae may be able to dissipate loads and may crack around the surface35. In the present study, we described the large osteocyte lacunae were confirmed in the DMM group particularly around micro-cracks but not so pronounced in the DMM þ walking group (Supplementary Fig. 1). Actually little is known about the mechanism of changes in osteocyte lacunae surface in musculo-skeletal pathologies, specifically regarding subchondral bone and exercise, our results indicate that increased osteocyte lacunae surface of subchondral bone in the DMM group may be associated with low mineral material strength that could cause OA development, and that gentle treadmill walking may prevent bone fragility via improvement of microenvironment surrounding osteocyte lacunae. Surprisingly, osteocyte apoptosis was not detected in the TUNEL staining even in the DMM and DMM þ walking groups which is consistent with a recent study reported by Boudenot et al.12 Several studies showed that necrotic bone was confirmed adjacent to osteoclastic bone resorption and degenerated cartilage4,36. These
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Fig. 8. Histological section of ALP staining in the medial tibia 4 weeks after surgery. ALP-stained cell (brown) was localized in hypertrophic chondrocytes or the subchondral bone marrow in the sham group (aeb). ALP-stained chondrocytes on the top of the subchondral cyst were diminished (arrow head) in DMM group (ced). Strongly ALP-stained osteoblasts were confirmed in subchondral bone cysts of the DMM group (d, black arrow) compared to the DMM þ walking group (eef). ALP-positive cells were increased in the osteophyte region in the DMM group (geh). AC, articular cartilage; SB, subchondral bone; BM, bone marrow; OP, osteophyte. The black box represents a magnification of the figure shown on the right. Scale bars ¼ 100 mm.
studies indicate that the cell death mechanisms might include factors other than apoptosis, such as osteonecrosis, and might not be affected by short-term exercise. To explain these cell death mechanisms in early OA warrant further studies. There are several limitations in the present study. Firstly, there was a lack of statistical power to determine the effects of treadmill walking; therefore, no significant differences, particularly regarding SBC growth and osteoclasts activity in subchondral bone region, were confirmed between the DMM and DMM þ walking groups. Secondly, the present study involved short-term intervention periods in the early stage of OA which is a transient period. Hence, we could not affirm the effectiveness of gentle treadmill exercise in late stage of OA. Therefore, the effects of long-term treadmill walking on cartilage and subchondral bone should be confirmed. Thirdly, we used an experimental rat model; thus, our findings may not translate directly to humans. Finally, there are
controversies regarding the initial structural changes in the DMM knee and whether these alterations first occur in the subchondral bone or the articular cartilage. In conclusion, short-term gentle treadmill walking suppressed increasing osteocyte deaths and had a tendency to prevent SBC growth generated by osteoclasts in the DMM knee. These changes coincide with the prevention of cartilage degeneration, including biomechanical softening. Interestingly, there was a positive correlation between cartilage degeneration and osteocyte deaths, indicating that biomechanical and biological links exist between cartilage and subchondral bone, and the preventive effects of treadmill walking on subchondral bone deterioration might be partly explained by the chondroprotective effect. Further researches are required for investigating the effects of gentle treadmill walking on subchondral bone changes in an instabilityinduced OA.
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Author contributions All authors have made substantial contributions to (1) the conception and design of the study, or acquisition of data, or analysis and interpretation of data; (2) drafting the article or revising it critically for important intellectual content; and (3) final approval of the version to be submitted. The specific contributions of the authors are as follows: (1) Conception and design of the study: HI, TA, AI, and SY. (2) Analysis and interpretation of the data: HI, TA, AI, MN, XZ, SY, and HK. (3) Drafting of the article: HI, TA, JT, and HK. (4) Critical revision of the article for important intellectual content: HI, TA, AI, MN, and HK. (5) Final approval of the article: HI, TA, and HK. (6) Statistical expertise: HI, TA, and JT. (7) Obtaining of funding: HK. (8) Collection and assembly of data: HI.
Role of the funding source This study was supported in part by a JSPS KAKENHI Grant-in-Aid for Scientific Research (A) (number 20240057) and a JSPS KAKENHI Grant-in-Aid for Challenging Exploratory Research (number 25560258). Conflict of interest The authors have no competing interests. Acknowledgements This study was supported in part by a JSPS KAKENHI Grant-inAid for Scientific Research (A) (no. 25242055) and a JSPS KAKENHI Grant-in-Aid for Challenging Exploratory Research (no. 25560258). Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.joca.2015.04.015. References 1. 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. Osteoarthritis Cartilage 2014;22:1077e89. 2. Pan J, Zhou X, Li W, Novotny JE, Doty SB, Wang L. In situ measurement of transport between subchondral bone and articular cartilage. J Orthop Res 2009;27:1347e52. 3. 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. Osteoarthritis Cartilage 2014;22: 1036e43. 4. McErlain DD, Ulici V, Darling M, Gati JS, Pitelka V, Beier F, et al. An in vivo investigation of the initiation and progression of subchondral cysts in a rodent model of secondary osteoarthritis. Arthritis Res Ther 2012;14:R26. 5. Bloecker K, Guermazi A, Wirth W, Benichou O, Kwoh CK, Hunter DJ, et al. Tibial coverage, meniscus position, size and damage in knees discordant for joint space narrowing e data from the Osteoarthritis Initiative. Osteoarthritis Cartilage 2013;21:419e27.
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